Inspection device package and inspection system

Abstract

To provide an inspection device package and an inspection system.SOLUTION: An inspection device package has: an inspection device for a detection target substance contained in an endoplasmic reticulum of a lipid bilayer membrane, and a sealing bag that seals the inspection device under reduced pressure. The inspection device has a base material, and a micro flow path provided inside the base material. The base material is made of a polymeric material. One end of the micro flow path has an inflow part opened on a surface of the base material. The other end of the micro flow path is airtightly closed. An inner wall of the micro flow path is subjected to surface modification with a first compound containing an antibody capable of binding to a membrane protein contained in the lipid bilayer membrane, and a second compound containing a quaternary ammonium ion structure. A gas permeability of the sealing bag is equal to or less than 10 g / m2 day atm.SELECTED DRAWING: Figure 2

Description

Technical Field

[0001] The present invention relates to an inspection device package and an inspection system.

Background Art

[0002] Cancer is one of the three leading causes of death in Japan. As the stage of cancer progresses, the 5-year survival rate decreases, so it is desirable to detect and treat cancer at an early stage through checkups and the like. However, cancer examinations require large-scale sophisticated equipment, skilled examination technicians, etc., so it takes time to obtain results, and as a result, the current situation is that the acceptance rate is low. In order to shorten the time until examination results are obtained and to promote regular medical checkups, an inexpensive and simple cancer diagnosis method and a biomarker that utilizes substances in the living body for diagnosis are expected.

[0003] As an inexpensive and simple diagnostic method, point-of-care testing (POCT) has attracted attention. POCT refers to an examination performed by medical staff beside the subject, and takes advantage of the shortening of the examination time and the subject's feeling of proximity to the examination, and is an examination that improves the quality of medical care and the QOL of the subject.

[0004] A biomarker is a substance in the living body that reflects the presence or progression of a disease in terms of its concentration, etc. Recently, extracellular vesicles (EV) such as exosomes, which include miRNAs and proteins expected as cancer biomarkers, have attracted attention as biomarkers. Exosomes are vesicles with a lipid bilayer membrane secreted from cells, and are expected as biomarkers because they reflect the presence of various diseases not only in their contents but also in their secretion amount, size, surface molecules, etc.).

[0005] Microfluidic chips (microchips) are known as devices that realize POCT (Point-of-Cost Testing). A microchip is a device that has microchannels with a width and depth of approximately 10 μm to 1 mm. In POCT, methods have been developed to analyze biomarkers using microdevices with microchannels. As such microdevices, microchips that do not require external power to deliver the sample into the microdevice have been proposed (see Patent Document 1). [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Japanese Patent Publication No. 2005-31070 [Overview of the initiative] [Problems that the invention aims to solve]

[0007] However, the device (microchip) described in Patent Document 1 requires degassing under vacuum immediately before use in order to deliver the sample. Since degassing requires equipment such as a vacuum chamber, it is unsuitable as a device for point-of-care testing (POCT).

[0008] Furthermore, when considering POCT operations using EVs, individual elemental technologies such as biomarker capture techniques for samples (EVs) and simple detection methods for captured biomarkers have already been developed. However, these individual elemental technologies have not been integrated, and improvement was needed.

[0009] This invention has been made in view of these circumstances, and aims to provide a biomarker testing device package and a biomarker testing system. [Means for solving the problem]

[0010] The present invention encompasses the following embodiments.

[0011] [1] The device comprises a detection device for a target substance contained in the vesicle of a lipid bilayer, and a sealing bag for sealing the detection device under reduced pressure, wherein the detection device comprises a substrate and a microchannel provided inside the substrate, the substrate is made of a polymer material, one end of the microchannel has an inlet opening to the surface of the substrate, the other end of the microchannel is hermetically closed, the inner wall of the microchannel is surface-modified with a first compound containing an antibody that has the ability to bind to membrane proteins contained in the lipid bilayer, and a second compound containing the structure of a quaternary ammonium ion, and the gas permeability of the sealing bag is 10 g / m 2 • Test device package with a minimum of 1 day·atm

[0012] [2] The structure of the quaternary ammonium ion is the inspection device package described in [1] having an epoxy group.

[0013] [3] The inspection device package according to [1], wherein the inspection device has a detection unit for the substance to be detected downstream of the location where the second compound is provided relative to the inlet, and the inner wall of the microchannel in the detection unit is surface-modified with a third compound that includes a portion that binds to the substance to be detected.

[0014] [4] The inspection device package according to [1], wherein the microchannel has a downward slope toward the downstream side from the inlet.

[0015] An inspection system comprising an inspection device package as described in any one of items [5][1] to [4], and a detection device for detecting the substance to be detected. [Effects of the Invention]

[0016] According to the present invention, a testing device package and testing system for detecting target substances can be provided. [Brief explanation of the drawing]

[0017] [Figure 1]FIG. 1 is a schematic perspective view of the inspection device package 100. [Figure 2] FIG. 2 is a sectional view taken along line II-II of FIG. 1. [Figure 3] FIG. 3 is a schematic diagram showing a part of the inner wall of the microchannel 4. [Figure 4] FIG. 4 is a schematic diagram showing a part of the inner wall of the microchannel 4.

Embodiments for Carrying Out the Invention

[0018] In the inspection device included in the inspection device package of the present embodiment, substances contained in the endoplasmic reticulum of the lipid bilayer are used as inspection target substances. Examples of the lipid bilayer include extracellular vesicles (EVs), enveloped viruses, and the like. Examples of the detection target substances contained in the endoplasmic reticulum of the lipid bilayer include miRNAs, proteins, and the like. Examples of the sample containing the lipid bilayer include blood, urine, interstitial fluid, and the like.

[0019] That is, according to the inspection device package of the present embodiment, the detection target substances contained in the endoplasmic reticulum of the lipid bilayer can be detected, and the measurement of the detection target substances becomes possible. As a result, the detection target substances can be treated as biomarkers.

[0020] Hereinafter, the inspection device package 100 according to the present embodiment will be described with reference to FIGS. 1 to 4. In all the following drawings, for the sake of easy viewing of the drawings, the dimensions, ratios, etc. of each component are appropriately different.

[0021] FIG. 1 is a schematic perspective view of inspection device package 100. FIG. 2 is a cross-sectional view taken along line II-II of FIG. 1. As shown in FIGS. 1 and 2, inspection device package 100 is composed of inspection device 1 and a sealing bag 2 that hermetically seals inspection device 1 under reduced pressure. In inspection device package 100, inspection device 1 is sealed inside the reduced-pressure sealing bag 2. Inspection device 1 is taken out of sealing bag 2 immediately before use, and a sample containing a lipid bilayer is added to inflow portion 41 for use. Details of sealing bag 2 will be described later.

[0022] (Inspection Device) Inspection device 1 has a substrate 3 and a microchannel 4 provided inside substrate 3. In inspection device 1 shown in FIG. 1, it is shown that there are three microchannels 4 extending in the longitudinal direction in plan view, but it is not limited to this, and the number of microchannels 4 can be appropriately changed. The microchannel 4 can be, for example, 100 μm in width and 25 μm in height, but is not limited to this.

[0023] (Substrate) Substrate 3 is composed of a first substrate 31 and a second substrate 32. First substrate 31 is a flat plate rectangular in plan view. Second substrate 32 is a member having a recess with a predetermined depth corresponding to microchannel 4. By overlapping the surface of second substrate 32 having recess 32a on the upper surface 31a of first substrate 31, microchannel 4, which is a space surrounded by upper surface 31a and recess 32a, is formed. The shape (plan view shape, depth) of recess 32a can be appropriately selected according to the detection target substance.

[0024] In FIGS. 1 and 2, it is shown that the plan view shape of first substrate 31 and the plan view shape of second substrate 32 coincide, and the outer periphery of first substrate 31 and the outer periphery of second substrate 32 coincide in substrate 3, but it is not limited to this. If microchannel 4 is formed when first substrate 31 and second substrate 32 are overlapped, the plan view shapes of first substrate 31 and second substrate 32 can adopt various shapes. For example, when first substrate 31 and second substrate 32 are overlapped, the outer periphery of first substrate 31 may be located outside the outer periphery of second substrate 32 in plan view.

[0025] When the inspection device 1 is placed on a horizontal surface, the upper surface 31a of the first substrate 31 may be parallel to the horizontal plane, or it may have a downward slope that inclines from the inlet 41 (described later) of the microchannel 4 toward the downstream side of the microchannel 4.

[0026] The substrate 3 is formed from a polymer material. More specifically, at least the portion of the substrate 3 facing the microchannel 4 is made of a polymer material. In particular, it is preferable that the substrate 3 be composed of a polymer material capable of dissolving a large amount of gas. Examples of "polymer materials capable of dissolving a large amount of gas" include rubber materials, which have a sparse solid microstructure and a large degree of freedom of motion for solid molecules. By making the substrate 3 from a polymer material capable of dissolving a large amount of gas, it becomes easier to aspirate the sample described later, making it easier to use.

[0027] In the base material 3, the material for the first base material 31, which is a flat plate, is preferably a light-transmitting polymer material or glass, and the material for the second base material 32 having a recess 32a is preferably a material that is easy to mold and has high gas solubility. A preferred material for such a second base material 32 is a light-transmitting elastomer, and PDMS (polydimethylsiloxane) is particularly preferred.

[0028] (Microfluidic channels) One end of the microchannel 4 has an inlet 41 that opens to the surface of the substrate 3 (the upper surface 32b of the second substrate 32). The other end of the microchannel 4 is hermetically sealed. As shown in Figure 2, the other end of the microchannel 4 may be an opening 42 that opens to the surface of the substrate 3. In that case, the opening 42 is hermetically sealed by a removable sealant 45. The other end of the microchannel 4 may be provided with a liquid reservoir space that is wider than the width and height of the microchannel.

[0029] Figures 3 and 4 are schematic diagrams showing a portion of the inner wall 43 of the microchannel 4. The microchannel 4 has a disruption section 47 that disrupts the lipid bilayer endoplasmic reticulum and causes the target substance to leak out from the endoplasmic reticulum. The inner wall 43 of the disruption section 47 is surface-modified with a first compound 5 having an antibody that specifically binds to the membrane proteins of the lipid bilayer endoplasmic reticulum, and a second compound 6 having the structure of a quaternary ammonium ion.

[0030] (first compound) The first compound 5 consists of an antibody 51 that specifically binds to membrane proteins of the endoplasmic reticulum of the lipid bilayer contained in the sample, and a polymer (linker site) 52 that is modified by the antibody 51 and binds to the inner wall 43.

[0031] The antibody 51 contained in the first compound 5 can be any antibody that specifically binds to the membrane proteins of the endoplasmic reticulum of the lipid bilayer contained in the sample, and can be appropriately selected depending on the type of membrane protein of the endoplasmic reticulum of the lipid bilayer to be detected. The antibody 51 may be the antibody itself that directly binds to the substance to be detected, or only the antigen-binding site within the antibody may be used.

[0032] Examples of polymer 52 include polymers having functional groups that can bind to antibody 51. Examples of "binding functional groups" include carboxyl groups that can condense with amino groups of antibody 51, and epoxy groups that undergo addition reactions with antibody 51. Examples of polymers having the above functional groups include acrylates and methacrylates.

[0033] (Second compound) The second compound 6 consists of a site (substituent) 61 having the structure of a quaternary ammonium ion and a polymer (linker site) 62 that includes the site having the structure of a quaternary ammonium ion and is bonded to the inner wall 43.

[0034] In this embodiment, the moiety 61 having the structure of a quaternary ammonium ion functions as a cationic surfactant that disrupts the vesicles of the lipid bilayer. Examples of polymer 62 include acrylates and methacrylates.

[0035] Poly-2-(dimethylamino)ethyl methacrylate (pDMAEMA) can be cited as a polymer 62 having the above-described site 61.

[0036] In the diagram, the antibody 51 of the first compound 5 and the site 61 having a quaternary ammonium ion structure of the second compound 6 are shown to be bound to separate polymers 52 and 62, respectively, but this is not limited to this arrangement. For example, the antibody 51 and the site 61 having a quaternary ammonium ion structure may be bound to the same polymer chain.

[0037] (Detection unit) The inspection device 1 may have a detection unit 48 for the substance to be detected located downstream of the location where the second compound 6 is provided with respect to the inlet 41. If a detection unit 48 is present, the inner wall 43 of the detection unit 48 may be surface-modified with a third compound 7 that has a site that specifically binds to the substance to be detected.

[0038] (Third compound) The third compound 7 consists of a site 71 that specifically binds to the substance to be detected, and a polymer (linker site) 72 that includes the site that specifically binds to the substance to be detected and binds to the inner wall 43. The site 71 that specifically binds to the substance to be detected can be appropriately selected by a person skilled in the art depending on the type of substance to be detected. Examples of the site 71 that specifically binds to the substance to be detected include various nucleic acid molecules such as DNA and their fragments such as oligonucleotides, proteins and peptides such as antigens and antibodies, lipids and sugars, or low molecular weight compounds such as drugs. Examples of polymers 72 include acrylates and methacrylates.

[0039] In addition, while the first compound 5, the second compound 6, and the third compound 7 are assumed to each have polymers (polymers 52, 62, and 72) that bind to the inner wall 43, they are not limited to this. For example, the first compound 5 does not need to have polymer 52 as long as it can introduce antibody 51 into the inner wall 43. If the first compound 5 does not have polymer 52, for example, a pretreatment may be performed to provide a "functional group that can bind to antibody 51" on the surface of the inner wall 43, and then the functional group provided on the surface of the inner wall 43 may be bound to antibody 51.

[0040] In the second compound 6, for example, a pretreatment may be performed to provide a functional group (e.g., a carboxyl group) that can bond to an amino group on the surface of the inner wall 43, and after introducing an amino group to the surface of the inner wall 43, the amino group may be converted into a quaternary ammonium ion structure.

[0041] In the third compound 7, for example, a pretreatment may be performed to provide a "functional group that can bond to the moiety 71" on the surface of the inner wall 43, and the functional group provided on the surface of the inner wall 43 may be bonded to the moiety 71.

[0042] The densities of the first compound 5, the second compound 6, and the third compound 7 (the ratio of polymer to the surface area of ​​the inner wall 43) should be set to a density that can break down the lipid bilayer and a density that can capture the target substance to a detectable degree, respectively. The density that can break down and capture should be set in advance through preliminary experiments, in accordance with the lipid bilayer and the target substance to be detected.

[0043] Because the inspection device 1 has a third compound 7, the target substance leaked by the first compound 5 and the second compound 6 can be fixed inside the microchannel 4 (detection unit 48) and then detected by a known method.

[0044] The target substance immobilized by the third compound 7 in the detection unit 48 may be detected by fluorescence detection.

[0045] When detecting target substances contained in EV, a known laminar dendritic amplification method (LFDA) may be employed. In this case, streptavidin fluorescein isothiocyanate from Streptomyces avidin can be used as the fluorescent reagent.

[0046] LFDA uses a bifurcated microchannel. The microchannel used in LFDA has two inlets at one end and one opening at the other end, which is sealed. The two channels extending from the two inlets merge and connect to each other midway before extending to the other end. For convenience, in the following explanation, the two inlets will be distinguished as the first inlet and the second inlet, the channel extending from the first inlet will be called the first channel, and the channel extending from the second inlet will be called the second channel.

[0047] First, a sample solution containing EV (sample solution) is flowed through the first channel, and a solution of an antibody capable of binding to the target substance (e.g., biotin-labeled anti-CD63 antibody) (antibody solution) is flowed through the second channel. At this time, by providing the first and second compounds described above on the inner wall of the first channel, the target substance contained in EV is released into the sample solution.

[0048] At the LFDA, the target substances are amplified as follows: In the microchannel, a laminar flow of the sample solution and antibody solution is formed downstream of the confluence of the first and second channels. By providing the aforementioned third compound on the inner wall at a position overlapping with the interface between the sample solution and the antibody solution, the third compound captures the target substance. Antibodies flowing through the second channel then bind to the captured target substance. Further target substances flowing from the first channel then bind to the antibodies bound to the target substance.

[0049] Thus, after the third compound captures the target substance, the antibody supplied from the second channel binds to the target substance, and the target substance supplied from the first channel binds to the antibody, and these bindings occur continuously and repeatedly in accordance with the supply from each channel. Due to these bindings, the target substance is amplified in a dendritic manner in the region where the third compound is located.

[0050] Next, a fluorescent label (e.g., fluorescently labeled streptavidin (F-SA)) is passed through the first channel, and an amplification reagent (e.g., biotin-labeled anti-streptavidin (Bio-anti-SA)) is passed through the second channel. This causes the amplification reagent and the fluorescent label to alternately bind to the antibody bound to the third compound, thereby amplifying the detection sensitivity.

[0051] In LFDA (Low-Level Food Detector), it is advisable to simultaneously perform a blank experiment in which only buffer without the sample is passed through, and to detect the target substance by comparing the fluorescence intensity with that when the sample solution is passed through.

[0052] In addition to fluorescence detection, the target substance may be detected using electrochemical methods. Known methods can be used for electrochemical detection.

[0053] (Sealed bag) The sealed bag 2 is a packaging bag made of a gas barrier film. The gas barrier film is a laminate having a base material and a gas barrier layer. The gas barrier film may further have a heat seal layer on top of the gas barrier layer.

[0054] Possible base materials include polyester films such as polyethylene terephthalate (PET) and polyethylene naphthalate (PEN), polyolefin films such as polyethylene and polypropylene, polyethersulfone (PES), polystyrene films, polyamide films, polyvinyl chloride films, polycarbonate films, polyacrylonitrile films, and polyimide films.

[0055] The thickness of the substrate should preferably be, for example, between 6 μm and 200 μm.

[0056] The surface of the substrate facing the gas barrier layer may be subjected to physical treatments such as corona treatment, plasma treatment, or flame treatment, or chemical treatments such as acid or alkali treatment, in order to improve adhesion with the gas barrier layer.

[0057] The gas barrier layer may employ a vapor-deposited film containing silicon oxide or aluminum oxide. The gas barrier layer may further contain at least one metal selected from the group consisting of zinc, tin, and iron, or an oxide of said metal.

[0058] The thickness of the gas barrier layer should be between 5 nm and 500 nm.

[0059] It is preferable that an anchor coat layer is provided between the substrate and the gas barrier layer to enhance adhesion between the substrate and the gas barrier layer. The material of the anchor coat layer is preferably a composite of an acrylic polyol containing a hydroxyl group and an isocyanate compound having at least two or more isocyanate groups in its molecule.

[0060] Acrylic polyols are polymeric compounds obtained by polymerizing (meth)acrylic acid derivative monomers having hydroxyl groups at their terminals and side chains, or polymeric compounds obtained by copolymerizing (meth)acrylic acid derivative monomers having hydroxyl groups at their terminals and side chains with other monomers. Acrylic polyols have hydroxyl groups at their terminals and side chains and react with the isocyanate groups of isocyanate compounds.

[0061] Examples of the above (meth)acrylic acid derivative monomers include hydroxyethyl (meth)acrylate and hydroxybutyl (meth)acrylate.

[0062] Other examples of monomers mentioned above include (meth)acrylic acid esters, styrene, cyclohexyl maleimide, and phenyl maleimide.

[0063] Examples of the above-mentioned isocyanate compounds include aromatic isocyanates such as tolylene diisocyanate (TDI), diphenylmethane diisocyanate (MDI), xylene diisocyanate (XDI), and tetramethylxylylene diisocyanate (TMXDI), as well as aliphatic isocyanates such as hexamethylene diisocyanate (HDI), bisisocyanate methylcyclohexane (H6XDI), isophorone diisocyanate (IPDI), and dicyclohexylmethane diisocyanate (H12MDI). Polymers or derivatives of these monomeric isocyanates can also be used.

[0064] The thickness of the anchor coat layer 12 should be between 50 nm and 500 nm.

[0065] The surface of the gas barrier layer may be provided with an overcoat layer in contact with the gas barrier layer to suppress cracking in the gas barrier layer. The overcoat layer contains a water-soluble polymer having a hydroxyl group or a water-soluble polymer having a carboxyl group.

[0066] Polyvinyl alcohol (PVA), polyacrylic acid (PAA), ethylene-vinyl alcohol copolymer, and polyvinylpyrrolidone (PVP) can be used as materials for the overcoat layer.

[0067] The thickness of the overcoat layer should be between 100 nm and 1000 nm.

[0068] The gas permeability of the gas barrier film, which is the material for sealing bag 2, is 10 g / m². 2 It is less than or equal to 10 g / m³. In other words, the gas permeability of sealed bag 2 is 10 g / m³. 2 It is less than or equal to the day and ATM.

[0069] The gas barrier film is formed into a bag shape with the heat-seal layer facing the inside of the sealed bag 2. Information about the contents (inspection device 1) may be printed on the outer surface of the substrate. A transparent barrier film (product name: GL BARRIER, manufactured by Toppan Printing Co., Ltd.) can be used as such a gas barrier film.

[0070] In the inspection device package 100, the inspection device 1 can be stored under reduced pressure for a long period of time by sealing it under reduced pressure using such a sealing bag 2. The inspection device 1, once removed from the sealing bag 2, remains under reduced pressure until immediately before removal.

[0071] The inspection device 1 can be removed from the sealed bag 2 immediately before use, and by adding the sample containing lipid bilayer vesicles to the inlet 41, the sample can be introduced into the microchannel 4 without requiring external power. The following will explain this in detail.

[0072] As described above, the inspection device 1 of the inspection device package 100 is sealed inside a degassed sealed bag 2. In the inspection device 1 under reduced pressure, dissolved gas (air) is released from the polymer material constituting the base material 3.

[0073] When the inspection device 1 is removed from the sealed bag 2 and the pressure returns from reduced pressure to atmospheric pressure, redissolution of air into the polymer material constituting the substrate 3 of the inspection device 1 begins. At this time, if a sample is added to the inlet 41 so as to block the inlet 41, the internal space of the microchannel 4 is closed from the outside (hereinafter referred to as the closed space). The air present in the closed space redissolves into the substrate 3 from the inner wall 43 of the microchannel 4. As the air in the closed space redissolves into the substrate 3, the pressure in the closed space decreases, and the sample is introduced into the microchannel 4.

[0074] When the sample is introduced into the microchannel 4, the lipid bilayer endoplasmic reticulum contained in the sample is specifically captured by the first compound 5, which surface-modifies the inner wall of the microchannel 4, and is further disrupted by the second compound 6, which is located near the first compound 5. Specifically, the lipid bilayer endoplasmic reticulum is specifically captured by the first compound 5, which has an antibody that specifically binds to the membrane protein of the lipid bilayer endoplasmic reticulum, and is disrupted by the second compound 6, which has a quaternary ammonium ion structure. The target substance leaks out from the disrupted lipid bilayer endoplasmic reticulum.

[0075] The obtained target substance is used for testing by various methods. If the detection unit 48 described above is provided in the microchannel 4, the target substance is captured by the third compound 7 and used for testing in the detection unit 48.

[0076] If the opening 42 of the testing device 1 is sealed with a removable sealant 45, a sample containing lipid bilayer vesicles can be introduced into the testing device 1, the target substance can be leaked out, and then the sealant 45 can be removed to recover the sample containing the target substance from the exposed opening 42. This allows for the recovery of miRNAs contained in lipid bilayer vesicles, for example, simplifying the pre-processing for PCR testing. Therefore, using the testing device package 100 can shorten the time required for PCR testing.

[0077] In this manner, the inspection device package 100, which includes an inspection device 1 that detects a target substance contained in the sample after the sample is introduced into the microchannel 4, can be used even in situations where equipment such as a vacuum chamber is unavailable, as the inspection device 1 is sealed under reduced pressure in the sealing bag 2.

[0078] [Manufacturing method for inspection device packages] (1) Method of manufacturing an inspection device (Manufacturing of substrates (microfluidic channels)) The second substrate 32 of the inspection device 1 is manufactured by the following soft lithography method. First, a mold having a shape complementary to the recess 32a of the second substrate 32 is fabricated by a known method, for example, by photolithography followed by etching on a metal material. The "complementary shape" of the mold can be a convex portion (for example, 100 μm wide and 25 μm high) corresponding to the recess 32a. Protrusions corresponding to the inlet 41 and opening 42 may be provided at the ends of the convex portion.

[0079] PDMS is poured into the completed mold to create a PDMS chip with a recess measuring 100 μm in width and 25 μm in height. A glass slide is attached to the recessed surface of the fabricated PDMS chip to form a microchannel.

[0080] In addition to the above method, the second substrate 32 may be manufactured by micro-injection molding. Alternatively, the second substrate 32 may be manufactured by patterning a flat plate using a known photolithography method and then dry etching it.

[0081] (Surface modification by the first and third compounds) The formed microchannel is filled with benzophenone (10% by mass acetone solution, 3 μL), which is the photopolymerization initiator, and left to stand at room temperature for about 5 minutes to allow the initiator to be supported in the channel. Next, the PDMS chip is ultrasonically cleaned with ultrapure water at room temperature for 5 minutes. After ultrasonic cleaning, the moisture is removed using an air duster and the chip is attached to a new glass slide.

[0082] Next, for example, 2-(dimethylamino)ethyl methacrylate (DMAEMA) (2.0 mol / L aqueous solution, 6.0 μL), a monomer having a tertiary amine, is passed through the channel. After passing the solution through, the inner wall of the channel can be modified with poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) by UV graft polymerization through UV irradiation (365 nm, 100 W) for 10 minutes.

[0083] The PDMS chip is ultrasonically cleaned with ultrapure water at room temperature for 5 minutes. After ultrasonic cleaning, the moisture is removed using an air duster, and the chip is attached to a new glass slide to prepare a PDMAEMA-modified PDMS chip.

[0084] Prepare a solution (2.0 μL) of an antibody (e.g., anti-integrin β1 antibody (0.0-1.40 mg / mL, PBS solution, 10 μL)) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (condensing agent, 4.5 mg / mL, 1.0 μL). Fill the microchannel of the PDMAEMA-modified PDMS chip with the obtained solution.

[0085] Next, the sample is left to stand at 37°C for 2 hours at 100% humidity to fix the anti-integrin β1 antibody.

[0086] Next, the second substrate is ultrasonically cleaned with ultrapure water at room temperature for 5 minutes. After removing the moisture using an air duster, the second substrate is attached to the first substrate (glass slide), and a microchannel can be fabricated in which the first compound is fixed to the inner wall of the channel.

[0087] By using a compound that corresponds to the "site that specifically binds to the target substance" of the third compound, instead of an antibody, it is possible to create a microchannel in which the third compound is immobilized on the inner wall of the channel.

[0088] (Surface modification by a second compound) A mixed solution of bromoethane (1.28 mol / L) and epichlorohydrin (1.28 mol / L) (DMF solution, 8.0 μL) is passed through a PDMAEMA-modified PDMS chip. After the solution is passed through, the inlet and outlet of the channel are sealed with Teflon® tape, and the chip is left to stand at 40°C for 12 hours to produce a quaternary amine-modified PDMS chip.

[0089] To confirm quaternization, polyT-TMR is used. At room temperature, the sample is ultrasonically washed with ultrapure water, degassed under vacuum, then POLYT-TMR (10 μmol / L carbonate buffer solution, 3.0 μL) is passed through, and ultrasonic washing is performed again. After that, carbonate buffer (5.0 μL, pH 9.5) is passed through, and fluorescence observation is performed. By examining the value of red fluorescence before and after the quaternization procedure, the quaternization of the tertiary amine can be confirmed.

[0090] (2) Method for manufacturing sealed bags A gas barrier film having a gas barrier layer can be formed by vacuum deposition of the above-mentioned gas barrier layer material onto a resin film (for example, a biaxially oriented PET film). It is preferable to pre-treat the surface on which the gas barrier layer will be formed with corona.

[0091] Alternatively, before forming the gas barrier layer, an anchor coat layer forming material may be applied to the corona-treated surface to form the anchor coat layer. Examples of anchor coat layer forming materials include a copolymer of hydroxyethyl methacrylate and methyl methacrylate (acrylic polyol (weight average molecular weight 10 × 10)). 3 A 5% methyl ethyl ketone solution can be used, with )) as the main component and an HDI nurate-type isocyanate curing agent added in an amount of 1 equivalent relative to the amount of hydroxyl groups in the main component. For the application method, any known printing method, such as gravure coating, can be appropriately employed. An anchor coat layer can be formed by drying the applied forming material.

[0092] Furthermore, an overcoat layer may be formed by applying an overcoat-forming material to the surface of the gas barrier layer. As the overcoat-forming material, an aqueous solution of the water-soluble polymer mentioned above can be used. The application method can be any known printing method, such as gravure coating. The overcoat layer can be formed by drying the applied forming material.

[0093] The resulting gas barrier film is molded into a bag shape using heat sealing or an adhesive as appropriate to produce a sealed bag.

[0094] (3) Method for manufacturing inspection device package A test device package can be manufactured by placing the above-mentioned test device in a sealed bag and sealing it under a reduced pressure environment. Before sealing it in the sealed bag, the test device should be stored under a reduced pressure environment until the gas contained in the second substrate 32 is sufficiently removed. Since the appropriate reduced pressure conditions (degree of reduced pressure, storage time under reduced pressure) for the second substrate will differ depending on the material, volume, etc. of the second substrate, it is advisable to conduct preliminary experiments to set the reduced pressure conditions that allow for proper removal of gas from the second substrate.

[0095] The manufacturing methods for the inspection device package 100, inspection device 1, and sealing bag 2 in this embodiment have been described above. However, the manufacturing methods are not limited to these, and an appropriate manufacturing method can be selected depending on the materials constituting the inspection device 1 and the materials of the sealing bag 2.

[0096] [Inspection System] The inspection system of this embodiment comprises the inspection device package 100 of this embodiment described above, and a detection device for detecting the target substance.

[0097] A fluorescence microscope (all-in-one fluorescence microscope, model BZ-8100, manufactured by Keyence Corporation) can be used as the detection device.

[0098] Such a testing system allows for the integration of a technique for capturing target substances contained in lipid bilayers with a simple detection technique for the captured target substances.

[0099] Preferred embodiments of the present invention have been described above with reference to the attached drawings, but the present invention is not limited to these examples. The shapes and combinations of the constituent members shown in the above examples are merely examples, and can be modified in various ways based on design, specifications, etc., without departing from the spirit of the present invention. [Industrial applicability]

[0100] The present invention, made in view of these circumstances, provides a testing device package for a target substance and a testing system for a target substance. [Explanation of symbols]

[0101] 100 Inspection Device Package 1. Inspection device 2 Sealed bag 31 First base material 32 Second base material 4 Microchannels 41 Inlet 42 Opening 43 (Inner wall of a microfluidic channel) 45 Sealing material 47 Crushing section 48 Detection unit 5 First compound 6 Second compound 7 Third compound

Claims

1. A testing device for detecting target substances contained in the endoplasmic reticulum of lipid bilayers, The inspection device has a sealing bag for depressurizing and sealing, The inspection device comprises a substrate and The substrate has a microchannel provided inside it, The aforementioned substrate is made of a polymer material, One end of the microchannel has an inlet opening that opens onto the surface of the substrate. The other end of the aforementioned microchannel is hermetically sealed. The inner wall of the microchannel contains a first compound which has the ability to bind to membrane proteins contained in the lipid bilayer, A second compound containing the structure of a quaternary ammonium ion is surface-modified with, The gas permeability of the aforementioned sealed bag is 10 g / m³. ２ A test device package that is below the daily ATM limit.

2. The structure of the quaternary ammonium ion is an epoxy group, as described in claim 1 of the inspection device package.

3. The inspection device has a detection unit for the substance to be detected downstream of the location where the second compound is provided relative to the inflow section, The inspection device package according to claim 1, wherein the inner wall of the microchannel in the detection unit is surface-modified with a third compound that includes a portion that binds to the substance to be detected.

4. The inspection device package according to claim 1, wherein the microchannel has a downward slope toward the downstream side from the inlet.

5. A test device package according to any one of claims 1 to 4, An inspection system comprising a detection device for detecting the target substance.

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