Lateral flow device

Abstract

To provide a highly homogeneous lateral flow device which is light-weight, inexpensive, and is suitable for mass production and image analysis.SOLUTION: A lateral flow device is obtained by pasting light-transmissive plastic plates together, at least one surface of which being textured with a hydrophobic material, on the textured surface(s) so as to secure a gap that may allow a liquid to spread by capillary action.SELECTED DRAWING: Figure 1-1

Description

【Technical Field】 【0001】 The present invention relates to a lateral flow device and a method for quantifying a detection target using the same. 【Background Art】 【0002】 The immunochromatography method is applied as an immunochromatography device in antigen detection kits such as pregnancy tests and influenza virus, and is widely used in the medical field. However, since conventional immunochromatography devices utilize capillary action for sample development, filters made of opaque materials such as glass fiber and nitrocellulose have been used (Patent Document 1). Therefore, the detection of the measurement target has mainly been performed visually, and quantification has been difficult. 【0003】 In order to solve the above problems, in recent years, immunochromatography devices using glass plates have been developed. For example, Patent Document 2 discloses a device composed of a rough glass plate. By wetting the glass plate with a sample solution, the translucent plate is made transparent or nearly transparent, and the measurement target in the sample is captured by an antigen fixed on the rough surface, and quantification of the measurement target is realized by digitizing it as the amount of transmitted light when irradiated from the outside to the device. 【0004】 However, the rough surface processing of the glass surface is processed by sandblasting or the like, so the unevenness of the glass surface becomes non-uniform between plates. As a result, even when trying to scan the glass surface or digitize the measurement target captured by image analysis after imaging, scattered light and reflected light vary for each device, resulting in variations in the obtained numerical values and making it difficult to perform highly accurate quantification with reproducibility. There was also a problem that glass is difficult to process finely, and when mass-producing homogeneous products, the manufacturing cost becomes high. Furthermore, it also included the problem that the specific gravity of the glass itself is large and it is not easy to transport or handle because it is vulnerable to impact. 【Prior Art Documents】 [Patent Documents] 【0005】 [Patent Document 1] Japanese Patent Publication No. 2011-257138 [Patent Document 2] Patent No. 6039965 [Overview of the Initiative] [Problems that the invention aims to solve] 【0006】 The present invention aims to develop and provide a lightweight, inexpensive, and highly homogeneous lateral flow device suitable for mass production and image analysis. 【0007】 The present invention aims to develop and provide a method for optically detecting and quantifying a target substance in a sample with high sensitivity and accuracy. [Means for solving the problem] 【0008】 The inventors have diligently conducted research and succeeded in solving the above problem by using a light-transmitting plastic plate with a hydrophobic, textured surface instead of the conventional rough-surfaced glass plate. Light-transmitting plastics such as PET resin have a lower specific gravity than glass, so the weight per device can be reduced to about 1 / 10 of that of glass. Furthermore, plastic plates are easy to mold and process, and the texture can be reproducibly formed in specific areas of the surface of each plate, making it possible to stably and mass-produce homogeneous devices. Moreover, by using this device, the target substance in a sample can be detected optically with higher sensitivity and quantified with higher accuracy compared to conventional methods. The present invention is based on the results of this development and provides the following. 【0009】 (1) A lateral flow device comprising light-transmitting plastic plates, each having a hydrophobic textured surface, bonded together on the textured surfaces to maintain gaps through which a liquid can spread by capillary action. (2) The lateral flow device according to (1), comprising an introduction section for introducing a sample, an expansion section for the introduced sample to spread between the sections, a fixing section for fixing a detection agent that reacts with the object to be detected in the sample to the surface of the texture, a detection section for detecting the reaction product of the object to be detected in the sample and the detection agent, and a recovery section for collecting sample waste liquid. (3) The lateral flow device according to (1) or (2), comprising a spacer that provides the gap between the light-transmitting plastic plates. (4) The lateral flow device according to any one of (1) to (3), wherein a portion of the light-transmitting plastic surface is textured. (5) The lateral flow device according to any one of (1) to (4), wherein the bonding surface of the light-transmitting plastic plate is made of a hydrophilic material. (6) The lateral flow device according to any one of (1) to (5), wherein the depth of the texture is 0.3 μm to 1.5 μm. (7) The lateral flow device according to any one of (3) to (6), wherein the thickness of the spacer is 5 μm to 30 μm. (8) The lateral flow device according to any one of (2) to (7), comprising two or more of the fixing parts on which different detection agents are fixed. (9) A lateral flow device according to any one of (2) to (8), wherein the detection agent is labeled. (10) A lateral flow device according to any one of (2) to (9), wherein the detection agent is a nucleic acid, peptide, protein, or low molecular weight compound. (11) The lateral flow device according to (10), wherein the nucleic acid is a nucleic acid aptamer or a nucleic acid fragment containing a nucleic acid binding domain recognition sequence. (12) The lateral flow device according to (10), wherein the peptide or protein is an antigen, an antibody or an active fragment thereof, a ligand, or a receptor or a ligand-binding region thereof. (13) The lateral flow device according to any one of (2) to (12), wherein the object to be detected is a nucleic acid, peptide, protein, or low molecular weight compound. (14) A method for quantifying a target substance in a sample using a lateral flow device described in any of (1) to (13), comprising: an introduction step of introducing a sample from an introduction section; a detection step of detecting a reaction product between the target substance and a detection agent in a detection section; and a measurement step of measuring the amount of the target substance in the sample based on the detection result in the detection step. (15) The quantitative method according to (14), wherein the detection of the reaction product is based on fluorescence, luminescence, or color development emitted from the reaction product. (16) The quantitative method according to (15), wherein the amount of the object to be detected is measured based on fluorescence intensity, luminescence intensity, or color density. [Effects of the Invention] 【0010】 The lateral flow device of the present invention provides a lightweight, homogeneous device suitable for image analysis and color density (absorbance) measurement, which can be provided stably and in large quantities at a low cost. [Brief explanation of the drawing] 【0011】 [Figure 1-1] This is a schematic diagram of the lateral flow device of the present invention. A is a top view, B is a bottom side view, and C is a side view. This diagram shows a basic embodiment in which the introduction section (0101), deployment section (0102), fixing section (0103), detection section (0104), and recovery section (0105) that constitute the lateral flow device (0100) exist independently on the device. [Figure 1-2] This is a schematic diagram showing another embodiment of the lateral flow device of the present invention. A is a top view, B is a bottom side view, and C is a side view. This diagram shows an embodiment in which the fixed part (0103) and the detection part (0104) of the lateral flow device (0100) are integrated into one unit. [Figure 2] This is a flowchart of the method for quantifying the target substance according to the present invention. [Figure 3]This is a schematic diagram showing the components of the lateral flow device (0300) used in the embodiment of the present invention. A shows a top view, B shows a bottom side view, and C shows a side view. The lateral flow device (0300) illustrated here is composed of a light-transmissive plastic plate (0301 / 0302) and a spacer (0304). One of the light-transmissive plastic plates (0302) has a ribbed pattern (0303). In addition, in FIG. 3, the introduction part (0101) and the recovery part (0105) shown in FIG. 1 are also illustrated. The lateral flow device (0300) shown in FIG. 3 has the fixing part and the detection part integrated as in the embodiment shown in FIGS. 1-2. 【Mode for Carrying Out the Invention】 【0012】 1. Lateral Flow Device 1-1. Overview The first aspect of the present invention is a lateral flow device. The lateral flow device of the present invention is characterized in that it is composed of a light-transmissive plastic plate whose surface is ribbed with a hydrophobic material. According to the present invention, it is possible to provide a homogeneous and low-cost device suitable for image analysis and absorbance measurement. 【0013】 1-2. Definitions The following terms used in this specification are defined. As used in this specification, the term "lateral flow device" (often abbreviated as "device" in this specification) refers to a sample measurement device that detects or analyzes a biological sample contained in a liquid sample by a capture body, a catalyst body, etc. fixed in the device during the process in which a liquid sample added to the device end is developed in the device by capillary action. Usually, since an antibody is fixed in the device as a capture body and is used for detecting an antigen in a sample by an antigen-antibody reaction, it is also called a device for immunochromatography or a test strip. However, in the device of this specification, it is not limited to an antibody and may include an antigen, an enzyme, a nucleic acid aptamer, etc. as a capture body or a catalyst. [[ID=二十一]] [[ID=二十二]] 【0014】 [[ID=二十三]] In this specification, "light-transmissive plastic" refers to a synthetic resin having the property of transmitting light. The light transmittance of the light-transmissive plastic in this specification is not limited as long as it transmits light, but it may be 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more. Usually, a semi-transparent resin to a transparent resin of 60% or more is selected. Specifically, for example, polymethyl methacrylate (PMMA: acrylic resin), polyethylene terephthalate (PET), polycarbonate (PC), polyvinyl chloride (PVC), polyacrylate (PA), polystyrene (PS), polyethylene (PE), and polypropylene (PP) etc. may be mentioned. The light-transmissive plastic in this specification may be either thermoplastic or thermosetting, but a material with easy moldability and processability is preferred. 【0015】 In this specification, "texture" refers to a fine uneven pattern applied to the surface of a material. 【0016】 In this specification, "texturing" refers to surface-processing a texture on a light-transmissive plastic plate. 【0017】 In this specification, "hydrophobic material" refers to a material with low affinity for water. Many plastics usually correspond to hydrophobic materials. The plastics exemplified in the above light-transmissive plastics are also hydrophobic. In this specification, hydrophobic plastics are referred to as hydrophobic plastics. 【0018】 In this specification, "hydrophilic material" refers to a material with high affinity for water. For example, glass etc. may be mentioned. On the surface of a hydrophilic material, water does not be repelled, but forms a film of water and spreads thinly. 【0019】 In this specification, "sample" refers to a test object to be analyzed or detected in the device of the present invention. The sample is not limited as long as it is a substance that can contain the detection object. For example, it includes cells, tissues, extracts obtained by extracting the detection object from body fluids, and includes sewage, seawater, lake water, etc. 【0020】 In this specification, "sample waste liquid" refers to a sample that has been subjected to the device of the present invention and has undergone analysis or detection. In principle, this refers to a sample that has passed through the detection unit of the device of the present invention. 【0021】 In this specification, "body fluid" refers to a biological sample in liquid form. Examples include blood (including serum, plasma, and interstitial fluid), cerebrospinal fluid, urine, lymph, digestive fluid (including saliva), sweat, nasal secretions, tears, semen, vaginal fluid, periradicular fluid, ascites, pleural fluid, and extracts of various tissues or cells. 【0022】 In this specification, "substance to be detected" refers to a substance to be detected by the lateral flow device of the present invention. The type of substance to be tested is not limited, but examples include biological substances that may be contained in the sample. 【0023】 In this specification, "biologically derived substances" refers to substances produced by the metabolism of living organisms. Examples include peptides, proteins, sugars, lipids, and low-molecular-weight compounds. 【0024】 In this specification, "detection agent" refers to a drug that binds to or reacts with the target substance in the sample. Examples include nucleic acids, peptides, proteins, or low-molecular-weight compounds. 【0025】 In this specification, "nucleic acid" generally refers to a biomolecule composed of nucleotides linked together by phosphodiester bonds. Typically, this refers to naturally occurring nucleic acids such as DNA, which is composed of deoxyribonucleotides having one of the bases adenine (A), guanine (G), cytosine (C), and thymine (T), and / or RNA, which is composed of ribonucleotides having one of the bases adenine, guanine, cytosine, and uracil (U), all of which are naturally occurring nucleotides. 【0026】 In this specification, "peptide" refers to an amino acid polymer in which 2 to 20 amino acids are linked together in a chain by peptide bonds. In this specification, it is synonymous with "oligopeptide." 【0027】 In this specification, "protein" refers to an amino acid polymer in which 21 or more amino acids are linked together in a chain by peptide bonds. In this specification, it is synonymous with polypeptide. 【0028】 In this specification, "nucleic acid-binding protein" refers to a protein that contains a nucleic acid-binding domain, recognizes a specific binding sequence present on a nucleic acid sequence such as genomic DNA, and binds to it. Examples include transcription factors. The nucleic acid-binding domain may be either a DNA-binding domain or an RNA-binding domain. Examples of specific structural motifs that constitute the nucleic acid-binding domain include zinc fingers, leucine zippers, β-hairpins, helix-turn helixes, helix-loop helixes, wing helixes, HMG boxes, RRM domains, and TAL effectors. 【0029】 In this specification, "low molecular weight compound" refers to a natural compound or chemically synthesized product with a molecular weight of approximately several hundred to several thousand. The molecular structure and type are not particularly limited. 【0030】 In this specification, "nucleic acid aptamer" refers to an aptamer composed of nucleic acids that has the ability to bind strongly and specifically to a target substance based on the three-dimensional structure formed based on the secondary and tertiary structures of a single-stranded nucleic acid molecule via hydrogen bonds, etc. In this specification, nucleic acid aptamers that bind strongly and specifically to a target substance (e.g., peptide or protein) contained in a sample are included. While RNA aptamers and DNA aptamers are generally known as nucleic acid aptamers, the nucleic acids constituting the nucleic acid aptamer in this specification are not particularly limited. It may include DNA aptamers, RNA aptamers, or aptamers composed of a combination of DNA and RNA. 【0031】 In this specification, "nucleic acid-binding domain recognition sequence" refers to a target base sequence that a nucleic acid-binding protein recognizes and binds to via its encompassing nucleic acid-binding domain. Nucleic acid-binding domain recognition sequences exist on the genome or RNA and are composed of specific base sequences such as palindromic sequences. Specific examples of nucleic acid-binding domain recognition sequences include transcription factor-binding sequences contained within promoters. The nucleic acid-binding domain includes a DNA-binding domain that binds to DNA and an RNA-binding domain that binds to RNA. Examples of specific structural motifs that constitute the nucleic acid-binding domain include, but are not limited to, zinc fingers, leucine zippers, β-hairpins, helix-turn helixes, helix-loop helixes, wing helixes, HMG boxes, RRM domains, and TAL effectors. 【0032】 In this specification, "nucleic acid-binding domain-recognizing nucleic acid" refers to the target nucleic acid of the nucleic acid-binding domain. 【0033】 1-3. Components The components of the lateral flow device of the present invention are shown in Figure 3. As shown in this figure, the lateral flow device (0300) of the present invention includes a light-transmitting plastic plate (0301 / 0302) as an essential component and a spacer (0304) as an optional component. Each component will be described in detail below. 【0034】 (1) Light-transmitting plastic plate In this specification, "light-transmitting plastic plate" (often abbreviated as "plate" in this specification) (0301 / 0302) means a plate-shaped member made of light-transmitting plastic that determines the external shape of the device of the present invention. 【0035】 In the device of the present invention, the plate may be composed of a single light-transmitting plastic material, or it may be composed of a combination of two or more different light-transmitting plastic materials. An example of the latter is a two-layer plate made of two different types of light-transmitting plastics. 【0036】 The overall shape of the plate is generally the shape of the device of the present invention. The specific shape is not particularly limited; it should be tailored to the shape required for the device. Examples include rectangles, squares, polygons, circles, ellipses, irregular shapes, or combinations thereof. Rectangles are generally preferred. 【0037】 While there are no restrictions on the size of the plate, it should be within the range of typical device sizes. For example, if the device is rectangular, it should be within the range of 20mm-100mm x 5mm-18mm, 30mm-80mm x 8mm-15mm, or 40mm-60mm x 10mm-15mm. 【0038】 The thickness of the light-transmitting plastic plate is not particularly limited, as long as it maintains the low cost, low weight, and homogeneity that are the effects of the device of the present invention. Typically, it may be 10 μm or more, 20 μm or more, 30 μm or more, 40 μm or more, 50 μm or more, 100 μm or more, 200 μm or more, 300 μm or more, 400 μm or more, 500 μm or more, 600 μm or more, 700 μm or more, 800 μm or more, 900 μm or more, or 1 mm or more, and may also be 4 mm or less, 3.5 mm or less, 3.0 mm or less, 2.5 mm or less, 2.0 mm or less, or 1.5 mm or less. 【0039】 As described in "1-4. Configuration" below, the device of the present invention generally comprises at least two plates. In the device of the present invention, the surface of at least one of these plates (0302) is textured (0303) with a hydrophobic material. The textured surface can be any textured surface known in the art. The device of the present invention is characterized by the homogeneity of each mass-produced device. Since the textured surface is a part that forms a channel that is directly related to its homogeneity, the processing method is not limited as long as it can copy the same or near-same shape of texture to each individual device. Usually, the textured surface formed on the mold during the molding of the plastic plate can be transferred to the surface, or the textured surface formed on the mold can be transferred to the surface of the molded plastic plate by press processing. 【0040】 The textured surface only needs to be applied to one or both sides of the plate, either entirely or partially. 【0041】 In the device of the present invention, the textured surface formed by the texturing process provides a rough surface to the plate. 【0042】 The shape of the texture on the plate is not limited. It may be an irregular shape such as wrinkles, or it may be a shape composed of multiple convex and / or concave parts having a specific shape such as a cube or hemisphere. Alternatively, it may be a shape in which a specific pattern consisting of multiple textures of the same and / or different shapes is repeated. 【0043】 The depth of the texture, that is, the distance between the highest point of the convex part and the lowest point of the concave part constituting the texture, may be, for example, 0.3 μm or more, 0.5 μm or more, 0.6 μm or more, 0.7 μm or more, 0.8 μm or more, or 0.9 μm or more, and may also be 1.5 μm or less, 1.4 μm or less, 1.3 μm or less, 1.2 μm or less, 1.1 μm or less, or 1.0 μm or less. 【0044】 If the texture is composed of a plurality of protrusions or recesses having a specific shape, the spacing between the protrusions or recesses may be 10 μm or more, 20 μm or more, 30 μm or more, 40 μm or more, 50 μm or more, 60 μm or more, 70 μm or more, 80 μm or more, 90 μm or more, or 100 μm or more, and may also be 1000 μm or less, 900 μm or less, 800 μm or less, 700 μm or less, 600 μm or less, 500 μm or less, 400 μm or less, 300 μm or less, or 200 μm or less. 【0045】 In the plate, at least the textured areas are made of hydrophobic plastic. While the surface of the hydrophobic plastic is water-repellent, peptides, proteins, and other substances can be adsorbed and immobilized on its surface by hydrophobic bonding without requiring any special pretreatment. 【0046】 On the other hand, the bonding surfaces of the plates, that is, the surfaces on which the plates face each other to construct the device of the present invention, may be made of a hydrophilic material in areas other than the textured areas. In the device of the present invention, each part, such as the introduction section described later, is formed in the narrow gap between the plates. If the bonding surface is made of a hydrophilic material, it is preferable because water-soluble biological samples such as urine can easily spread within that gap. The hydrophilic material may be a hydrophilic light-transmitting plastic (for example, polyethylene terephthalate), or a hydrophilic coating formed on the surface of a hydrophobic light-transmitting plastic. For example, a silica-based coating such as glass can be used. 【0047】 (2) Spacer In this specification, "spacer" (0304) is a selective component in the device of the present invention that provides a gap between the plates through which a liquid can spread by capillary action. The spacer is placed between the plates to be bonded and provides the gap by its own thickness. 【0048】 The thickness of the spacer is not limited as long as it is thick enough for the liquid to spread by capillary action. For example, thicknesses of 5μm to 30μm, 6μm to 28μm, 7μm to 26μm, 8μm to 24μm, 9μm to 22μm, 10μm to 20μm, 12μm to 18μm, or 14μm to 16μm are acceptable. It is preferable that the thickness of the spacer be uniform throughout, but for example, there may be a gradient in thickness from the introduction section to the detection section, as described later. 【0049】 The shape of the spacer is not particularly limited as long as it can provide a uniform gap throughout the device of the present invention. For example, to ensure a flow path for the sample in the device, it may be linear in shape along the edges of the device. As a specific example, if the device is rectangular, the spacer may be string-like in shape along its long side. The entire spacer does not need to be of equal width. 【0050】 The material of the spacer is not particularly limited, as long as it does not dissolve or denature due to the sample. It also does not need to be light-transmitting. For example, plastic (including epoxy resin), metal, glass, etc., would suffice. Double-sided tape can be used as a specific example of a spacer. 【0051】 1-4. Structure The lateral flow device of the present invention has a structure in which opposing light-transmitting plastic plates are bonded together while maintaining a gap that allows liquid to spread by capillary action. This gap forms a flow path in the device and also houses a fixing section, a spreading section, a detection section, and a recovery section, which will be described later. In this case, the textured surface of the plate faces the inside of the gap. 【0052】 The plates constitute the main body of the device. The number of plates that make up the device is not limited. Typically, two plates of the same or nearly identical shape are placed facing each other to form the device, but it may also be composed of one or three or more light-transmitting plastic plates. In the case of one plate, for example, one example is a configuration in which the plate is folded in half with the textured surface facing inward. In the case of three plates, one example is a configuration in which two plates are bonded to one plate, facing each other. This applies to cases where two devices are formed on a single plate, for example. In the latter case, the shapes of the opposing plates are different. 【0053】 Figure 1 shows a schematic diagram of one embodiment of the lateral flow device of the present invention. As shown in this figure, the device (0100) of the present invention includes an introduction section (0101), an deployment section (0102), a fixing section (0103), a detection section (0104), and a recovery section (0105). Each section will be described below. 【0054】 1-4-1. Introduction The "introduction section" (0101) is a section in the device of the present invention for introducing a sample, and is configured as a section that is open to the outside. 【0055】 While there are no restrictions on the placement of the introduction section in the device, it is preferable to place it near the end of the device, considering that the device detects or analyzes a biological sample during the process of spreading a liquid sample by capillary action. 【0056】 The configuration of the introduction section is not limited. It may be a hole provided in a plate constituting the top surface of the device, penetrating from the outside to the gap, or it may be the edge of the gap in a bonded plate. The shape of the introduction section is not limited. For example, in the case of the hole, it may be square, circular, polygonal, or any other shape. 【0057】 The size of the entry point is also not limited. It can be determined appropriately considering the size of the device, the type and / or volume of the sample to be introduced, etc. If the entry point is the end of the gap as described above, the size of the opening will be the entry point. If the entry point is the hole as described above, the diameter or the length of the long side may be 1 / 2, 1 / 3, or 1 / 4 of the short side of the device. 【0058】 The detection and / or analysis of the target substance in the sample is initiated when a liquid sample is introduced into the gap between the devices from the introduction section. 【0059】 1-4-2. Development Section The "deployment section" (0102) is the section for which the sample introduced into the device expands between the various parts, and is configured as the main flow path within the device. The "various parts" referred to here refer to the spaces between other parts, excluding the said deployment section, such as between the introduction section and the fixing section, between the fixing section and the detection section, and between the detection section and the recovery section. 【0060】 The development area consists of a region within the gap between plates that does not constitute other parts. When the sample is an aqueous solution, it is preferable that the development area be made of a hydrophilic material so that the sample can spread smoothly through the gap. 【0061】 The sample introduced from the introduction section is spread by capillary action through the expansion section, passing through the fixing section and detection section to the recovery section. 【0062】 1-4-3. Fixed part The "fixing portion" (0103) is a portion having a textured surface in the device of the present invention, and by fixing the detection agent to the surface of the texture, it provides a reaction site between the target substance in the introduced sample and the detection agent. Therefore, in the device of the present invention, the texture is present in at least the region corresponding to the fixing portion, and the detection agent is fixed only to the texture in that region. In this case, the recesses of the texture constitute a flow path in the fixing portion. 【0063】 In the device, the fixed part is positioned between the introduction part and the detection part. As mentioned above, the deployment part is interposed between the introduction part and the fixed part, and between the fixed part and the detection part, but the fixed part and the detection part may be adjacent to each other or integrated as described later. 【0064】 As mentioned above, a detection agent is a drug that can specifically capture or detect a target substance in a sample by binding to or reacting with it. Examples include nucleic acids, peptides, proteins, or small molecule compounds. More specifically, for nucleic acids, examples include nucleic acid aptamers or nucleic acid fragments containing nucleic acid binding domain recognition sequences, and for peptides or proteins, examples include antigens, antibodies or their active fragments, ligands, receptors or their ligand-binding regions. 【0065】 The detection agent fixed to the fixed part may be labeled as needed. Depending on the type of detection agent, labeling substances known in the relevant art may be used. 【0066】 The labeling site of the detection agent can be appropriately determined according to the type of detection agent and the characteristics and intended use of the labeled substance. For example, if the detection agent is a nucleic acid, the phosphate group, sugar, and / or base can be labeled. The 5' end and / or 3' end are particularly suitable as labeling sites. If the detection agent is a peptide or protein, the constituent amino acid residues can be labeled. For peptides or proteins, the side chains of each amino acid residue, the amino group of the N-terminal amino acid residue, or the carboxyl group of the C-terminal amino acid residue are suitable as labeling sites. Furthermore, if the detection agent is a low-molecular-weight compound, the functional groups it contains are suitable as labeling sites. 【0067】 Any substance known in the field can be used as the labeling material. Examples include radioisotopes, fluorescent substances, quenchers, chemiluminescent substances, DIG, biotin, or magnetic beads. 【0068】 A "radioactive isotope" refers to an isotope with a different mass number that emits radiation. For example, 32 P, 3 H, 14 C is one example. 【0069】 A "fluorescent substance" is a substance that becomes excited by absorbing excitation light of a specific wavelength and emits fluorescence when it returns to its original ground state. Examples include FITC, rhodamine, fluorescein, fluorescein, Texas Red®, Cy3, Cy5, Cy7, FAM, HEX, JOE, ROX, TET, Bodipy493, NBD, TAMRA, Quasar®, CAL Fluor® Red610, SYBR Green®, Eva Green®, and SYTOX Green®. 【0070】 A "quencher" refers to a substance that has the property of absorbing the excitation energy of the fluorescent substance and suppressing fluorescence. Examples include AMRA, DABCYL, BHQ-1, BHQ-2, or BHQ-3. 【0071】 A "chemiluminescent substance" is a substance that, after being excited by a chemical reaction, releases the difference in energy as light when it returns to its ground state. Examples include acridinium esters. 【0072】 The detection agent can be labeled at multiple locations with the same or two or more different labeling substances. The target substance bound to the detection agent labeled with the labeling substance can serve as an indicator when detecting the complex or reaction resulting from the binding reaction or chemical reaction between the target substance and the detection agent in the sample using the detection unit described later. 【0073】 The method for immobilizing the detection agent onto the texture is not particularly limited. As mentioned above, it can be immobilized via hydrophobic coupling, or it can be immobilized using immobilization methods known in the art. For example, it can be immobilized using the silane coupling method. 【0074】 The device of the present invention may include two or more fixed parts. In this case, the detection agent fixed to the texture of each fixed part may be the same or different. Furthermore, the arrangement of each fixed part in the device is not limited. They may be arranged in parallel between the introduction part and the detection part, or in series. Alternatively, a first fixed part may be arranged between the introduction part and the detection part, and a second fixed part and a subsequent second detection part may be arranged between the detection part and the recovery part. 【0075】 If the sample introduced from the introduction section contains the target object, the target object will react specifically with the detection agent fixed to the texture as the sample passes through the texture of the fixing section. 【0076】 The reaction between the target substance and the detection agent at the fixed site includes binding reactions or chemical reactions. Binding reactions include, but are not limited to, antigen-antibody reactions (immune reactions), ligand-receptor binding reactions, nucleic acid-peptide (protein) binding reactions, etc. Chemical reactions include, but are not limited to, catalytic reactions. Catalytic reactions include, for example, decomposition reactions, phosphorylation reactions, luminescence reactions, fluorescence reactions, or color development reactions. 【0077】 A reaction product is generated by the reaction between the substance to be detected and the detection agent. 【0078】 1-4-4. Detection Unit The "detection unit" (0104) is a unit that detects reaction products generated by the reaction between the target substance in the sample and the detection agent at the fixed unit. Detection of the reaction products in the detection unit makes it possible to determine the presence or absence of the target substance in the sample and / or to quantify it. 【0079】 The detection unit is configured to visualize or measure the reaction products. For example, it may include a substrate substance that is activated by the reaction products and emits light or changes color. 【0080】 The detection of reaction products can be achieved by detecting or measuring indicators that directly or indirectly indicate their presence or abundance. These "indicators" are not limited to, but include, for example, dye intensity, luminescence intensity, fluorescence intensity, absorbance, and radiation intensity. 【0081】 In the device, the detection unit is basically positioned between the fixed unit and the recovery unit, with a deployment unit between them. However, as mentioned above, the detection unit may be adjacent to or integrated with the fixed unit. An example of integration is when the indicator is immediately generated in the fixed unit by a reaction between the object to be detected and the detection agent. 【0082】 1-4-5. Collection Section The "recovery unit" (0105) is a unit that recovers the sample waste liquid. By recovering the unwanted sample waste liquid, the recovery unit prevents backflow of the sample waste liquid and maintains the spreading force and spreading direction of the sample generated by capillary action within the device. 【0083】 The device of the present invention functions by the fact that a liquid sample introduced through the introduction section gains a spreading force through capillary action. This capillary action disappears when the sample fills the gaps within the device, and at the same time, the spreading force of the sample is lost. 【0084】 In the recovery section, the sample waste liquid, after analysis or reaction is complete, is taken in by capillary action, allowing the sample to continue to be subjected to dispersive force even after the gaps within the device are filled with the sample. By determining the position of the recovery section within the device, the flow direction of the sample from the introduction section to the recovery section can be controlled. Normally, in order to make the most efficient use of the gaps within the device, the introduction section and the recovery section are placed at the furthest points from each other within the device, for example, at opposite ends, although this is not a limitation. 【0085】 The collection section is filled with a highly absorbent material to collect and retain the sample waste liquid. Examples include aggregates of plant fibers and / or animal fibers, and superabsorbent polymers (e.g., sodium polyacrylate). 【0086】 The recovery unit can be configured to be detachable from the device as needed. If the recovery unit exceeds the sample waste liquid recovery capacity, the sample dispersal force due to capillary action within the device can be maintained by replacing it with a new recovery unit. 【0087】 2. Method for quantifying the detected substance 2-1. Overview A second aspect of the present invention is a method for quantifying a target substance. In the method of the present invention, the target substance in a sample is quantified using the lateral flow device described in the first aspect. According to the method for quantifying a target substance of the present invention, by using the homogeneous device described in the first aspect, which is composed of a light-transmitting plastic plate, it is possible to quantify the target substance optically with high accuracy. 【0088】 2-2.Process Figure 2 shows a flowchart of the method for quantifying a target substance according to the present invention. As shown in this figure, this method includes an introduction step (S0201), a detection step (S0202), and a measurement step (S0203) as essential steps. Each step will be explained below. 【0089】 2-2-1.Introduction process The "introduction step" (S0201) is a step of introducing a sample from the device introduction section described in the first embodiment. 【0090】 The sample to be introduced is, in principle, a liquid, preferably an aqueous solution. However, even if the sample is in solid form, it can be used as a sample for this method by dissolving or suspending it in a solvent such as water to make it a liquid. 【0091】 Sample introduction can be achieved by inserting or dropping the sample into the introduction area using a pipette, syringe, or the like. 【0092】 2-2-2. Detection Process The detection step (S0202) is a step in which the reaction product of the target substance and the detection agent in the detection unit is detected. The sample introduced from the introduction unit reaches the fixed unit via the deployment unit inside the device. In the fixed unit, the detection agent fixed to the texture reacts with the target substance in the sample, producing a reaction product. In this step, the reaction product is detected through the detection unit of the device. Detection may be performed, for example, based on fluorescence, luminescence, or color development directly or indirectly emitted from the reaction product, or based on the intensity of transmitted light irradiated onto the detection unit from the outside. 【0093】 The detection method is not limited. The indicator may be detected by visual inspection, or it may be detected using a detection element such as an image sensor or a photodiode. The device of the present invention is composed of a light-transmitting plastic plate, and the detection unit is also light-transmitting. Therefore, a method in which transmitted light that has passed through the detection unit is detected by an imaging device such as a camera or luminometer that includes the detection element is particularly preferred. 【0094】 2-2-3.Measurement process The measurement step (S0203) is a step in which the amount of the target substance in the sample is measured based on the detection results in the detection step. 【0095】 In this step, the detection results obtained in the detection step are quantified using a measuring device. The measuring device can be the imaging device or a spectrophotometer. The quantification may be an absolute value obtained by measuring the absolute amount of the reaction product, or it may be calculated as a relative value based on the intensity of the detected reaction product, such as fluorescence intensity, emission intensity, or color density. In the method of the present invention, quantification as a relative value is preferable. In the case of relative values, it is preferable to obtain control values ​​for a negative control and / or a positive control. [Examples] 【0096】 The present invention will be specifically illustrated by the following embodiments. However, the embodiments of the present invention are not limited to the scope of the following embodiments. 【0097】 <Example 1> (the purpose) The lateral flow device of the present invention is fabricated, and its quantitative ability to detect substances in a sample is verified. (method) For the light-transmitting plastic plates, an embossed PET sheet (0.125 mm thick) and a transparent PET sheet were used. Purified human hemoglobin A1c (in-house purified) was immobilized on a portion of the embossed area corresponding to the texture, forming the immobilization area. After blocking the other areas of the plate surface with 1% BSA (Nacalai Tesque Co., Ltd.), both plates were dried. Next, the two plates were bonded together using 5 μm thick double-sided tape as a spacer, with the immobilization area facing inward. This constituted the lateral flow device of the present invention, having a 5 μm gap. An absorbent material (cellulose powder) was placed as a recovery section at one of the gap openings, which are the flow path ends of the fabricated lateral flow device, and a sample consisting of a mixture of gold colloid-labeled anti-human hemoglobin A1c monoclonal antibody (homemade) and human hemoglobin A1c was applied from the other opening as an inlet. Once the sample had flowed through the device, the concentration of the colored line in the detection section (integrated with the fixed section in this device) was measured. Specifically, the transmitted light from the light irradiated onto the detection section was photographed with a digital camera, and the entire detection section that captured the target substance was quantified and measured as an integrated value. 【0098】 (result) The obtained measurements are shown in Table 1 as integrated values. 【0099】 [Table 1] 【0100】 As shown in Table 1, the lateral flow device of the present invention makes it possible to quantify the target substance with high accuracy. 【0101】 <Example 2> (the purpose) The quantitative ability of the lateral flow device of the present invention to detect a target substance in a sample will be verified using different samples. (method) The basic procedure was carried out in accordance with Example 1. In this example, goat anti-mouse IgG (antiserum manufactured in-house by Protein Purification Industry Co., Ltd. and then self-purified) was immobilized on the fixed part of the device, and the device was fabricated in the same manner as in Example 1. First, mouse IgG solution (self-purified) was applied from the inlet, and after it had flowed completely through the channel inside the device, gold colloid-labeled anti-goat IgG solution (antiserum manufactured in-house by Protein Purification Industry Co., Ltd. and then self-purified and labeled with gold colloid) was applied from the inlet. When the sample had flowed completely through the channel inside the device, the concentration of the colored line in the detection unit was measured in the same manner as in Example 1. 【0102】 (result) The obtained measurements are shown in Table 2 as integrated values. 【0103】 [Table 2] 【0104】 Similar to Example 1, it was demonstrated that the lateral flow device of the present invention can be used to quantify the target substance with high accuracy, regardless of its type. 【0105】 <Example 3> (the purpose) The accuracy of measurement values ​​for the target substance in the same sample will be verified by using multiple lateral flow devices of the present invention and multiple conventional immunochromatography devices. (method) The lateral flow device of the present invention used a device fabricated by the method of Example 1. For the preparation of the conventional device, a fully frosted slide glass for cell diagnostics (Matsunami Glass Industry Co., Ltd.), which has a fine mesh and good reproducibility, was used as one of the device plates. Human hemoglobin A1c was immobilized on a portion of the mesh, and the other areas of the glass plate surface were blocked with BSA. The glass plate was dried. Next, with the fixed portion facing inward, the glass plate was bonded together with the transparent PET sheet prepared in Example 1 using 5 μm thick double-sided tape. This was used as a conventional immunochromatography device with a 5 μm gap. Using six devices, a sample containing a mixture of gold colloid-labeled anti-human hemoglobin A1c antibody and human hemoglobin A1c was applied through the introduction section, similar to Example 1. Once the sample had flowed through the device, the concentration of the colored line in the detection section was measured, similar to Example 1. The average value, standard deviation, and coefficient of variation of the integral values ​​obtained from each device were calculated, and the variability of the measurements between devices was compared. 【0106】 (result) Table 3 shows the measurement results for a device with a rough-surfaced glass plate using the conventional method, and Table 4 shows the measurement results for a device with a light-transmitting plastic plate according to the present invention. 【0107】 [Table 3] 【0108】 [Table 4] 【0109】 As is clear from the results in Tables 3 and 4, the coefficient of variation, which represents the variability of measurements between devices, was 13.11 for the conventional rough-surface glass device, while it was only 3.588 for the lateral flow device of the present invention, indicating a difference of more than 3.5 times. From these results, it is clear that the lateral flow device of the present invention exhibits less variability in measurements between devices and enables more accurate quantitative analysis compared to conventional devices.

Claims

[Claim 1] A lateral flow device comprising multiple light-transmitting plastic plates, each having a hydrophobic textured surface, bonded together on the textured surfaces so as to maintain gaps that allow liquid to spread by capillary action, An introduction section for introducing the sample, The unfolding section for unfolding the sample between its various parts. A fixing part in which a detection agent that reacts with the target substance in the sample is fixed to the surface of the textured surface, A detection unit for detecting the reaction product of the target substance in the sample and the detection agent, and It is equipped with a collection unit for collecting sample waste liquid, The aforementioned lateral flow device. [Claim 2] The lateral flow device according to claim 1, comprising a spacer that provides the gap between the light-transmitting plastic plates. [Claim 3] The lateral flow device according to claim 1 or 2, wherein a portion of the surface of the light-transmitting plastic plate is textured. [Claim 4] The lateral flow device according to any one of claims 1 to 3, wherein the bonding surface of the light-transmitting plastic plate is made of a hydrophilic material. [Claim 5] The lateral flow device according to any one of claims 1 to 4, wherein the depth of the texture is 0.3 μm to 1.5 μm. [Claim 6] A lateral flow device according to claim 2 or any one of claims 3 to 5 relating to claim 2, wherein the thickness of the spacer is 5 μm to 30 μm. [Claim 7] A lateral flow device according to any one of claims 1 to 6, comprising two or more of the fixing parts on which different detection agents are fixed. [Claim 8] A lateral flow device according to any one of claims 1 to 7, wherein the detection agent is labeled. [Claim 9] The lateral flow device according to any one of claims 1 to 8, wherein the detection agent is a nucleic acid, peptide, protein, or low molecular weight compound. [Claim 10] The lateral flow device according to claim 9, wherein the nucleic acid is a nucleic acid aptamer or a nucleic acid fragment containing a nucleic acid binding domain recognition sequence. [Claim 11] The lateral flow device according to claim 9, wherein the peptide or protein is an antigen, an antibody or an active fragment thereof, a ligand, or a receptor or a ligand-binding region thereof. [Claim 12] The lateral flow device according to any one of claims 1 to 11, wherein the object to be detected is a nucleic acid, peptide, protein, or low molecular weight compound. [Claim 13] A method for quantifying a target substance in a sample using a lateral flow device according to any one of claims 1 to 12, An introduction process in which the sample is introduced from the introduction section. A detection step in which the reaction product of the target object and the detection agent is detected in the detection unit, and A measurement step that measures the amount of the target substance in the sample based on the detection results in the above detection step. The quantitative method, including the method described above. [Claim 14] The quantitative method according to claim 13, wherein the detection of the reaction product is based on fluorescence, luminescence, or color development emitted from the reaction product. [Claim 15] The quantitative method according to claim 14, wherein the measurement of the amount of the object to be detected is based on fluorescence intensity, luminescence intensity, or color density.

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