Examination device including reaction unit coated with reactive substance

Abstract

The present invention provides an examination device comprising a substrate that has a reaction unit having a surface coated with a reactive substance, the examination device being characterized in that: the reactive substance is applied to the substrate surface in the reaction unit in a state in which the reactive substance is embedded in a hardly-volatile hydrophilic compound at room temperature; and the amount of the hydrophilic compound in the reaction unit is equivalent to 0.1-10.0 μL per 1 cm2. Consequently, the reactive substance is stably held when not in use and is quickly and uniformly dispersed during the reaction, and thus the accuracy of the measurement results obtained using the examination device can be improved.

Description

Test device having a reaction section coated with a reactive substance

[0001] The present invention relates to a testing device such as a microchip used for analyzing or testing components in biological samples or environmental samples, and a method for manufacturing the same, and more particularly to a testing device such as a microchip having a reaction portion whose surface is coated with a reactive substance, and a method for manufacturing the same.

[0002] Known examples of microchips used for analyzing or testing components in biological samples or environmental samples include those that have a reaction section containing a reactive substance such as an antibody or nucleic acid that binds to a substance to be measured in the sample, and that detect and analyze the reaction between the substance to be measured in the sample and the reactive substance by introducing the sample into the reaction section. For example, Patent Document 1 discloses a microchip for nucleic acid amplification reactions in which multiple reagents required for the reaction are layered and fixed in a predetermined order in a well that serves as a reaction field for the nucleic acid amplification reaction.

[0003] Patent Publication No. 2011-160728

[0004] As described above, microchips in which a reactive substance is fixed onto a substrate to form a reaction zone are known. However, when a liquid sample is added to the reaction zone to carry out a reaction, problems such as the dispersibility of the reactive substance can cause the reaction to not occur uniformly, resulting in variations in the measurement results.

[0005] The present invention aims to improve the accuracy of measurement results by stably retaining the reactive substance when not in use, while dispersing the reactive substance quickly and uniformly during reaction, in a testing device having a reaction section with a reactive substance such as a protein, sugar chain, or nucleic acid applied to the surface.

[0006] The present inventors have conducted extensive research to solve the above problems, and as a result, have found that, in producing a test device having a reaction portion in which a reactive substance is applied to the surface of a substrate, the reactive substance can be embedded in a hydrophilic compound that is hardly volatile at room temperature, such as glycerol, in the region of the substrate surface that will become the reaction portion, and applied in a state containing a certain amount of moisture, thereby stably maintaining the reactive substance when not in use (storage), and also efficiently dispersing the reactive substance when a liquid is added or poured in to cause a reaction, allowing the reaction to occur uniformly and improving the accuracy of the measurement results, thereby completing the present invention.

[0007] One aspect of the present invention is an inspection device including a substrate having a reaction section coated with a reactive substance on its surface, wherein the reactive substance is coated on the surface of the substrate in a state where it is embedded in a hydrophilic compound that is hardly volatile at room temperature, and the amount of the hydrophilic compound in the reaction section is 1 cm ２ The present invention relates to a test device characterized in that the amount of the hydrophilic compound is equivalent to 0.1 to 10.0 μL per sample, and the water content of the hydrophilic compound is 5 to 80 mass %.

[0008] Another aspect of the present invention is a method for producing a substrate having a region that will become a reaction site, the method comprising the steps of: preparing a substrate having a region that will become a reaction site; preparing a solution or dispersion in which a reactive substance is dissolved or dispersed in an aqueous solution of a hydrophilic compound that is hardly volatile at room temperature; adding the solution or dispersion of the reactive substance to the region that will become the reaction site of the substrate; and placing the substrate under an environmental relative humidity of 10 to 90% to adjust the water content of the hydrophilic compound contained in the reaction site to 5 to 80 mass % by water equilibrium. ２ The present invention relates to a method for producing a test device having a volume equivalent to 0.1 to 10.0 μL per sample.

[0009] According to the test device of the present invention, a reactive substance is embedded in a hydrophilic compound that is hardly volatile at room temperature and then applied to the reaction section of the test device in a state containing a certain amount of water, thereby making it possible to stably maintain the reactive substance while suppressing scattering and deterioration over time, and when a liquid sample is added and reacted, the reactive substance is efficiently dispersed, allowing the reaction to occur uniformly and improving the accuracy of the measurement results. Furthermore, embedding the reactive substance in a hydrophilic compound that is hardly volatile also has the effect of preventing the reactive substance from freezing and protecting its activity.

[0010] Graph showing the evaluation results of the variation in pigment distribution due to the addition of glycerol. Graph showing the evaluation results of the time required for pigment dispersion due to the addition of glycerol. Photographs showing the state of pigment dispersion when liquid is added without (left) and with (right) the addition of glycerol. Graph showing the evaluation results of the time required for pigment dispersion when diethylene glycol (top) or polyethylene glycol (bottom) is used. Graph showing the change over time in water content due to water equilibrium of each hydrophilic compound at an environmental humidity of 50%. Graph showing the effect of glycerol, etc. on thrombin activity after frozen storage. Graph with the water content in glycerol on the vertical axis and the relative humidity of the environment on the horizontal axis.

[0011] The test device of the present invention is a test device including a substrate having a reaction part coated on its surface with a reactive substance, wherein the reactive substance is coated on the surface of the substrate in the reaction part in a state where it is embedded in a hydrophilic compound that is hardly volatile at room temperature, and the amount of the hydrophilic compound in the reaction part is 1 cm ２ The amount of the solution is equivalent to 0.1 to 10.0 μL per unit volume, and the water content is 5 to 80% by mass.

[0012] The type of test device is not particularly limited as long as it includes a substrate having a reactive substance applied to its surface and a reaction area to which a liquid sample can be added, but preferred examples include microchips and test strips.

[0013] The substrate of the testing device can be made of metal, glass, plastic, silicone, etc., but from the viewpoint of detecting the reaction by light emission, color development, or visual inspection, a transparent material is preferred, and transparent plastic is more preferred. Examples include polyethylene, polypropylene, polystyrene, polymethyl methacrylate, cycloolefin polymer, cycloolefin copolymer, polyphenylene oxide, polyethylene terephthalate, polyethylene naphthalate, polycarbonate, polyamide, polyimide, phenolic resin, epoxy resin, polyvinylidene chloride, polyvinyl chloride, ABS resin, poly(2-methoxyethyl acrylate) (PMEA) resin, etc.

[0014] The water contact angle of the surface of the substrate to which the solution of a hydrophilic compound containing a reactive substance that is not volatile at room temperature is applied is not particularly limited, but is preferably 10 to 90°, more preferably 20 to 80°, and even more preferably 50 to 70°. In particular, when the substrate is flat, a substrate having a water contact angle of 50 to 70° is preferably used. In this case, it is sufficient that at least the portion of the substrate surface to which the reactive substance is applied has a water contact angle of 50 to 70°. By using a substrate having a surface with a water contact angle of 50 to 70°, the solution of a hydrophilic compound containing a reactive substance that is not volatile at room temperature can be efficiently spotted, and by allowing the solution to reach a water equilibrium state under constant humidity conditions, a homogeneous thin film containing the reactive substance embedded in the hydrophilic compound and retaining a constant amount of moisture can be more effectively formed. Among substrates made of the above-mentioned materials, substrates with a water contact angle within this range may be used, or those that have been surface-treated so that the water contact angle falls within the above range may be used. Here, preferred surface treatments include application of a reagent that changes the water contact angle of the surface, plasma treatment, excimer treatment, or corona treatment. Examples of reagents that change the water contact angle of a surface include Beamset 1461 (Arakawa Chemical Industries, Ltd.), RX-6-AQ P-2200 (Nippon Shokubai Co., Ltd.), and HFC-ASL8 (Harima Chemicals Co., Ltd.). Methods for changing the water contact angle of a portion of a substrate surface include masking a portion of the substrate and performing plasma treatment, excimer treatment, or corona treatment, or masking a portion of the substrate and applying a hydrophilizing reagent or a hydrophobic reagent. The masked region may be a portion to which a solution of a hydrophilic compound that contains a reactive substance and is not volatile at room temperature is applied, or a portion to which a solution of a hydrophilic compound that contains a reactive substance and is not volatile at room temperature is not applied.

[0015] In another embodiment, the substrate portion coated with the reactive substance embedded in a hydrophilic compound that is hardly volatile at room temperature has a water contact angle of 90° or less on the surface of the substrate portion, and the substrate portion can be an inspection device surrounded by one or more selected from a portion of the substrate surface having a water contact angle of more than 90° and an outflow prevention wall that prevents the outflow of the reactive substance. In this case, the portion hydrophilized to a water contact angle of 90° or less preferably has a water contact angle of 30° or less, and more preferably a water contact angle of 20° or less. In particular, by having a water contact angle of 20° or less, the applied hydrophilic compound (e.g., a glycerol solution) can be applied more uniformly.

[0016] Specifically, as one example, the substrate portion to which the reactive substance is applied in a state where it is embedded in a hydrophilic compound that is hardly volatile at room temperature can be an inspection device in which the surface of the substrate portion has a water contact angle of 90° or less, and the outside of the substrate portion is surrounded by a substrate portion having a surface water contact angle of more than 90°. As another example, the substrate portion to which the reactive substance is applied in a state where it is embedded in a hydrophilic compound that is hardly volatile at room temperature can be an inspection device in which the surface of the substrate portion has a water contact angle of 90° or less, and the outside of the substrate portion is surrounded by a substrate portion having a surface water contact angle of more than 90° and an outflow prevention wall that prevents the outflow of the reactive substance.

[0017] The test device may also be one in which the substrate portion to which the reactive substance is applied in a state where it is embedded in a hydrophilic compound that is hardly volatile at room temperature is surrounded by an outflow prevention wall that prevents the outflow of the reactive substance.

[0018] The test device of the present invention is preferably configured for analysis by adding or introducing a liquid sample into a reaction chamber, causing a reaction between the target substance in the liquid sample and the reactive substance. The reactive substance may be any substance that reacts with the target component (detection target substance) in the sample and can be appropriately selected depending on the type of target component. The reactivity of the reactive substance can be a biological reaction or a chemical reaction, including binding reactions. Binding reactions include not only covalent bond formation but also hydrogen bond formation, hydrophobic interactions, and binding between a substrate and a receptor or enzyme. Reactive substances include proteins (including peptides), sugars, nucleic acids, low-molecular-weight compounds, and the like. Examples include substances such as antibodies that specifically bind to the target substance, enzyme proteins that use the target substance as a substrate, and blood coagulation factors such as PT reagents. Furthermore, when the target substance is a nucleic acid, nucleic acid probes or polymerases (nucleic acid amplification enzymes) that amplify nucleic acids may be used. The nucleic acid amplification reagent may be a PCR reagent or a LAMP (Loop-mediated Isothermal Amplification) reagent. The reactive substance may also be a drug susceptibility test medium. Examples of drug susceptibility test medium include media containing antibiotics. The reactive substance may also be a general biochemical test reagent. The general biochemical test reagent may include, for example, various enzymes such as AST (GOT), ALT (GPT), LDH (lactate dehydrogenase) and its isozymes, ALP (alkaline phosphatase) and its isozymes, CK (creatine kinase) and its isozymes, amylase (Amy) and its isozymes, lipase, γ-GTP (γ-glutamyl transpeptidase), cholinesterase (ChE), sodium (Na), potassium (K), chloride (Cl), calcium (Ca), phosphorus (P) [inorganic phosphorus (I)], and the like. Examples of the components of the serum include iron (Fe), magnesium (Mg), and other electrolytes and metals; proteins such as total protein (TP) and serum protein fraction (PF); nitrogen-containing components such as urea nitrogen (BUN), creatinine (Cr), uric acid (UA), bilirubin (Bil), and ammonia; lipids such as cholesterol, HDL-cholesterol (HDL-C), LDL cholesterol (LDL-C), and triglycerides (TG); and sugar-related substances such as blood glucose (BS, GLU) and glycated hemoglobin (HbA1c).

[0019] The reactive substance is embedded in a hydrophilic compound that is hardly volatile at room temperature and is applied to a region of the substrate having a surface with a water contact angle of 50 to 70°, which will become the reaction site. For example, the reactive substance is dissolved or dispersed in an aqueous solution of a hydrophilic compound that is hardly volatile at room temperature, and the resulting solution or dispersion is spotted (added) to the region of the substrate that will become the reaction site. The reactive substance is then allowed to reach moisture equilibrium under conditions of an ambient relative humidity of 10 to 90% (preferably 10 to 50%, more preferably 10 to 30%), whereby the reactive substance is embedded in the hydrophilic compound and applied to the reaction site in a state containing a certain amount of moisture. The certain amount of moisture content can be 5 to 80% by mass, but is preferably 5 to 40% by mass, more preferably 5 to 20% by mass, and particularly preferably 5 to 10% by mass. The viscosity of the coating containing the hardly-volatile hydrophilic compound and the reactive substance in the reaction zone after application and allowing it to reach moisture equilibrium is, for example, in the range of 1 to 4000 mPa s, preferably 40 to 4000 mPa s, and more preferably 100 to 4000 mPa s at 4° C. to 30° C. The environmental relative humidity refers to the relative humidity in a room where work or the like is performed.

[0020] The hydrophilic compound that is hardly volatile at room temperature is preferably a hydrophilic aliphatic hydrocarbon compound that is liquid at room temperature and normal pressure, is hardly volatile, and contains an oxygen atom and has a molecular weight of 80 to 1000, preferably 80 to 600. Examples of such a hydrophilic compound include alcohol compounds and ether compounds, and linear compounds are preferred. It is preferable that the hydrophilic compound does not exhibit a denaturing effect on reactive substances such as proteins, but rather exhibits a stabilizing effect on reactive substances such as proteins.

[0021] Examples of hydrophilic compounds that are hardly volatile at room temperature include water-soluble polyethers such as polyethylene glycol, polyhydric alcohols such as ethylene glycol and glycerol, ethylene oxide adducts of polyhydric alcohols such as ethylene oxide adducts of glycerol, and partial ester compounds of polyhydric alcohols such as glycerol monoesters and glycerol diesters. More preferred are glycerol, diethylene glycol, and polyethylene glycols having a molecular weight of 100 to 600 (low molecular weight PEGs).

[0022] Hydrophilic compounds that are hardly volatile at room temperature, such as glycerol, diethylene glycol, and polyethylene glycol with a molecular weight of 100 to 600, hardly volatilize at room temperature and normal pressure and exist in liquid form, but they have a strong affinity for water and can maintain moisture through water equilibrium in response to environmental humidity. For example, when an aqueous solution containing glycerol is left standing at room temperature and normal pressure, the water evaporates, leaving only the glycerol. However, the remaining glycerol contains moisture according to the water equilibrium, and it is thought that this trace amount of moisture has a stabilizing effect on reactive substances and an effect of promoting the dispersion of reactive substances when a liquid sample is added.

[0023] In the test device of the present invention, the amount of the hydrophilic compound in the reaction zone on the surface of the substrate is 1 cm ２ The amount is equivalent to 0.1 to 10.0 μL per 1 cm. ２ The amount is preferably 1.0 to 5.0 μL per cm ２ The amount is preferably 1.0 to 3.0 μL per cm ２ This is equivalent to 1.3 to 2.5 μL per 1 cm ２ The equivalent of 0.1 to 10.0 μL per cm ２ In the case of glycerol, if the density of glycerol is 1.26 mg / μL, the amount of the hydrophilic compound present in 1 cm is 0.1 to 10.0 μL. ２ Similarly, in the case of glycerol, the concentration is preferably 1.26 to 6.3 mg / cm ２ (1-5 μL / cm ２ ), more preferably 1.26 to 3.78 mg / cm ２ (1-3 μL / cm ２ ), and more preferably 1.638 to 3.15 mg / cm ２ (1.3-2.5μL / cm ２ ) In the case of diethylene glycol and polyethylene glycol, the weight can be converted from the density in the same way.

[0024] By setting the amount of hydrophilic compound within the above range, the amount of residual moisture due to water equilibrium in the hydrophilic compound layer in which the reactive substance in the reaction section is embedded falls within a suitable range, making it possible to form a thin film that efficiently retains the reactive substance, preventing the reactive substance from scattering during storage or transportation and retaining it stably, and also providing excellent dispersibility of the reactive substance when a liquid sample is added.

[0025] In the test device of the present invention, the reaction section may be recessed to allow the addition or inflow of a liquid sample. The test device preferably has a channel (inlet channel) connected to the reaction section and capable of supplying a liquid sample to the reaction section. Such a channel can be fabricated by providing a groove on the surface of the substrate that is connected to the recess that serves as the reaction section. The test device may also have a channel (outlet channel) connected to the reaction section and capable of allowing a liquid sample to flow out of the reaction section. The test device may also include a member such as a film that covers the reaction section and the channel. Transparent plastic is preferred as the material for the film; for example, polyethylene terephthalate (PET) resin, cycloolefin polymer (COP) resin, cycloolefin copolymer (COC) resin, polystyrene (PS) resin, polycarbonate (PC) resin, or polymethyl methacrylate (PMMA) resin is more preferred. The substrate and film can be bonded together using an adhesive, pressure-sensitive adhesive, or the like.

[0026] The testing device of the present invention can be used by adding or flowing a liquid sample into the reaction section and causing the target substance in the liquid sample to react with a reactive substance. For example, by adding the liquid sample to the reaction section, the reactive substance is dispersed within the reaction section, and the target substance in the liquid sample reacts with the reactive substance. By detecting this reaction, the target substance in the liquid sample can be detected, and by quantifying the reaction, the target substance can be quantified.

[0027] The test device of the present invention can be manufactured, for example, by the following steps: The method for manufacturing the test device of the present invention includes the steps of: preparing a substrate having a region that will become a reaction zone; preparing a solution or dispersion in which a reactive substance is dissolved or dispersed in an aqueous solution of a hydrophilic compound that is hardly volatile at room temperature; adding the solution or dispersion of the reactive substance to the region of the substrate that will become the reaction zone; and placing the substrate under conditions in which the relative humidity in the environment is 10 to 90% (preferably 10 to 50%, more preferably 10 to 30%), and adjusting the water content of the hydrophilic compound contained in the reaction zone to 5 to 80% by mass (preferably 5 to 40% by mass, more preferably 5 to 20% by mass, particularly preferably 5 to 10% by mass) by water equilibrium.

[0028] The substrate may be any of the substrates described above. The water contact angle of the substrate surface is not particularly limited, but is preferably 10 to 90°, more preferably 20 to 80°, and even more preferably 50 to 70°.

[0029] To prepare a solution or dispersion in which a reactive substance is dissolved or dispersed in an aqueous solution of a hydrophilic compound that is hardly volatile at room temperature, an aqueous solution of the hydrophilic compound that is hardly volatile at room temperature may first be prepared, and then the reactive substance may be dissolved or dispersed in the aqueous solution, or the reactive substance may be dissolved or dispersed in water, and then the hydrophilic compound that is hardly volatile at room temperature may be dissolved in the aqueous solution.

[0030] The concentration of the hydrophilic compound in the solution or dispersion is finally determined by adding the above-mentioned predetermined amount (1 cm ２ Equivalent to 0.1 to 10.0 μL per cm, preferably 1 cm ２ per cm, preferably 1.0 to 5.0 μL equivalent ２ per 1 cm, more preferably 1.0 to 3.0 μL ２ The concentration may be any concentration suitable for disposing the hydrophilic compound in an amount of 1.3 to 2.5 μL per 1000 μL of the solution or dispersion, but is preferably 0.2 to 20 mass %. The concentration of the reactive substance in the solution or dispersion can be adjusted appropriately based on the amount required for the reaction.

[0031] The method for adding the solution or dispersion to the region that will become the reaction zone of the testing device is not particularly limited, but it is preferably added dropwise using a dispenser, inkjet printer, pipette, etc. The amount added is preferably 0.5 to 50 μL. By setting the amount within this range, the reactive substance can be efficiently fixed to the reaction zone in a state where it is embedded in the hydrophilic compound.

[0032] The step of adjusting the water content of the hydrophilic compound contained in the reaction zone to 5 to 80% by mass can be performed, for example, by achieving water equilibrium between the water content of the hydrophilic compound and the water content of the environment under constant humidity conditions (ambient relative humidity 10 to 90%). The method for achieving water equilibrium between the water content of the hydrophilic compound and the water content of the environment is not particularly limited, but for example, leaving the hydrophilic compound at room temperature and pressure under constant humidity conditions is preferred. In the reaction zone, the hydrophilic compound maintains a water content of 5 to 80% by mass through water equilibrium. Maintaining such a state containing a trace amount of water makes it possible to form a homogeneous thin film containing a reactive substance and to uniformly disperse the reactive substance when a liquid sample is added. The step of adjusting the water content of the hydrophilic compound contained in the reaction zone to 5 to 80% by mass can also be performed while the test device is sealed in a moisture-proof packaging material. For example, a method of sealing the test device in a moisture-proof packaging material and leaving it standing in a humidity-controlled environment can be used. As a result, the water content of the hydrophilic compound reaches water equilibrium with the water content of the gas within the moisture-proof packaging material.

[0033] The microchip, which is one aspect of the testing device of the present invention, will be described below by citing preferred embodiments, although the testing device of the present invention is not limited to the following embodiments.

[0034] In the test device of the present invention, the method for applying the reactive substance in a state where it is embedded in a hydrophilic compound that is hardly volatile at room temperature is not particularly limited, but examples thereof include the following two methods.

[0035] In the first method, when a portion of a flat substrate is to be hydrophilized, the water contact angle of the portion other than the portion to be coated with the reactive substance is set to 70° or more, and the contact angle of the portion to be coated with the reactive substance is set to less than 50°, and then the reactive substance and the hydrophilic compound are applied. The combination of the water contact angle of the portion other than the portion to be coated with the reactive substance and the hydrophilic compound and the water contact angle of the portion to be coated with the reactive substance and the hydrophilic compound is preferably 80° or more for the portion other than the portion to be coated with the reactive substance (the former) and less than 30° for the portion to be coated with the reactive substance (the latter). It is more preferable that the former be 90° or more and the latter be less than 20°. By reducing the latter to 20° or less, the applied liquid wets and spreads throughout the hydrophilized region. Here, hydrophilization can be achieved by surface treatment such as aqua plasma, atmospheric pressure plasma, vacuum plasma, excimer, corona, etc., or by applying a commercially available hydrophilization reagent. The reactive substance and the hydrophilic compound can be applied after being diluted with water, alcohol, a hydrophilic solvent, etc. The alcohol and the hydrophilic solvent are preferably volatile compounds.

[0036] A second method involves forming a shape that creates a physical flow barrier around the application area (application area) where the reactive substance and the hydrophilic compound are applied, such as by forming a concave (depressed) shape, thereby preventing the applied liquid from flowing outside the application area. In this case, it is also preferable that the application area be hydrophilized. The degree of hydrophilization is preferably such that the water contact angle of the application area is 50° or less, more preferably 20° or less. This facilitates uniform wetting and spreading of the liquid containing the reactive substance and the hydrophilic compound throughout the application area. In this case, the viscosity of the liquid containing the reactive substance and the hydrophilic compound is preferably 10 to 4000 mPa·s, more preferably 20 to 200 mPa·s, and even more preferably 30 to 100 mPa·s.

[0037] A microchip, which is one aspect of the testing device of the present invention, has a reaction region in which a reactive substance is embedded in a hydrophilic compound that is hardly volatile at room temperature and is applied in a state containing a certain amount of moisture, an inlet-side flow path connected to the reaction region and for introducing a liquid into the reaction region, and an outlet-side flow path connected to the reaction region and for discharging a liquid from the reaction region.

[0038] The microchip may have the reaction section, the inlet-side channel, and the outlet-side channel inside. That is, the microchip may be used to analyze components in a sample by passing a liquid sample through a channel formed inside the microchip and reacting a substance to be measured in the liquid sample with a reactive substance applied to the reaction section in the channel. In such a case, an inlet may be provided at the beginning of the inlet-side channel to supply liquid to the inlet-side channel, and an outlet may be provided at the end of the outlet-side channel to discharge the liquid to the outside. In another embodiment, a waste liquid reservoir for accumulating liquid may be provided at the end of the outlet-side channel.

[0039] Such a microchip having the reaction section, the inlet-side channel, and the outlet-side channel therein may be a microchip obtained by bonding a substrate and a film together. In such a case, an example is a microchip obtained by bonding a film to a substrate having a depression on its surface that serves as the reaction section and grooves that serve as channels (inlet-side channel and outlet-side channel) with an adhesive or the like. Regarding the inlet and outlet, through-holes may be provided at the starting end of the inlet-side channel and the terminal end of the outlet-side channel in the substrate, respectively, to serve as the inlet and outlet, or holes may be provided at corresponding positions on the film side. A reactive substance embedded in a hydrophilic compound that is hardly volatile at room temperature is applied to the reaction section, and the reactive substance embedded in the hydrophilic compound may also be applied to the bottom surface of the depression that serves as the reaction section in the substrate.

[0040] The reaction section of the test device of the present invention may be partially or entirely covered or sealed with a moisture-permeable film or a film having a breathable portion. The outside of the reaction section may further be sealed with a moisture-proof packaging material. The moisture content and viscosity of the reactive component are maintained within a certain range by the reactive component reaching moisture equilibrium due to the humidity inside the moisture-proof packaging material. The test device of the present invention may be provided as a package sealed with a moisture-proof packaging material, which may be opened before use. This allows the moisture retention state of the reactive substance embedded in the hydrophilic compound to be stably maintained. Examples of moisture-proof packaging materials include aluminum pouches.

[0041] Instead of packaging with a moisture-proof packaging material, the hydrophilizing compound (glycerol) may be brought into equilibrium at a moisture content of 5 to 80% by mass in an environment of 2 to 22°C and a relative humidity of 10 to 90%, and then the test device may be placed in a state where no water vapor passes inside or outside the test device (for example, the portions of the reaction section of the test device that ventilate with the outside air, such as the inlet or outlet, are sealed with a moisture-proof film, and the test device is formed from a moisture-proof material). In particular, in the case of a test device in which a flow channel is formed by joining a film and a flow channel molded product (for example, a microchip), it is preferable to use a moisture-proof film as the film. In this case, when moisture equilibrium is reached, the ratio of the volume of the microchip flow channel to the total volume of the reactive substance, hydrophilic compound, and solvent or dispersion medium such as water is preferably 100:1 or less (i.e., the volume of the microchip flow channel is 100 times or less the volume of the hydrophilic compound), more preferably 50:1 or less (i.e., the volume of the microchip flow channel is 50 times or less the volume of the hydrophilic compound), and even more preferably 10:1 or less (i.e., the volume of the microchip flow channel is 10 times or less the volume of the hydrophilic compound). When the storage environment of the test device becomes cold, the relative humidity of the gas in the packaging material and flow channel increases, and the hydrophilic compound (e.g., glycerol) absorbs surrounding moisture, resulting in an increase in the moisture content of the hydrophilic compound. When the test device is stored in a sub-freezing environment such as in a cold region, the ratio of the volume of the microchip to the volume of the hydrophilic compound is set to 100:1 or less, i.e., the volume of the flow path of the microchip is set to 100 times or less the volume of the hydrophilic compound, thereby reducing the amount of water absorption by the hydrophilic compound when the temperature drops to low, and limiting the increase in the water content of the hydrophilic compound.

[0042] An example of a manufacturing method for the test device of the present invention includes the following steps: preparing a substrate having a region that will become a reaction site; preparing a solution or dispersion in which a reactive substance is dissolved or dispersed in an aqueous solution of a hydrophilic compound that is not volatile at room temperature; adding or applying the solution or dispersion of the reactive substance to the region of the substrate that will become the reaction site; and adjusting the water content of the hydrophilic compound contained in the reaction site to 5 to 80% by mass. Examples of the step of preparing a substrate having a region that will become a reaction site include, as described above, methods such as partially changing the water contact angle of the substrate or providing a physical barrier. Examples of the step of preparing a solution or dispersion in which a reactive substance is dissolved or dispersed in an aqueous solution of a hydrophilic compound that is not volatile at room temperature include a method of mixing the reactive substance and the hydrophilic compound with a diluting medium or dispersing medium such as water so that the concentration of the hydrophilic compound is 10% by mass or less. Examples of the step of adding or applying a solution or dispersion of the reactive substance and the hydrophilic compound to the region of the substrate that will become the reaction zone include a method of applying 0.5 μL to 10 μL of the solution or dispersion. Examples of the step of adjusting the water content of the hydrophilic compound contained in the reaction zone to 5 to 80% by mass include a method of drying the hydrophilic compound in a temperature- and humidity-controlled environment so that the water content in the hydrophilic compound is 5 to 80% by mass, and a method of drying the hydrophilic compound in a temperature- and humidity-controlled environment (for example, an absolute humidity of 0 to 40 g / m ３ ) and moisture-proof (e.g., 10 μg / m ２ 1. A method in which the solution or dispersion is sealed in a packaging material having a water vapor transmission rate of 1000 rpm or less (water vapor transmission rate of 1000 rpm or less / h) and a moisture equilibrium is reached between the solution or dispersion and the gas in the packaging material, so that the moisture content in the hydrophilic compound is 5 to 80 mass %.

[0043] By adjusting the water content of the hydrophilic compound contained in the reaction zone to 5 to 80% by mass, moisture equilibrium is reached between the solution or dispersion and the gas in the packaging material, enabling the reactive substance to be immobilized at a concentration of 80 to 95% by mass of the hydrophilic compound (e.g., glycerol). The film thickness of the solution or dispersion after moisture equilibrium is preferably 500 μm or less, more preferably 200 μm or less, and even more preferably 100 μm or less. Applying a hydrophilic compound diluted or dispersed in a dilution medium or dispersion medium and then drying or allowing it to reach moisture equilibrium reduces the film thickness of the solution or dispersion. The thinner the film thickness, the faster the redissolution rate of the reactive substance when using the testing device. When applying the solution or dispersion, the concentration of the hydrophilic compound in the solution or dispersion is preferably 0.1 to 50% by mass, more preferably 0.2 to 30% by mass, and even more preferably 0.2 to 10% by mass. The concentration of the hydrophilic compound in the solution or dispersion in the moisture-proof packaging material is preferably 20% by mass to 95% by mass, more preferably 80% by mass to 95% by mass, and even more preferably 90% by mass to 95% by mass.

[0044] The present invention will be specifically described below with reference to examples, although the present invention is not limited to the embodiments of the examples.

[0045] Evaluation was performed using a microchip made of COP (cycloolefin polymer), which has a surface water contact angle of 70°, as the substrate. 0-2 μL of glycerol was added to 12 μL of an aqueous solution containing a coloring substance, and the solution was applied to a 5 mm diameter area on the substrate surface. The water was then removed by natural drying. After reaching moisture equilibrium, the dye distribution in the reagent-applied area was measured using an absorbance meter, and the variation in the reagent distribution (coefficient of variation: CV) was calculated. The moisture content of the glycerol after natural drying was 25% by mass.

[0046] The results are shown in Figure 1. Adding a small amount of glycerol suppressed the variation in reagent distribution within the coating surface.

[0047] 200 μL of physiological saline was added to the thin film formed on the substrate in Example 1, and the thin film was left standing at 37° C., and the time required for the applied reagent (colored substance) to be dispersed over the entire spot was measured.

[0048] The results are shown in Figure 2. Adding 1.3 μL or more of glycerol per square centimeter of application area significantly reduced the time required for dispersion.

[0049] Figure 3 shows the appearance of the colored substance-coated surface without and with glycerol (2 μL per square cm of coated area) (the surface was left to dry at 22°C for 24 hours). Without glycerol, the colored substance precipitated and dried, but with glycerol, the colored substance adhered uniformly to the coated surface as a thin liquid film.

[0050] Substrates with surface water contact angles of 90°, 70°, and 50° were prepared, and solutions containing coloring substances and various amounts of glycerol were applied to them and allowed to dry naturally. The adhesion and scattering properties of the solutions were evaluated. The results are shown in Table 1.

[0051] The adhesion / scattering of the applied reagent depends on the contact angle between the solution and the substrate surface and the amount of glycerol added, and good adhesion of the glycerol-added reagent was observed when the contact angle was in the range of 50 to 70°.

[0052]

[0053] An experiment similar to Example 2 was carried out using diethylene glycol and polyethylene glycol (PEG200) with a molecular weight of 200 as hydrophilic compounds that are hardly volatile at room temperature instead of glycerol. The results are shown in Figure 4. The effect of shortening the time required to disperse the coloring substance was observed even when diethylene glycol and polyethylene glycol (PEG200) with a molecular weight of 200 were used.

[0054] The moisture content and moisture equilibrium of each hydrophilic compound were examined at an environmental relative humidity of 50%. Specifically, 10 μL of each of glycerol, diethylene glycol, dipropylene glycol, 3-ethoxypropane-1,2-diol, and polyethylene glycol (PEG2000) with a molecular weight of 200 Da was dispensed into different containers and allowed to stand at 22°C and a relative humidity of 50%. The mass was measured immediately after dispensing and after 13 hours, 22 hours, and 109 hours, and the moisture content (moisture absorption rate) was calculated using the following formula: Moisture content [%] = [(liquid mass after standing - liquid mass immediately after dispensing) ÷ liquid mass immediately after dispensing] × 100

[0055] The results are shown in Figure 5. As a result, it was found that glycerol, diethylene glycol, dipropylene glycol, 3-ethoxypropane-1,2-diol, and low-molecular-weight PEG (PEG2000) all rapidly absorb moisture at an ambient humidity of 50% and can stably maintain a moisture content of 5 to 40% by mass for a long period of time. Among these, glycerol, diethylene glycol, and low-molecular-weight PEG were able to stably maintain a moisture content of 15 to 30% by mass for a long period of time at an ambient humidity of 50%, demonstrating that they are more preferable substances with low moisture volatilization and long-term stability.

[0056] Thrombin Protection: 0.1 units of thrombin were added to a 2 mL Simport tube, and 10 μL of a 10% by weight solution (1 μL / 10 μL) of glycerol, diethylene glycol, propylene glycol, or PEG200 was added and applied. The tube was allowed to reach moisture equilibrium at 50% relative humidity. After storage at room temperature for 3 days, thrombin activity was measured. A control sample was the same solution frozen at -30°C. The frozen samples were returned to room temperature and thrombin activity was measured. The results are shown in Figure 6. While thrombin activity was nearly inactivated within 3 days at room temperature without additives, the addition of glycerol maintained 80% or more of its activity even after 3 days at room temperature. Figure 7 shows the relationship between the relative humidity and the moisture content of the glycerol.

Claims

1. A test device comprising a substrate having a reaction part coated with a reactive substance, wherein the reactive substance is coated on the reaction part in a state of being embedded in a hardly volatile hydrophilic compound at normal temperature, and the amount of the hydrophilic compound in the reaction part is 0.1 to 10.0 μL equivalent per cm ２ per area, and the water content of the hydrophilic compound is 5 to 80% by mass. A test device characterized by this.

2. The amount of the hydrophilic compound in the reaction part is 1.0 to 3.0 μL equivalent per 1 cm ２ The inspection device according to claim 1, wherein the amount is equivalent to 1.0 to 3.0 μL per cm 3. The hydrophilic compound is at least one selected from the group consisting of glycerol, diethylene glycol, and polyethylene glycol having a molecular weight of 100 to 600, and the inspection device according to claim 1.

4. The substrate is a substrate made of at least one selected from cycloolefin polymer, polyethylene, polypropylene, polystyrene, polymethyl methacrylate, cycloolefin polymer, polyphenylene oxide, polyethylene terephthalate, polyethylene naphthalate, polycarbonate, polyamide, polyimide, phenol resin, epoxy resin, polyvinylidene chloride, polyvinyl chloride, ABS resin, and poly(2-methoxyethyl acrylate) (PMEA) resin, and the inspection device according to claim 1.

5. The reactive substance is a protein, a saccharide, or a nucleic acid, and the inspection device according to claim 1.

6. The reactive substance is a gene amplification reagent, a drug sensitivity test medium, or a general biochemistry test reagent, and the inspection device according to claim 1.

7. The inspection device according to claim 1 is a microchip.

8. The microchip has a flow path connected to the reaction part and allowing a liquid to flow into the reaction part, and the inspection device according to claim 7.

9. The inspection device according to claim 8 further includes a film adhered to the substrate and covering the flow path and the reaction part of the substrate.

10. For the substrate part coated with the reactive substance in a state embedded in a hydrophilic compound that is hardly volatile at normal temperature, the water contact angle of the surface of the substrate part is 50 to 70°, and the inspection device according to claim 1.

11. For the substrate part coated with the reactive substance in a state embedded in a hydrophilic compound that is hardly volatile at normal temperature, the water contact angle of the surface of the substrate part is 30 to 90°, and the substrate part is surrounded by at least one selected from a part of the substrate surface having a water contact angle exceeding 90° and an outflow prevention wall for preventing the outflow of the reactive substance, and the inspection device according to claim 1.

12. The inspection device according to any one of claims 1 to 11, packaged with a moisture-proof packaging material. Step of preparing a substrate having a region to be a reaction part, step of preparing a solution or dispersion in which a reactive substance is dissolved or dispersed in an aqueous solution of a hydrophilic compound that is hardly volatile at room temperature, step of adding the solution or dispersion of the reactive substance to the region to be the reaction part of the substrate, step of adjusting the water content of the hydrophilic compound contained in the reaction part to 5 to 80% by mass, and a reaction part having a reactive substance coated on the surface in a state of being embedded in a hydrophilic compound that is hardly volatile at room temperature, and the amount of the hydrophilic compound in the reaction part is 1 cm ２ A method for manufacturing a test device, which is equivalent to 0.1 to 10.0 μL per hit.

14. The amount of the added dissolution liquid or dispersion liquid is 10 to 50 μL, and the method for manufacturing the inspection device according to claim 13.

15. The water contact angle of the part where the reactive substance is coated in a state embedded in a hydrophilic compound that is hardly volatile at normal temperature is 50° to 70°, and the method for manufacturing the inspection device according to claim 13.

16. A method for manufacturing the inspection device according to claim 13, wherein the inspection device is the inspection device according to any one of claims 1 to 12.

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