Examination device equipped with reaction portion coated with reactive substance

Abstract

This examination device comprises a first base material having a recessed portion in a part thereof, a second base material covering the recessed portion, and a reaction portion formed by the recessed portion of the first base material and the second base material, characterized in that: in the reaction portion, the reaction portion is coated with a reactive substance in a state embedded in an immobilizing agent; the immobilizing agent contains a hydrophilic compound; the amount of the hydrophilic compound in the reaction portion is equivalent to 0.1-10.0 μL per 1 cm2 of the coated area of the reaction portion when converted to 100% of the hydrophilic compound; the moisture content of the hydrophilic compound is 5-80 % by mass; and the examination device has a water vapor permeability of 0.001 g / m2 / day or less under conditions of a temperature of 8°C and a relative humidity of 100%.

Description

Inspection device equipped with a reaction section coated with a reactive substance.

[0001] The present invention relates to an inspection device such as a microchip used for analyzing or testing components in biological samples or environmental samples, and more particularly to an inspection device such as a microchip equipped with a reaction section on which a reactive substance is coated.

[0002] Examples of microchips used to analyze or test components in biological or environmental samples include those having a reaction section containing reactive substances such as antibodies or nucleic acids that bind to the target substance in the sample, and in which the sample is introduced into the reaction section to detect and analyze the reaction between the target substance and the reactive substance in the sample. For example, Patent Document 1 discloses a nucleic acid amplification reaction microchip in which multiple reagents necessary for the reaction are stacked and fixed in a predetermined order within a well that serves as the reaction site for the nucleic acid amplification reaction.

[0003] Japanese 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 the reaction, problems such as the dispersibility of the reactive substance can lead to uneven reaction and variability in measurement results. In addition, since it takes time from the fabrication of a microchip containing a reactive substance to its actual use, there is also the problem of difficulty in stably maintaining the reactive substance in the reaction zone for a long period of time.

[0005] The present invention relates to a testing device equipped with a reaction section coated with a reactive substance such as a protein, carbohydrate, or nucleic acid on its surface. The objective is to improve measurement accuracy by rapidly and uniformly dispersing the reactive substance during the reaction, while stably retaining the reactive substance for a long period of time when not in use.

[0006] The inventors diligently conducted research to solve the above problems. As a result, they found that in fabricating an inspection device having a first substrate having a recess in part, a second substrate covering the recess, and a reaction section formed by the recess of the first substrate and the second substrate, by embedding the reactive substance in a fixed state containing a certain amount of moisture with an immobilizing agent containing a hydrophilic compound that is not easily volatile at room temperature, such as glycerol, in the region that forms the reaction section formed by the first substrate and the second substrate, when a liquid is added or introduced to carry out a reaction between the reactive substance and the liquid, the reactive substance is efficiently dispersed in the liquid and the reaction can be carried out uniformly. Furthermore, they found that at least the portion of the second substrate covering the recess has a water vapor transmission rate of 0.001 g / m³ under the temperature and humidity conditions (temperature: 8°C, relative humidity: 100%) under which the inspection device is intended to be stored. 2 By achieving moisture resistance of less than / day, the moisture content of the hydrophilic compound is kept constant, and the reactive substance is stably retained when not in use (storage). Furthermore, by adding sugars in addition to the hydrophilic compound as an immobilizing agent, the activity of the reactive substance can be stably maintained, and thus the present invention was completed.

[0007] One aspect of the present invention is an inspection device having a first substrate having a recess in part, a second substrate covering the recess, and a reaction section formed by the recess of the first substrate and the second substrate, wherein a reactive substance is coated onto the reaction section in a state in which it is embedded with an immobilizing agent, the immobilizing agent contains a hydrophilic compound, and the amount of the hydrophilic compound in the reaction section is such that it covers a 1 cm² area of ​​the reaction section. ２ The amount is equivalent to 0.1 to 10.0 μL of the hydrophilic compound per unit, the water content of the hydrophilic compound is 5 to 80% by mass, and at least the portion of the second substrate covering the recess has a water vapor transmission rate of 0.001 g / m² under conditions of temperature: 8°C and relative humidity: 100%. 2 This relates to a testing device characterized by having a lifespan of less than or equal to / day.

[0008] According to the inspection device of the present invention, reactive substances can be stably held while suppressing scattering and deterioration over time, and when a liquid sample is added and reacted, the reactive substances are efficiently dispersed, allowing for a uniform reaction. On the other hand, the reaction section has a water vapor transmission rate of 0.001 g / m³. 2 By sealing the material with a moisture-resistant substrate (less than 1 / day), it can be stored stably for a long period of time. Furthermore, by embedding the reactive substance in an immobilizing agent containing a hydrophilic compound and carbohydrates that are not easily volatile at room temperature, the reactive substance can be stored stably while maintaining its activity.

[0009] A graph showing the evaluation results of the variation in dye distribution due to the addition of glycerol. A graph showing the evaluation results of the time required for dye dispersion due to the addition of glycerol. A photograph showing the dispersion of dyes during liquid addition with and without glycerol (left) and with glycerol (right). A graph showing the evaluation results of the time required for dye dispersion when using polyethylene glycol (top) or diethylene glycol (bottom) with a weight-average molecular weight of 200. A graph showing the time change in water content due to water equilibrium of each hydrophilic compound at an ambient humidity of 50%. A graph showing the range of fluctuation in water content of reagents sealed inside the chip with COP film (left) and moisture-proof film (right). A graph showing the protective effect on thrombin coated with glycerol. A graph showing the protective effect on thrombin coated with polyethylene glycol 200.

[0010] The inspection device of the present invention comprises a first substrate having a recess in part, a second substrate covering the recess, and a reaction section formed by the recess of the first substrate and the second substrate, wherein a reactive substance is coated onto the reaction section in a state in which it is embedded with an immobilizing agent, the immobilizing agent contains a hydrophilic compound, and the amount of the hydrophilic compound in the reaction section is such that it covers a 1 cm² area of ​​the reaction section. ２ The amount is equivalent to 0.1 to 10.0 μL of the hydrophilic compound per unit, the water content of the hydrophilic compound is 5 to 80% by mass, and at least the portion of the second substrate covering the recess has a water vapor transmission rate of 0.001 g / m² under conditions of temperature: 8°C and relative humidity: 100%. 2 It is characterized by being less than or equal to / day.

[0011] The inspection device is not particularly limited in type as long as it has a first substrate having a recess in part, a second substrate covering the recess, and a reaction section formed by the recess of the first substrate and the second substrate, to which a reactive substance is applied and to which a liquid sample can be added, but preferred examples include microchips and test pieces.

[0012] A recess is provided in a part of the first substrate. The area and depth of the recess are determined as appropriate depending on the type of reactive substance and the purpose of the inspection, but for example, the area is 0.2 to 5 cm². ２ Preferably 0.5 to 2 cm ２ The recess can be 0.1 to 10 mm deep, preferably 0.5 to 5 mm deep. The shape of the recess is not particularly limited and can be, for example, a square or circular shape.

[0013] The first substrate material of the inspection device can be metal, glass, plastic, silicone, etc., but a transparent material is preferred from the viewpoint of detecting the reaction by light emission, color development, or visual inspection, 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, and poly-2-methoxyethyl acrylate (PMEA) resin.

[0014] The surface area of ​​the substrate to which the solution of the immobilizing agent containing the reactive substance is applied preferably has a water contact angle of 10 to 90°, more preferably 20 to 80°, and even more preferably 50 to 70°. In particular, when the substrate is flat, a substrate surface with 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 with a surface having a water contact angle of 50 to 70°, the solution of the immobilizing agent containing the reactive substance can be efficiently spotted, and by reaching a moisture equilibrium state under constant humidity conditions, a homogeneous thin film containing the reactive substance embedded in the immobilizing agent and retaining a certain amount of moisture can be formed more effectively.

[0015] Furthermore, among the substrates of the materials described above, those with a water contact angle within this range may be used, or substrates that have been surface-treated to have a water contact angle within the above range may be used. Here, preferred surface treatments include coating with 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 the surface include Beamset 1461 (Arakawa Chemical Industries, Ltd.), RX-6-AQ P-2200 (Nippon Shokubai Co., Ltd.), and HFC-ASL8 (Harima Chemicals, Ltd.). Methods for changing the water contact angle of a portion of the 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 hydrophobicizing reagent.

[0016] The testing device of the present invention is preferably one that allows a liquid sample to be added to or flowed into a reaction section and reacts the target substance in the liquid sample with the reactive substance for analysis. The reactive substance can be any substance that reacts with the target component (target substance) in the sample, and can be appropriately selected depending on the type of target component. The reactivity of the reactive substance includes biological reactions and chemical reactions, and biological reactions include binding reactions. The binding reactions include not only the formation of covalent bonds, but also the formation of hydrogen bonds, hydrophobic interactions, and binding of substrates to receptors or enzymes. Reactive substances include proteins (including peptides), carbohydrates, nucleic acids, and low molecular weight compounds. For example, the reactive substance may be an antibody that specifically binds to the target substance, or an enzyme protein that uses the target substance as a substrate (such as thrombin, ecarin, t-PA (tissue plasminogen activator, urease, etc.) or a blood coagulation factor such as PT reagent). If the target substance is a nucleic acid, a nucleic acid probe or a nucleic acid amplification polymerase (nucleic acid amplification enzyme) may also 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 testing medium. Examples of drug susceptibility testing media include media containing antibiotics.

[0017] Furthermore, the reactive substance may be a general biochemical test reagent. General biochemical test reagents include, for example, 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) and various other enzymes, sodium (Na), potassium (K), chloride (Cl), calcium (Ca), phosphorus (P) [inorganic phosphorus (I)]. Examples include electrolytes and metals such as protein (P), iron (Fe), and magnesium (Mg); proteins such as total protein (TP) and serum protein fraction (PF); nitrogenous components such as blood 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).

[0018] The reactive substance is embedded in an immobilizing agent containing a hydrophilic compound and applied to the reaction area on the substrate. For example, the reactive substance is dissolved or dispersed in an aqueous solution of the immobilizing agent containing a hydrophilic compound, and the resulting solution or dispersion is spot-added to the reaction area on the substrate. By bringing the mixture to moisture equilibrium under environmental relative humidity conditions of 10-90% (preferably 10-50%, more preferably 10-30%), the reactive substance is embedded in the immobilizing agent containing the hydrophilic compound and applied to the reaction area while containing a certain amount of moisture. The certain amount of moisture can be 5-80% by mass. If the moisture content is less than 5% by mass, the dispersibility of the reactive substance may decrease, and if it exceeds 80% by mass, the coating liquid may flow too much, making it difficult to keep it in the designated reaction area. Therefore, it is set to 5-80% by mass. Preferably, it is 5-40% by mass, more preferably 5-20% by mass, and particularly preferably 5-10% by mass. In this specification, the water content of a hydrophilic compound refers to the value (mass%) obtained by dividing the mass of water contained in the immobilizing agent composition by the total mass of the immobilizing agent composition after the immobilizing agent composition applied to the reaction area has reached water equilibrium, and can be calculated by known methods such as weight change. Reaching water equilibrium means, for example, the state in which the mass change of the immobilizing agent composition becomes less than 1 mass% per hour when left standing in a constant humidity environment. The viscosity of the coating containing the immobilizing agent and reactive substance in the reaction area after application and reaching water 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 ambient relative humidity refers to the relative humidity of the room in which work is performed.

[0019] In this specification, "immobilizing agent" refers to a composition for embedding and holding a reactive substance on a substrate or within a reaction area. Embedding means a state in which the reactive substance is dispersed and held in a matrix of a hydrophilic compound alone or a hydrophilic compound + carbohydrate. The immobilizing agent includes a hydrophilic compound. Here, examples of hydrophilic compounds include hydrophilic compounds that are not volatile at room temperature (e.g., 20-30°C). In this specification, not volatile means the property of not substantially volatilizing under normal temperature and pressure (20°C, 1 atm). Examples of hydrophilic compounds that are not volatile at room temperature include aliphatic hydrocarbon compounds that are liquid at normal temperature and pressure, are not volatile, and have a molecular weight of 80-1000, preferably 80-600, containing hydrophilic oxygen atoms. Examples include alcohol compounds and ether compounds, and it is preferable that they are linear compounds. 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.

[0020] Examples of hydrophilic compounds that are not highly 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 preferably, glycerol, diethylene glycol, and polyethylene glycol (low molecular weight PEG) with a molecular weight of 100 to 600 are used.

[0021] Hydrophilic compounds that are not highly volatile at room temperature, such as glycerol, diethylene glycol, and polyethylene glycol with molecular weights of 100 to 600, hardly volatilize at room temperature and pressure and exist in a liquid state. However, they have a strong affinity for water and can retain moisture through moisture equilibrium in response to ambient humidity. For example, when an aqueous solution containing glycerol is left standing at room temperature and pressure, the water evaporates, leaving behind glycerol. However, a small amount of water remains in the remaining glycerol according to the moisture equilibrium, and this trace amount of water is thought to contribute to the stabilization effect of reactive substances and the dispersion-promoting effect of reactive substances when a liquid sample is added.

[0022] The immobilizing agent used for embedding the reactive substance may contain a saccharide in addition to the hydrophilic compound. That is, the reactive substance may be embedded in a mixture of the hydrophilic compound and the saccharide. Here, examples of the saccharide include monosaccharides, polysaccharides, sugar alcohols, preferably saccharides (including monosaccharides and disaccharides), and more preferably disaccharides. Sucrose is particularly preferable. Specific examples include sucrose, mannose, mannitol, sorbitol, dextran, trehalose and the like. Two or more of these saccharides may be used. By using these saccharides, the activity of the encapsulated reactive substance can be maintained at a higher level than when the hydrophilic compound is used alone. In particular, when a low-molecular-weight PEG is used as the hydrophilic compound, even when the low-molecular-weight PEG alone cannot maintain the activity of the reactive substance, the activity of the encapsulated reactive substance can be maintained at a higher level by using it in combination with a saccharide. The amount of these saccharides used is, for example, 10 to 100 mg / mL.

[0023] In the test device of the present invention, the amount of the hydrophilic compound in the reaction section is per 1 cm ２ of the application area of the reaction section, which corresponds to 0.1 to 10.0 µL in terms of 100% of the hydrophilic compound. If the amount of the hydrophilic compound is less than 0.1 µL / cm ２ , the effect of preventing drying may be insufficient, and if it exceeds 10.0 µL / cm ２ , the hydrophilic compound may inhibit the reaction when a liquid sample is added. Preferably, it is 1.0 to 5.0 µL per 1 cm ２ , more preferably 1.0 to 3.0 µL per 1 cm ２ , and still more preferably 1.3 to 2.5 µL per 1 cm ２ .

[0024] 0.1 to 10.0 µL per 1 cm ２ corresponds to 0.1 to 10.0 µL per 1 cm of the application area in the reaction section ２the amount of 100% of the hydrophilic compound per is 0.1 to 10.0 µL, and when the hydrophilic compound contains glycerol, if the density of glycerol is 1.26 mg / µL, the coating area of the reaction part is 1 cm ２ converted to 100% glycerol per, it is 0.126 to 12.6 mg. Similarly, in the case of glycerol, it is preferably 1.26 to 6.3 mg / cm ２ (1 to 5 µL / cm ２ ), more preferably 1.26 to 3.78 mg / cm ２ (1 to 3 µL / cm ２ ), still more preferably 1.638 to 3.15 mg / cm ２ (1.3 to 2.5 µL / cm ２ ). The same weight conversion based on density can be performed for diethylene glycol, polyethylene glycol, and the like.

[0025] By setting the amount of the hydrophilic compound within the above range, the residual water content due to water equilibrium in the hydrophilic compound layer embedding the reactive substance in the reaction part falls within a suitable range, which makes it possible to efficiently form a thin film retaining the reactive substance. During storage and transportation, the reactive substance can be stably retained while preventing scattering or the like thereof, and the dispersibility of the reactive substance is also excellent when a liquid sample is added.

[0026] The reaction part in the test device of the present invention is sealed with a second base material, preferably a film with low water permeability (moisture-proof film). This allows the water content in the reaction part to be maintained within a certain range, enabling long-term storage. Furthermore, by using a moisture-proof film as the second base material, the test device can be further reduced in size, and the bonding between the first base material and the second base material can be facilitated. Regarding the thickness of the second base material, from the viewpoint of the strength and flexibility of the second base material, the thickness is preferably 20 to 300 µm, more preferably 50 to 150 µm. The water vapor permeability of a film depends on temperature, and decreases as the temperature becomes lower. The water vapor permeability of the moisture-proof film used for sealing is such that the water vapor permeability of a COP film is about 1 g / m ２ / day, whereas it is preferably 0.01 g / m ２ / day or less at a storage temperature of 8°C, more preferably 0.001 g / m２ It is more preferable that it be less than or equal to / day. The water vapor transmission rate of the film at 8°C and 100% relative humidity should be 0.001 g / m². ２ By keeping the moisture content below 1 / day, fluctuations in moisture content during refrigerated storage over one year can be suppressed to within 10%. The water vapor permeability of film changes with temperature, with lower temperatures resulting in lower water vapor permeability. Most commercially available film catalog values ​​for water vapor permeability are given at 40°C; for example, when laminated to a chip, the water vapor permeability at 8°C is 0.001 g / m². ２ To achieve a value of less than / day, the water vapor transmission rate at 40°C must be 0.06 g / m³. ２ You can select options with a value of / day or less. Note that the water vapor transmission rate of films, etc., can be measured using a method compliant with JIS Z 0208.

[0027] As a film with low moisture permeability (moisture-proof film), there is the Aclar UltrRx3000 manufactured by Honeywell Japan (water vapor transmission rate of 0.06 g / m² at a temperature of 40°C and relative humidity of 100%). ２ At a temperature of 8°C and a relative humidity of 100%, the water vapor transmission rate is 0.001 g / m³. ２ Materials with low water vapor permeability, such as ( / day), can be used. As for the material of the moisture-proof film, fluorine-based resins such as polychlorotrifluoroethylene resin (PCTFE) and polytetrafluoroethylene resin (PTFE) are preferred because they have high moisture resistance even when thin and also have high transparency. The film can be attached to the part of the first substrate that will be the reaction part using an adhesive or adhesive. For example, a film with a larger area than the recess can be prepared, an adhesive or adhesive can be applied to the part surrounding the recess, and then it can be placed on the recess of the first substrate.

[0028] The inspection device preferably has a channel (inflow channel) that is connected to the reaction section and can supply a liquid sample to the reaction section. Such a channel can be made by providing a groove on the surface of the first substrate that is connected to a recess that will become the reaction section. The inspection device may also have a channel (outflow channel) that is connected to the reaction section and can discharge the liquid sample from the reaction section.

[0029] Thus, when the first substrate has a recess that serves as a reaction area and a groove that serves as a flow channel connected to the recess, the second substrate covers the recess and groove of the first substrate to form the reaction area and the flow channel connected to the reaction area. By using a material with low moisture permeability as the second substrate, it is possible to prevent moisture from permeating into the flow channel and the reaction area, thereby maintaining a constant moisture content within the inspection device. For example, when the inspection device has a reaction area and a flow channel for supplying a solution to the reaction area, a reactive substance embedded in a hydrophilic compound (such as glycerol) may be embedded in the recess that serves as the reaction area, and after bringing the hydrophilic compound (such as glycerol) to 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%, the groove that serves as the flow channel may be sealed together with the recess that serves as the reaction area using a moisture-proof film as described above.

[0030] The testing device of the present invention can be used by adding or introducing a liquid sample into the reaction section and reacting the target substance in the liquid sample with a reactive substance. For example, by adding a 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.

[0031] The inspection device of the present invention can be manufactured, for example, by the following steps. The method for manufacturing the inspection device of the present invention is a method for manufacturing an inspection device comprising: preparing a first substrate having a region (recess) that will be a reaction part in part; preparing a solution or dispersion by dissolving or dispersing a reactive substance in an aqueous solution of an immobilizing agent containing a hydrophilic compound (an aqueous solution of an immobilizing agent containing a hydrophilic compound and a sugar may also be used); adding the solution or dispersion of the reactive substance to the recess of the substrate; placing the first substrate under conditions where the relative humidity of 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 recess 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; and covering the reaction part with a moisture-proof second substrate as described above.

[0032] As the first substrate, the substrate described above can be used. The water contact angle of the recessed surface of the substrate is not particularly limited, but is preferably 10 to 90°, more preferably 20 to 80°, and even more preferably 50 to 70°.

[0033] For the preparation of a solution or dispersion obtained by dissolving or dispersing a reactive substance in an aqueous solution of an immobilizing agent containing a hydrophilic compound (or an aqueous solution of an immobilizing agent containing a hydrophilic compound and a carbohydrate), one may first prepare an aqueous solution of an immobilizing agent containing a hydrophilic compound (or an aqueous solution of an immobilizing agent containing a hydrophilic compound and a carbohydrate), and then dissolve or disperse the reactive substance in it, or one may dissolve or disperse the reactive substance in water first, and then dissolve the hydrophilic compound (and carbohydrates if necessary).

[0034] The concentration of the hydrophilic compound in the solution or dispersion is ultimately the predetermined amount in the recess (reaction area 1 cm²). ２ An amount equivalent to 0.1 to 10.0 μL, preferably 1 cm, when converted to 100% of the hydrophilic compound per unit. ２ An amount equivalent to 1.0 to 5.0 μL per unit, more preferably 1 cm ２ An amount equivalent to 1.0 to 3.0 μL per unit, more preferably 1 cm ２The concentration should be suitable for arranging a hydrophilic compound in an amount equivalent to 1.3 to 2.5 μL per unit, but it is preferably 0.2 to 20% by mass. On the other hand, the concentration of the reactive substance in the solution or dispersion can be appropriately adjusted based on the amount required for the reaction.

[0035] The method of adding the dissolving solution or dispersion to the reaction area of ​​the testing device is not particularly limited, but it is preferable to add it dropwise using a dispenser, inkjet, pipette, etc. The amount added is preferably 0.5 to 50 μL. By using this range, the reactive substance can be efficiently embedded in the immobilizing agent containing the hydrophilic compound and fixed to the reaction area.

[0036] The step of adjusting the water content of the hydrophilic compound contained in the reaction section to 5 to 80% by mass can be, for example, a method of bringing the water content of the hydrophilic compound and the water content of the environment into water equilibrium under constant humidity conditions (ambient relative humidity of 10 to 90%). The method of bringing the water content of the hydrophilic compound and the water content of the environment into water equilibrium is not particularly limited, but for example, it is preferable to leave it standing at room temperature, atmospheric pressure, and constant humidity conditions. In the reaction section, the water content of the hydrophilic compound is maintained at 5 to 80% by mass by water equilibrium. By maintaining such a state containing a small amount of water, a homogeneous thin film containing reactive substances can be formed, and the reactive substances can be uniformly dispersed when a liquid sample is added.

[0037] The process of covering the reaction section with a moisture-resistant second substrate can be carried out by joining the moisture-resistant second substrate to the surface of the first substrate having recesses or grooves that serve as flow channels using an adhesive or bonding agent.

[0038] The following describes a preferred embodiment of a microchip, which is one aspect of the inspection device of the present invention. However, the inspection device of the present invention is not limited to the following embodiments.

[0039] A microchip, which is one embodiment of the inspection device of the present invention, has a reaction section in which a reactive substance is embedded in an immobilizing agent containing a hydrophilic compound (which may be a hydrophilic compound and a sugar) and coated while containing a certain amount of moisture; an inflow channel connected to the reaction section for introducing liquid into the reaction section; and an outflow channel connected to the reaction section for discharging liquid from the reaction section, wherein the reaction section is sealed with a moisture-proof film.

[0040] The microchip may have the reaction section, the inflow channel, and the outflow channel inside. That is, it may be a microchip for analyzing the components of a sample by passing a liquid sample through the channels formed inside and reacting the substance to be measured in the liquid sample with a reactive substance applied to the reaction section in the channels. In such a case, an inlet can be provided at the starting end of the inflow channel to supply liquid to the inflow channel, and an outlet can be provided at the end of the outflow channel to discharge the liquid to the outside. In another embodiment, a waste liquid storage section for accumulating liquid may be provided at the end of the outflow channel. Preferably, the inlet and outlet are sealed with an openable sealing material. The sealing material may be completely removable, or a part of the sealing material may be bonded to the first or second substrate. Furthermore, the sealing material has a water vapor transmission rate of 0.001 g / m³ at a temperature of 8°C and a relative humidity of 100%. ２ It is preferable that it be less than or equal to / day.

[0041] The present invention will be specifically described below with reference to examples. However, the present invention is not limited to the embodiments described in the examples.

[0042] The evaluation was performed using a microchip made of COP (cycloolefin polymer) with a surface water contact angle of 70° as the substrate. To 12 μL of an aqueous solution containing a coloring substance (new coccine (CAS number: 2611-82-7)), 0 to 2 μL of glycerol was added and applied to a φ5 mm area on the substrate surface, and the moisture was removed by natural drying. After reaching moisture equilibrium, the pigment distribution in the reagent-coated area was measured using an absorbance meter, and the variability of the reagent distribution (coefficient of variation: CV) was determined. The moisture content of the glycerol after reaching moisture equilibrium by natural drying was 25% by mass.

[0043] The results are shown in Figure 1. Adding a small amount of glycerol suppressed variations in the reagent distribution within the coated surface.

[0044] In Example 1, 200 μL of physiological saline was added to the thin film formed on the substrate and allowed to stand at 37°C. The time required for the coating reagent (coloring substance) to disperse throughout the entire spot was measured.

[0045] The results are shown in Figure 2. φ5mm (area ≈ 0.196cm²) ２ When 0.26 μL of glycerol is added to the area of ​​) 1 cm ２ This corresponds to approximately 1.3 μL per square centimeter of coating area. By adding 1.3 μL or more of glycerol per square centimeter of coating area, the time required for dispersion was significantly reduced.

[0046] Figure 3 shows the appearance of the colored substance coated surface with and without glycerol addition (2 μL per square cm of coating area) (after drying at 22°C for 24 hours). When glycerol was not added, the colored substance precipitated and dried, but when glycerol was added, the colored substance adhered uniformly to the coated surface as a thin film of liquid.

[0047] Substrates with surface water contact angles of 90°, 70°, and 50° were prepared, and solutions containing a coloring agent and the respective amounts of glycerol were applied to COP films and allowed to air dry. Afterward, to investigate whether the coating would detach upon impact, the COP films were flicked with fingers to evaluate whether the coating remained attached to the COP film or whether it scattered from the COP film due to the impact of the flick. The results are shown in Table 1.

[0048] The adhesion / scattering properties of the applied reagent depended on the contact angle between the solution and the substrate surface and the amount of glycerol added. Strong adhesion of the glycerol-added reagent was observed in the contact angle range of 50–70°. ２ When the amount of glycerol applied per unit was as small as 0.3 μL, strong adhesion was observed even at a contact angle of 90°.

[0049]

[0050] The same experiment as in Example 2 was conducted using diethylene glycol and polyethylene glycol with a weight-average molecular weight of 200 (PEG200) as hydrophilic compounds that are not easily volatile at room temperature, instead of glycerol. The results are shown in Figure 4. Even when using diethylene glycol and polyethylene glycol with a weight-average molecular weight of 200 (PEG200), a reduction in the time required for the dispersion of the coloring substance was observed.

[0051] For each hydrophilic compound, the water content and moisture equilibrium were investigated at an ambient relative humidity of 50%. Specifically, 10 μL each of glycerol, diethylene glycol, dipropylene glycol, 3-ethoxypropane-1,2-diol, and polyethylene glycol (PEG200) with a weight-average molecular weight of 200 were dispensed into different containers and left to stand at a temperature of 22°C and a relative humidity of 50%. The mass was measured immediately after dispensing and after 13, 22, and 109 hours, and the water content (moisture absorption rate) was calculated using the following formula: Formula: Water content [%] = [(Mass of liquid after standing - Mass of liquid immediately after dispensing) ÷ Mass of liquid immediately after dispensing] × 100

[0052] The results are shown in Figure 5. The results showed that glycerol, diethylene glycol, dipropylene glycol, 3-ethoxypropane-1,2-diol, and low molecular weight PEG (PEG200) all rapidly absorbed moisture at an ambient humidity of 50%, and could stably maintain a moisture content of 5-40% by mass for a long period. In particular, glycerol, diethylene glycol, and low molecular weight PEG were able to stably maintain a moisture content of 15-30% by mass for a long period at an ambient humidity of 50%, indicating that they exhibited less water volatilization and long-term stability, making them more preferable substances.

[0053] To investigate the film conditions necessary to suppress fluctuations in the water content of reagents sealed within a chip to a certain range, a recess was created inside a polystyrene chip, a reagent thin film containing 1 μL of glycerol was prepared, the recess was sealed with film, and the change in the water content of the reagent was measured by weight change when left standing under saturated water vapor conditions. The flow channel area of ​​the chip was 0.2 cm². ２ , 1.0 cm ２ , 2.2 cm ２ Three types were used, and the film used for sealing was either COP film (Zeon ZF14-100, 100 μm thick) or moisture-proof film (Honeywell Aclar UltRx3000, 76 μm thick). The water vapor transmission rate of the COP film and moisture-proof film at a temperature of 40°C and 100% relative humidity was 1.1 g / m², respectively. ２ / 24h, 0.06 g / m ２ The test period was 24 hours. The relative humidity during the storage test was set to 100%, and the ambient temperature was set to three different temperatures: -30°C, 8°C, and 22°C. The test results are shown in Figure 6.

[0054] When encapsulated in COP film, the range of moisture content fluctuation (Figure 6 left) increased in a time-dependent manner, and the rate of increase in moisture content depended on the ambient temperature, becoming slower at lower temperatures. Moisture content fluctuations exceeding 10% occurred after 10 days at 22°C and 50 days at 8°C, while no increase in moisture content was observed at -30°C. On the other hand, when encapsulated in moisture-proof film (Figure 6 right), the range of moisture content fluctuation was significantly suppressed compared to COP film encapsulation, with moisture content fluctuations exceeding 10% after 40 days at 22°C and 12 months at 8°C. Furthermore, under freezing conditions at -30°C, no change in moisture content was observed for either COP film or moisture-proof film. Specifically, the water vapor transmission rate at 40°C was 0.06 g / m³. ２ / 24h or less (water vapor transmission rate at 8°C is 0.001 g / m³) ２ By sealing the reagents in a moisture-proof film (less than 24 hours), the fluctuation range of the reagent's moisture content can be suppressed to within ±10% for 12 months at 8°C.

[0055] Investigation of Thrombin Protection Effect (1) 0.1 units of thrombin were added to a 2 mL moisture-proof tube manufactured by Simport, and 10 μL of glycerol was added to form a thin film on the bottom surface of the moisture-proof tube. The mixture was brought to moisture equilibrium at a temperature of 22°C and an ambient relative humidity of 50%. After storage at a temperature of 22°C and a relative humidity of 5%, the thrombin activity was measured. For control, thrombin prepared immediately before use from dispensed and frozen thrombin was used. Note that under the above conditions, when a mixture of 0.1 units of thrombin and 10 μL of ultrapure water was used as the coating solution without using glycerol, the thrombin was completely inactivated.

[0056] The results are shown in Figure 7. In Figure 7, the names on the horizontal axis represent the following: Glycerol only: Results obtained using a mixture of 0.1 units of thrombin and 10 μL of glycerol as the coating solution. 10%PEG5000: Results obtained using a mixture of 0.1 units of thrombin, 10 μL of glycerol, and 10 μL of a 10% by mass aqueous solution of polyethylene glycol with a weight-average molecular weight of 5000 as the coating solution. 10%PVA500P: Results obtained using a mixture of 0.1 units of thrombin, 10 μL of glycerol, and 10 μL of a 10% by mass aqueous solution of polyvinyl alcohol as the coating solution. Lipidure BL103: Results obtained using a mixture of 0.1 units of thrombin, 10 μL of glycerol, and 10 μL of "Lipidure BL103" (NOF Co., Ltd.) as the coating solution. Lipidure BL203: Results obtained using a mixture of 0.1 units of thrombin, 10 μL of glycerol, and 10 μL of "Lipidure BL203" (NOF Co., Ltd.) as the coating solution. Lipidure BL403: Results obtained using a mixture of 0.1 units of thrombin, 10 μL of glycerol, and 10 μL of "Lipidure BL403" (NOF Oil & Fats Co., Ltd.) as a coating solution. Lipidure BL405: Results obtained using a mixture of 0.1 units of thrombin, 10 μL of glycerol, and 10 μL of "Lipidure BL405" (NOF Oil & Fats Co., Ltd.) as a coating solution. Lipidure BL702: Results obtained using a mixture of 0.1 units of thrombin, 10 μL of glycerol, and 10 μL of "Lipidure BL702" (NOF Oil & Fats Co., Ltd.) as a coating solution. Lipidure BL802: Results obtained using a mixture of 0.1 units of thrombin, 10 μL of glycerol, and 10 μL of "Lipidure BL802" (NOF Oil & Fats Co., Ltd.) as a coating solution. Lipidure BL803: Results obtained using a mixture of 0.1 units of thrombin, 10 μL of glycerol, and 10 μL of "Lipidure BL803" (NOF Oil & Fats Co., Ltd.) as a coating solution. Lipidure BL1003: Results obtained when a mixture of 0.1 units of thrombin, 10 μL of glycerol, and 10 μL of "Lipidure BL1003" (NOF Co., Ltd.) was used as a coating solution. Lipidure BL1201: Results obtained when a mixture of 0.1 units of thrombin, 10 μL of glycerol, and 10 μL of "Lipidure BL1201" (NOF Co., Ltd.) was used as a coating solution. LipidureNH01: Results obtained using a mixture of 0.1 units of thrombin, 10 μL of glycerol, and 10 μL of "Lipidure NH01" (NOF Co., Ltd.) as the application solution. HSA 100 μg / drop: Results obtained using a mixture of 0.1 units of thrombin, 10 μL of glycerol, and 100 μg of human serum albumin as the application solution. Glycine: Results obtained using a mixture of 0.1 units of thrombin, 10 μL of glycerol, and 10 μL of 200 mg / mL glycine solution as the application solution. Treharose: Results obtained using a mixture of 0.1 units of thrombin, 10 μL of glycerol, and 10 μL of 0.5 mol% trehalose solution as the application solution. Sucrose: Results obtained using a mixture of 0.1 units of thrombin, 10 μL of glycerol, and 10 μL of 0.5 mol% sucrose solution as the application solution. CRT: Activity test results before accelerated testing of 0.1 unit of dispensed and frozen thrombin. All of the above Lipidures are derivatives of 2-methacryloyloxyethyl phosphorylcholine polymer.

[0057] Figure 7 shows that when only glycerol was added, thrombin activity decreased to approximately 20% after 30 days at a temperature of 22°C and a relative humidity of 5%, but when glycerol was added along with carbohydrates such as trehalose or sucrose, thrombin activity of 75% or more could be maintained. Furthermore, Figure 7 shows that even in a harsh test of 30 days of storage at a temperature of 22°C and a relative humidity of 5%, 50-60% of thrombin activity could be maintained when using a mixture of 10 μL of glycerol and 10 μL of "Lipidure NH01", a mixture of 10 μL of glycerol and one drop of HSA (human serum albumin) 100 μg / drop, and a mixture of 10 μL of glycerol and 10 μL of 200 mg / mL glycine solution.

[0058] Investigation of Thrombin Protective Effect (2) 0.1 units of thrombin were added to a 2 mL moisture-proof tube manufactured by Simport, and 10 μL of a 10 vol% aqueous solution of polyethylene glycol (PEG200) with a weight-average molecular weight of 200 was added to prepare a coating solution. This coating solution was placed in a 2 mL moisture-proof tube to form a thin film on the bottom surface, and moisture equilibrium was reached at a temperature of 22°C and a relative humidity of 50%. After that, it was stored at a temperature of 22°C and a relative humidity of 5% for 30 days, and then thrombin activity was measured. For the control, thrombin prepared immediately before use from dispensed and frozen thrombin was used.

[0059] The results are shown in Figure 8. In Figure 8, the names listed on the horizontal axis represent the following: Sorbitol: Results obtained using a mixture of 0.1 units of thrombin, 10 μL of 10% aqueous solution of PEG200, and 10 μL of 0.5 mol% sorbitol solution as the coating solution. Maltose: Results obtained using a mixture of 0.1 units of thrombin, 10 μL of 10% aqueous solution of PEG200, and 10 μL of 0.5 mol% maltose solution as the coating solution. Mabnitol: Results obtained using a mixture of 0.1 units of thrombin, 10 μL of 10% aqueous solution of PEG200, and 10 μL of 0.5 mol% mannitol solution as the coating solution. Glucose: Results obtained using a mixture of 0.1 units of thrombin, 10 μL of 10% aqueous solution of PEG200, and 10 μL of 0.5 mol% glucose solution as the coating solution. Mannose: Results obtained using a mixture of 0.1 units of thrombin, 10 μL of 10% aqueous solution of PEG200, and 10 μL of 0.5 mol% mannose solution as the coating solution. Raffinose: Results obtained using a mixture of 0.1 unit thrombin, 10 μL of 10% aqueous solution of PEG200, and 10 μL of 0.5 mol% raffinose solution as the coating solution. Dextran40000: Results obtained using a mixture of 0.1 unit thrombin, 10 μL of 10% aqueous solution of PEG200, and 10 μL of dextran solution with a weight-average molecular weight of 40000 at 100 mg / mL as the coating solution. Sucrose: Results obtained using a mixture of 0.1 unit thrombin, 10 μL of 10% aqueous solution of PEG200, and 10 μL of 0.5 mol% sucrose solution as the coating solution. PEG200 only: Results obtained using a mixture of 0.1 unit thrombin and 10 μL of 10% aqueous solution of PEG200 as the coating solution. CRT: Results of the activity test of 0.1 unit thrombin dispensed and frozen before the accelerated test.

[0060] As shown in Figure 8, polyethylene glycol with a weight-average molecular weight of 200 (PEG200) has a weak denaturing effect, and as the "PEG200 only" results indicate, enzymes such as thrombin and physiologically active substances tend to lose activity when encapsulated for extended periods. However, polyethylene glycol, which exhibits fluidity at a temperature of 22°C and a relative humidity of 5%, showed activity-protective effects against enzymes such as thrombin and physiologically active substances when used with carbohydrates such as sorbitol, maltose, glucose, dextran, and sucrose. Among the carbohydrates, sorbitol showed particularly excellent activity-protective effects.

Claims

1. An inspection device comprising a first substrate having a recess in part, a second substrate covering the recess, and a reaction section formed by the recess of the first substrate and the second substrate, wherein a reactive substance is coated onto the reaction section in a state in which it is embedded with an immobilizing agent, the immobilizing agent contains a hydrophilic compound, and the amount of the hydrophilic compound in the reaction section is such that it is measured in the amount of the hydrophilic compound in the coating area of ​​the reaction section per 1 cm² ２ The amount is equivalent to 0.1 to 10.0 μL of the hydrophilic compound per unit, the water content of the hydrophilic compound is 5 to 80% by mass, and at least the portion of the second substrate covering the recess has a water vapor transmission rate of 0.001 g / m² under conditions of temperature: 8°C and relative humidity: 100%. 2 A testing device characterized by having a value of less than / day.

2. The inspection device according to claim 1, wherein the hydrophilic compound is one or more selected from the group consisting of glycerol, diethylene glycol, and polyethylene glycol having a molecular weight of 100 to 600.

3. The inspection device according to claim 1, wherein the immobilizing agent contains the hydrophilic compound and a carbohydrate.

4. The testing device according to claim 3, wherein the carbohydrate is one or more selected from the group consisting of sucrose, mannose, mannitol, sorbitol, dextran, and trehalose.

5. The inspection device according to claim 1, wherein the first substrate is a substrate made from one or more materials selected from cycloolefin polymer, polyethylene, polypropylene, polystyrene, polymethyl methacrylate, cycloolefin copolymer, polyphenylene oxide, polyethylene terephthalate, polyethylene naphthalate, polycarbonate, polyamide, polyimide, phenolic resin, epoxy resin, polyvinylidene chloride, polyvinyl chloride, ABS resin, and poly-2-methoxyethyl acrylate (PMEA) resin.

6. The testing device according to claim 1, wherein the reactive substance comprises a protein, a carbohydrate, or a nucleic acid.

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

8. The inspection device according to claim 7, wherein the microchip is connected to the reaction section and has a channel for introducing liquid into the reaction section.

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