WO2016152702A1 - 分析デバイス - Google Patents
分析デバイス Download PDFInfo
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- WO2016152702A1 WO2016152702A1 PCT/JP2016/058447 JP2016058447W WO2016152702A1 WO 2016152702 A1 WO2016152702 A1 WO 2016152702A1 JP 2016058447 W JP2016058447 W JP 2016058447W WO 2016152702 A1 WO2016152702 A1 WO 2016152702A1
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- microfluidic device
- antibody
- flow path
- specific binding
- resin
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- G—PHYSICS
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
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- B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J19/0046—Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
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- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502761—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54306—Solid-phase reaction mechanisms
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N37/00—Details not covered by any other group of this subclass
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- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
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- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
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- B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
Definitions
- the present invention relates to a test device, a test kit and a test system using specific binding.
- the present invention also relates to a method for manufacturing the device.
- the present invention relates to a microfluidic device including a microchannel capable of analyzing a plurality of items at once.
- Analytical methods utilizing specific binding such as immunoassays that utilize the affinity of antibodies for antigens, have been widely used in clinical examinations and drug discovery fields. Since the amount of sample and reagent used can be small and the time required for the assay can be shortened, instead of an assay using a plate such as a so-called 96-well plate, the reaction system has a small volume. Assays using devices have been developed.
- the present inventors have also disclosed a microfluidic device for immunoassay arranged in a flow path as a pillar-like structure in which beads in which an antibody is solid-phased are uniformly dispersed and held in a photocured hydrophilic resin. (Patent Document 1, Non-Patent Document 1).
- FIG. 8A shows a microfluidic device in which a primary antibody is solid-phased on polystyrene fine beads having a diameter of about 1 ⁇ m, and pillar-shaped microstructures are provided in a flow path with a photocurable resin.
- a microbead in which a primary antibody is solid-phased is suspended in a hydrophilic photocurable resin solution, and a liquid filled in the flow path in a suspension state is patterned and cured by exposure treatment and cured. It is an analysis device of one item.
- This device is used as follows. First, a sample such as serum or urine is filled in the flow path and incubated to bind the antigen contained in the sample to the primary antibody on the beads. After the specimen is washed with a washing solution, a fluorescently labeled antibody is injected and incubated to bind to the antigen to be detected bound to the primary antibody. Next, after washing the fluorescently labeled antibody not bound with the washing solution, the fluorescence of the labeled antibody is detected by a fluorescence detector.
- FIG. 8B shows a three-item analysis device that can detect three different antigens in a single assay. This device is fixed by mixing microbeads in which three kinds of antibodies recognizing different antigens are solid-phased and suspending them in a photocurable resin, and similarly by exposing them to curing in a flow path. Yes.
- the single item analysis device shown in FIG. 8A only needs to use a detector that detects one fluorescence, the detector is also small and can detect with high sensitivity in a short time. There was a problem that could not be done. Further, in the case of the three-item analysis device shown in FIG. 8B, since it is necessary to select a light source whose excitation spectrum is sufficiently separated, a maximum of three types of antigens can be detected by one device. In addition, an optical system that matches the type of fluorescent dye is required, and an optical system switching device for switching a plurality of optical filters is also required. Therefore, it is difficult to reduce the size and price of the fluorescence detector.
- a bead on which a single primary antibody A is solid-phased is suspended in a photocurable resin solution to fill the flow path.
- the flow path is covered with a photomask having a hole so that only a portion where the resin is to be cured can be exposed, and the resin is cured by irradiating UV.
- the uncured resin is washed and discharged from the flow path, whereby the A antibody-immobilized beads are fixed in the flow path (FIG. 9A right).
- the bead on which the B antibody is immobilized is suspended in a photocurable resin solution to fill the channel (FIG. 9B).
- the flow path is covered with a photomask in which only the portion where the resin is to be cured can be exposed, and the B antibody-immobilized beads are cured together with the photocurable resin by UV irradiation. After photocuring the resin, the uncured resin is washed.
- FIG. 9C shows the result of measurement using a multi-item device prepared using beads coated with an antibody by this method.
- the multi-item device in FIG. 9D was manufactured as follows. Anti-CRP antibody and anti-CEA antibody were coated with polystyrene beads, respectively.
- Photocurable resins include a photopolymerizable prepolymer solution (MI-1, manufactured by Kansai Paint Co., Ltd.), a photocuring initiator solution (PIR-1, manufactured by Kansai Paint Co., Ltd.), and purification, having polyethylene glycol as a basic skeleton. A resin mixed solution in which water was mixed was used.
- FIG. 9C it is considered that the beads coated with the anti-CRP antibody are caught in the first photocured region.
- the cured photocurable resin had a porous structure. Therefore, in the process of manufacturing the device, the beads get caught in the pores of the cured resin, and it is considered that multiple types of antibody-immobilized beads are included in one pillar-like structure in order to fit into the structure. It is done. That is, it is considered that the antibody-immobilized beads are fitted into the pores of the structure in the process of washing the uncured resin.
- the use of a microarray instead of a microchannel system can prevent contamination when a plurality of antibodies are immobilized.
- microarrays using photo-curing resins have been disclosed, but in all cases, a resin is applied, and a protein solution such as an antibody is applied thereon, followed by photo-curing and solidification.
- Non-Patent Documents 2 and 3 Therefore, the reaction field where the specific binding reagent such as an antibody reacts with the detection target is limited to the surface of the photocurable resin on which the specific binding reagent is applied.
- the reaction field is narrow and the detection sensitivity is low.
- there is a problem that the error between lots is large because there is a difference in the amount of the specific binding reagent immobilized between lots in order to immobilize the specific binding reagent by coating on the surface. .
- the present invention has been made in order to solve the above-mentioned problems, and maintains the advantage of a device having a micro flow channel that a detection result can be obtained with high sensitivity in a short time, while maintaining a single optical system even if there are multiple items It is an object to provide a microfluidic device that can be detected at once. Specifically, a method for individually fixing a specific binding reagent to a plurality of structures in a flow path is provided.
- the present invention relates to the following microfluidic device, analysis kit, analysis system for microfluidic device, and method for manufacturing the microfluidic device.
- the substrate is provided with at least one flow path, In each channel, A microfluidic device in which at least one kind of specific binding reagent mixed in a photocured hydrophilic resin or one or more microstructures in which one kind of specimen is held by crosslinking is arranged.
- a microfluidic device of (1) above A microfluidic device in which different specific binding reagents and / or analytes are held by crosslinking in the plurality of microstructures.
- microfluidic device (3) The microfluidic device according to (1) or (2) above, The microfluidic device, wherein the specific binding reagent is any one or more of an antibody, an antigen, avidin, streptavidin, and biotin.
- the microfluidic device according to any one of (1) to (3), A microfluidic device in which the specimen includes a cell, a cell mass, a cell membrane, an organelle, and an exosome.
- An analysis kit comprising the microfluidic device according to any one of (1) to (4) and a labeled reagent that specifically binds to a detection target.
- the analysis kit according to (5) An analysis kit in which the labeled reagent can be detected by a single optical system.
- a system used for analysis of the microfluidic device according to any one of (1) to (4), A measurement start means for performing the measurement; Detection means for detecting a single fluorescence that measures fluorescence intensity while scanning over a microfluidic device; An analysis system for a microfluidic device, comprising: display means for displaying fluorescence intensity as a numerical value.
- a method of manufacturing a microfluidic device A substrate preparation step of preparing a substrate including at least one flow path; A filling step of filling the flow path with a solution obtained by mixing one kind of specific binding reagent or one kind of specimen and a hydrophilic photocurable resin; An exposure step in which a part of the hydrophilic photocurable resin filled in the flow path is exposed using a photomask to photocur the resin; A cleaning step of cleaning and removing uncured resin from the flow path; A method of manufacturing a microfluidic device.
- a method of manufacturing the microfluidic device according to (8) After the washing step, A refilling step of filling the flow path with a solution obtained by mixing a specific binding reagent or sample different from the specific binding reagent or sample with a hydrophilic photocurable resin; A re-exposure step for exposing the hydrophilic photo-curable resin filled in the flow path using a photomask capable of exposing a portion where an uncured photo-curable resin exists; A re-washing step for washing and removing uncured resin from the flow path; A microfluidic device manufacturing method in which a plurality of different specific binding reagents and / or specimens are fixed in a flow path by repeating a refilling process and a rewashing process.
- FIG. 2A and 2B schematically show a method of manufacturing a microfluidic device using the photocurable resin of the present invention.
- FIG. 2C shows a photograph of the substrate and the position of the microchannel.
- FIG. 4A shows the measurement result of fluorescence intensity.
- FIG. 4B shows fluorescence micrographs obtained by performing the reaction by changing the antigen concentration using a device in which CRP is immobilized.
- FIG. 5A shows the results obtained by changing the CEA concentration
- FIG. 5B shows the results obtained by changing the CRP concentration.
- FIG. 6A shows a control
- FIG. 6B shows an anti-EGFR antibody immobilized on streptavidin
- FIG. 6C shows an example in which an anti-EGFR antibody is directly immobilized on the test.
- FIG. 8A shows typically the manufacturing method of the microfluidic device by a conventional method
- FIG. 8A shows a single item analysis device
- FIG. 8B shows a three item analysis device.
- FIGS. 9A to 9C are schematic views showing a solid phase immobilization process of plural kinds of fine beads.
- FIG. 9D shows the detection result.
- the microfluidic device of the present invention specifically binds to a substance to be detected, such as an antibody, an antigen, an aptamer, DNA, RNA, or a cell lysate. Any of them may be immobilized as a specific binding reagent.
- the antibody may be an antibody molecule itself, or only a region that specifically binds to an antigen, such as Fab or Fab 2 .
- the antigen may be used as the whole antigen molecule, or may include only the epitope region.
- a reagent that specifically binds to the antibody such as protein A or protein G, may be immobilized as a specific binding reagent.
- streptavidin can be immobilized on the device of the present invention, and a molecule such as an antibody that specifically binds to the detection target can be biotinylated and immobilized on the device.
- any sample that may contain a test substance may be used.
- body fluids such as blood, serum, plasma, urine and saliva, and extracts obtained by extracting cells, tissues, and scraped specimens with a solvent such as physiological saline or buffer can be used.
- a prefilter may be provided at the entrance of the flow path so as to allow filtration.
- it is a fine cell piece and a cell membrane it is also possible to confirm a coupling
- any sample can be used as the solid phase, but it is preferable to select a sample that can be concentrated in terms of sensitivity.
- a cell, a cell mass, a cell membrane, an organelle, an exosome, etc. can be mentioned.
- By directly immobilizing a sample containing these it is very useful when, for example, it is desired to check whether the object to be detected is actually contained in the sample.
- a cell is mixed with a resin, cured, confined in the resin, and a fluorescently labeled antibody is added, it is possible to examine what kind of membrane protein exists in the cell. Therefore, it can be a very effective tool when using the device for research purposes.
- obtain information useful for device fabrication such as creating a device with multiple antibodies recognizing the same antigen immobilized and selecting antibodies with good detection sensitivity, or obtaining antibody combinations for sandwich assays. be able to.
- the microfluidic device system of the present invention since detection is highly sensitive, it is preferable to use a fluorescence detector.
- the microfluidic device system of the present invention only needs to detect one type of fluorescence, it does not require an apparatus for switching fluorescence and can be miniaturized.
- a mounting table on which a microfluidic device is placed By providing a mounting table on which a microfluidic device is placed and measuring the fluorescence intensity while scanning the device and expressing it as a numerical value, a complicated optical system unlike a microscope is not required. Cost reduction, size reduction, and weight reduction can be achieved.
- the prototype device actually weighs about 1 kg and is designed to be able to be driven by dry batteries, so it can be carried anywhere. Further, by using a fluorescence detector as the detection means, it is not necessary to have skill as in the case of microscope operation.
- the fluorescence detector is preferably provided with a memory mechanism, and it is preferable to input calibration curve data in which the concentration of the specimen with respect to the fluorescence intensity is measured in advance. Immediately after the fluorescence measurement, the concentration of the detection object is calculated and displayed by the display means, so that the user can know the concentration of the detection object in the sample.
- any fluorescent label may be used, but it is desirable that the wavelength does not overlap with the autofluorescence of the substrate or resin.
- organic compound type fluorescent labels those having an excitation wavelength of around 600 nm, such as Dylight650 (trademark), do not overlap with the autofluorescence of the substrate, so that the background can be kept low.
- inorganic compound type fluorescent labels can also be used.
- quantum dots have a very long fluorescence lifetime and are convenient for observation.
- fluorescent labels of biomolecule type such as protein can also be used.
- any hydrophilic photocurable resin may be used.
- one having an azide-based photosensitive group or one having at least two ethylenically unsaturated bonds in one molecule can be used.
- Water-soluble photocurable resins having at least two ethylenically unsaturated bonds per molecule generally have a number average molecular weight in the range of 300 to 30000, preferably 500 to 20000, and are homogeneous in aqueous media
- a sufficient ionic or nonionic hydrophilic group dispersed in the substrate such as a hydroxyl group, an amino group, a carboxy group, a phosphoric acid group, a sulfonic acid group, an ether bond, etc., and a wavelength in the range of about 250 to about 600 nm
- Those which are cured to become a water-insoluble resin when irradiated with the above light are preferably used (see Patent Documents 2 to 5).
- Examples of the compound having an ethylenically unsaturated bond that can be photopolymerized at both ends of the polyalkylene glycol include, but are not limited to, the following compounds.
- a photopolymerization initiator is included in the hydrophilic photocurable resin as necessary.
- This photopolymerization initiator serves as a polymerization initiating species and causes a cross-linking reaction between resins having a polymerizable unsaturated group.
- ⁇ -carbonyls such as benzoin and acyloin ethers such as benzoin ethyl ether
- polycyclic aromatic compounds such as naphthol, ⁇ -substituted acyloins such as methylbenzoin, and azoamide compounds such as 2-cyano-2-butylazoformamide.
- the use ratio of the hydrophilic photocurable resin and the photopolymerization initiator is not strictly limited, and can be varied over a wide range depending on the type of each component. In general, it is appropriate to use the photopolymerization initiator in a proportion of 0.1 to 5 parts by mass, preferably 0.3 to 3 parts by mass with respect to 100 parts by mass of the hydrophilic photocurable resin.
- AWP Advanced-unit Pendant Water-soluble Photopolymer
- any resin that can crosslink an amino group can be suitably used.
- AWP Adivalent-unit Pendant Water-soluble Photopolymer
- a higher resin concentration can produce a structure that is less likely to be washed away during cleaning, but the sensitivity of the device is reduced.
- the optimal AWP concentration may be selected depending on the affinity of the specific binding reagent for the target.
- an antibody When an antibody is used as a specific binding reagent, it is mixed with a resin at an antibody concentration of 1 ⁇ g / ml to 10 mg / ml. The higher the antibody concentration, the more sensitive the device can be made.
- the antibody concentration may be appropriately selected depending on antibody affinity and detection sensitivity.
- the antibody and AWP may be mixed at a volume ratio of 2: 1, but the mixing ratio can be appropriately selected according to the antibody used and the sensitivity of the antibody to the detection target.
- any ultraviolet irradiation apparatus having an irradiation intensity of about 20 mW / cm 2 in the vicinity of a wavelength of 310 nm may be used, and the curing is performed in 1 second to 3 minutes. Curing time depends on the concentration of AWP, specific binding reagent and analyte. The higher the AWP concentration, the shorter the curing time. Moreover, hardening of resin can be confirmed using a phase contrast microscope or a differential interference microscope.
- the shape of the structure of the photocurable resin mixed with the specific binding reagent and the specimen may be any shape depending on the shape of the photomask, such as a cylindrical pillar shape or a rectangular parallelepiped wall shape. In general, a shape having a large specific surface area, which is a ratio of the surface area of the reaction field to the volume of the specimen, is preferable because the detection sensitivity becomes higher.
- any substrate may be used, but since a photocurable resin is used, a substrate having a high light transmittance is preferable.
- a highly transparent material or a material that does not emit autofluorescence near the detection wavelength is suitable.
- a cyclic olefin polymer substrate or a cyclic olefin copolymer substrate has high processing accuracy by injection molding and is suitable for producing a micro flow path by fine processing. Further, it is sufficient that at least one microchannel is provided on the substrate.
- a washing solution used after fixing a specific binding reagent a washing solution or a blocking solution generally used for immunoassay can be used.
- a buffer solution such as a phosphate buffer solution, a Tris buffer solution, a carbonate buffer solution, PBS (phosphate buffered saline), or TBS (Tris buffered saline) can be used as the buffer solution.
- a protein such as an antibody is immobilized on the device
- a protein such as BSA (bovine serum albumin), skim milk, bovine serum, or albumin can be used as a blocking agent in order to prevent non-specific adsorption.
- nucleic acids such as DNA
- what is usually used for blocking nucleic acids such as salmon sperm DNA may be used as a blocking agent.
- the surfactant Triton X-100, Tween 20, Briji 35, Nonidet P-40, SDS or the like can be used.
- the microfluidic device is manufactured as shown in FIG.
- a substrate on which a flow path is formed is prepared.
- a photocurable resin and a specific binding reagent or specimen are mixed to fill the channel of the device.
- the device is covered with a photomask designed to transmit light only at a desired location, and the resin mixed with the specific binding reagent is photocured by irradiating ultraviolet rays (FIG. 2A).
- the uncured resin is sucked out and washed with a cleaning solution.
- FIG. 2B When a plurality of items of specific binding reagents and the like are immobilized on the device, this procedure is repeated, and the specific binding reagents and the like are fixed together with the photocurable resin in the flow path (FIG. 2B).
- a protein such as an antibody is solid-phased, it is stable for about one year if it is stored at 4 ° C. under moisture so
- FIG. 3 shows a procedure in the case where detection is performed with the antibody immobilized on the device. Since the channel of the device is filled with a buffer solution such as a cleaning solution or a blocking solution at the time of production, the buffer solution is removed. Next, the specimen is placed in the flow path and incubated. When the channel size is about 1000 ⁇ m ⁇ 6500 ⁇ m ⁇ 50 ⁇ m, 0.5 to 1.5 ⁇ l of sample is injected into one channel. Depending on the type of specimen and the concentration of the detection target, it is usually sufficient to incubate at room temperature for about 10 minutes.
- a buffer solution such as a cleaning solution or a blocking solution
- the detection target and the specific binding reagent can be bound in a shorter time.
- the reaction time may be appropriately set in the range of about 1 minute to 24 hours depending on the affinity of the specific binding reagent immobilized on the device to the detection target and the concentration of the detection target.
- the sample is sucked out, the cleaning solution is put in, and left to stand for about 1 minute. Replace the cleaning solution and repeat the same operation for cleaning. Usually, it is completely cleaned by repeating the cleaning operation about 5 times.
- the cleaning may be performed by replacing the cleaning liquid immediately without performing standing, and replacing the cleaning liquid about 7 to 8 times. What is necessary is just to adjust the frequency
- cleaning time suitably with the detection target and the specific binding reagent currently solid-phased.
- the secondary antibody that binds to the detection target is put into the flow path. Although depending on the concentration of the secondary antibody and the concentration of the detection target, the reaction is completed in about 30 seconds. Aspirate secondary antibody and add wash solution. Repeat the same washing procedure as above. Next, a tertiary antibody labeled with a fluorescent label or the like is placed in the channel and incubated. Usually, it is sufficient to incubate for about 30 seconds like the secondary antibody. Thereafter, washing is performed in the same manner to detect the label. When a tertiary antibody labeled with a fluorescent label is used, it may be observed with a fluorescence detector or a fluorescence microscope. The time required for the assay is 30 minutes or less.
- the “labeled reagent that specifically binds to the detection target” refers to a labeled secondary antibody that binds to the detection target, or a secondary antibody and a secondary antibody that specifically binds to the detection target.
- the combination may include a labeled reagent that can detect the detection target, such as a set of labeled tertiary antibodies that bind.
- an immunoassay in which an antibody is immobilized in a flow path has been described.
- detection may be performed with a labeled antibody that specifically binds to the detection target, or specific to the detection target. Detection may be performed by combining a binding antibody and a labeled antibody that recognizes the antibody. Further, when the nucleic acid is immobilized, it may be detected by hybridization according to a conventional method.
- the microfluidic device produced by the method of the present invention can immobilize a plurality of different specific binding reagents without intermingling with each microstructure, but can be used to immobilize a single specific binding reagent. Needless to say, you can.
- a microchip substrate 70 mm ⁇ 30 mm ⁇ 1.25 mm
- a cyclic olefin polymer (BS-X2194, manufactured by Sumitomo Bakelite) was used (see FIG. 2C, photograph).
- the substrate is provided with 40 cuboid (1000 ⁇ m ⁇ 6500 ⁇ m ⁇ 50 ⁇ m) microchannels.
- the diameter of the inlet and outlet of the microchannel is 1.0 mm.
- PSA prostate cancer marker
- CRP inflammation marker CRP
- CEA tumor marker CEA
- Anti-PSA antibody Abcam, ab10189, 2 mg / ml
- Antigen Human PSA (Acris Antibodies, P117-7)
- Secondary antibody Anti-PSA antibody (manufactured by Cell Signaling Technology, 5365)
- Tertiary antibody DyLight650 TM -labeled goat anti-rabbit IgG (Abcam, ab96902)
- All the primary antibodies were mixed at a concentration of 2 mg / ml with the photocurable resin AWP so that the antibody was 1 with respect to the resin 1 in a volume ratio, filled the flow path, and irradiated with ultraviolet rays for 5 seconds by an ultraviolet irradiation device. And photocured.
- Each antigen was diluted to a desired concentration with PBS containing 1% BSA, filled in the flow path, and incubated at room temperature for 10 minutes. Thereafter, the washing was performed 7 to 8 times using 10 ⁇ l of washing solution. Specifically, washing was performed as follows. The antigen is sucked out with an aspirator. Take 10 ⁇ l of wash solution into a micropipette and fill with approximately 1.3 ⁇ l of wash solution to fill the channel. The washing liquid was sucked out with an aspirator and filled with the washing liquid repeatedly, and washing was performed using 10 ⁇ l of the washing liquid per one channel. The washing solution used was PBS supplemented with 0.5% BSA (bovine serum albumin) and 0.5% Tween20.
- BSA bovine serum albumin
- the secondary antibody and the tertiary antibody were diluted with PBS containing 1% BSA so as to be 50 ⁇ g / ml, filled in the flow path for 30 seconds, respectively, and then washed with 10 ⁇ l of washing solution as described above.
- Fluorescence images were taken with a fluorescence microscope (Ni-E, Nikon Corporation). As shown in FIG. 4A, it is clear that both antigens can be detected at very low concentrations.
- the detection limits of PSA, CRP, and CEA calculated from the results were 2.29 ng / ml, 1.61 ng / ml, and 0.49 ng / ml, respectively. Since the cutoff value of PSA for prostate disease is 4 ng / ml, the cutoff value of CRP as an arteriosclerosis marker is 10 ng / ml, and the cutoff value of CEA as a cancer marker is 5 ng / ml, It is clear that it can be applied to the diagnosis of diseases. Each disease marker could be measured to a detection limit sufficient for practical use. In addition, the time required from specimen injection to detection was a very short time of 15 minutes.
- FIG. 4B shows a microchannel observed in a microchannel using a CRP device similar to that described above, reacting by changing the antigen concentration from 0 to 1610 ng / ml, then reacting with a secondary antibody and a tertiary antibody.
- the photograph of the structure to which the photocurable resin and the antibody are fixed is shown.
- a high pressure mercury lamp is used as a light source
- a Cy5 filter is used as a filter
- a digital CCD camera ORCA-R2 manufactured by Hamamatsu Photonics is used for photographing. Almost no background is observed, and it is observed that the fluorescence intensity increases depending on the analyte concentration. It can be detected by fluorescence microscope observation that CRP of 1.61 ng / ml or more can be detected.
- the device produced by the production method of the present invention is directly mixed and fixed with a specific binding reagent such as an antibody in a photocurable resin. Less compared to the case of using beads.
- anti-CEA antibody was mixed with AWP resin to fill the flow path provided on the substrate.
- the desired region (hereinafter referred to as the first region) is covered with a photomask designed to irradiate light, and the resin and antibody mixture is photocured by irradiating with ultraviolet rays, and a wall is formed in the channel. A rectangular parallelepiped structure was produced. Uncured resin was sucked out and washed with a cleaning solution.
- the anti-CRP antibody was mixed with AWP resin to fill the flow path.
- Resin and anti-CRP antibody are covered with a photomask designed to irradiate light to a region different from the first region where the anti-CEA antibody is fixed (hereinafter referred to as second region), and then irradiated with ultraviolet rays.
- the mixture was photocured.
- a device for assaying a composite item in which an anti-CEA antibody was immobilized in the first region and an anti-CRP antibody was immobilized in the second region was prepared.
- Example 2 The same antigens and antibodies as those used in Example 1 were used. Each antigen was filled into the flow path at various concentrations and reacted, and anti-CRP antibody and anti-CEA antibody were mixed and reacted so that the respective concentrations were 50 ⁇ g / ml. Since the secondary antibody is a rabbit antibody, the tertiary antibody was detected with DyLight650 (trademark) -labeled goat anti-rabbit IgG. For detection, a fluorescence microscope (Ni-E, manufactured by Nikon Corporation) was used. The results are shown in FIG.
- FIG. 5 shows the results of measurement using the multi-item assay device prepared above with different concentrations of CEA antigen and CRP antigen.
- the measurement results in the region where the anti-CEA antibody of the first region is immobilized are indicated by ⁇ , and the measurement results of the region where the anti-CRP antibody of the second region is immobilized are indicated by ⁇ .
- FIG. 5A shows the fluorescence intensity measured by changing the concentration of CEA antigen
- FIG. 5B shows the fluorescence intensity measured by changing the concentration of CRP antigen.
- the CEA antigen in the CEA antigen, an increase in fluorescence intensity is observed depending on the concentration in the first region ( ⁇ ) where the anti-CEA antibody is immobilized.
- the fluorescence intensity equivalent to the background level was observed in the second region ( ⁇ ) where the anti-CRP antibody was immobilized.
- the fluorescence intensity at the background level is measured in the first region ( ⁇ ) where the anti-CRP antibody is not immobilized. It was only done. That is, only the anti-CEA antibody is immobilized on the first region, and only the anti-CRP antibody is immobilized on the second region. Further, as shown in FIG. 5, all antigens can be detected up to the detection limit of very low concentration similar to the single item assay device shown in FIG.
- Example 2 since the same sample can be assayed at the same time, it is suitable for the measurement of items that are desirable to be assayed simultaneously such as anti-CRP antibody and anti-CEA antibody.
- a composite assay of two types of antibodies is shown, but any number of types can be incorporated into a microfluidic device by immobilizing antibodies to be detected simultaneously.
- Streptavidin was used as a specific binding reagent immobilized on the microchannel. Streptavidin was dissolved in PBS (pH 7.4) at a concentration of 10 mg / ml and mixed at a volume ratio of 1 to AWP1. After mixing, it was cured by ultraviolet rays in the same manner as in Example 1.
- the primary antibody biotin-modified anti-EGFR antibody, manufactured by Abcam, ab113645
- a streptavidin-biotin binding reaction was performed at room temperature for 1 hour.
- the anti-EGFR antibody was sucked out with an aspirator, washed with 10 ⁇ l of a washing solution, and used for the assay.
- a specimen containing an antigen a pleural effusion sediment from a lung cancer patient dissolved in a lysis buffer (Cell Signaling Technology, 9803) was used.
- the secondary antibody is an anti-L858R gene variant EGFR antibody (Cell Signaling Technology, 3197), the tertiary antibody is DyLight650 (trademark) labeled rabbit anti-goat IgG (Abcam, ab102343), each in PBS containing 1% BSA. Diluted to 50 ⁇ g / ml before use.
- FIG. 6A and 6B are fluorescence micrographs of the region where the anti-EGFR antibody is immobilized on streptavidin and FIG. 6C is the region where the anti-EGFR antibody is directly immobilized.
- FIG. 6A shows a subsequent assay without using a sample as a control
- FIGS. 6B and 6C show the same assay after incubation with the sample.
- the EML4-ALK fusion protein that causes lung cancer was detected.
- the EML4-ALK fusion protein is an abnormal protein in which the EML4 gene and the ALK gene are fused, and about half of the amino terminal side of the EML4 protein is fused with the intracellular region of the ALK receptor tyrosine kinase.
- Cell line H3122 expressing EML4-ALK fusion protein and cell line H358 not expressing EML4-ALK fusion protein were used.
- the cell lysate was prepared by dividing the collected cultured cells into cells and supernatant by centrifugation, removing the supernatant, and adding lysis buffer (Cell Signaling Technology, 9803) to the cells.
- AWP and a cell lysate prepared from two cell lines were mixed at a volume ratio of 1: 1, filled in microchannels, exposed to ultraviolet rays, and photocured. Washing was performed with a washing solution, and a device including a microstructure in which each cell lysate was solid-phased was produced.
- Detection was performed as follows. A mouse antibody that specifically binds to the EML4-ALK fusion protein (manufactured by Santa Cruz, SC-57024) was introduced into the channel and incubated for 30 seconds. After washing with the washing solution, DyLight650-labeled anti-mouse IgG antibody (Abcam, ab98797) was introduced into the flow path and incubated for 30 seconds. The sample was washed with a washing solution and observed with a fluorescence microscope and a bright field. The results are shown in FIG.
- the detection target is included by directly immobilizing the specimen. Further, since the specific binding of the antibody to the specimen can be confirmed, it is useful when selecting a highly specific antibody or a highly binding antibody used in the assay.
- the detector can be made small even when a plurality of items are inspected. Therefore, it is very useful for POCT (Point-of-care testing) in which a test is performed at the bedside of a patient or when a test result is required in a short time. Further, by directly immobilizing the specimen, it can be confirmed whether or not the detection target is included, and it can be confirmed in a very short time that the antibody specifically binds to the detection target.
- POCT Point-of-care testing
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Abstract
Description
(1)基板には少なくとも1つ以上の流路が設けられ、
個々の流路内には、
光硬化した親水性樹脂中に混合された一種類の特異的結合試薬又は一種類の検体が架橋により保持された微小構造物が少なくとも一つ以上配置されているマイクロ流体デバイス。
(2)前記(1)のマイクロ流体デバイスであって、
前記複数の微小構造物には夫々異なる特異的結合試薬及び/又は検体が架橋により保持されているマイクロ流体デバイス。
(3)前記(1)又は(2)記載のマイクロ流体デバイスであって、
前記特異的結合試薬が、抗体、抗原、アビジン、ストレプトアビジン、ビオチンのいずれか1つ以上であるマイクロ流体デバイス。
(4)前記(1)~(3)のいずれか1記載のマイクロ流体デバイスであって、
前記検体が、細胞、細胞塊、細胞膜、オルガネラ、エクソソームを含むものであるマイクロ流体デバイス。
(5)前記(1)~(4)のいずれか1記載のマイクロ流体デバイスと
検出対象に特異的に結合する標識された試薬を含む分析キット。
(6)前記(5)記載の分析キットであって、
前記標識された試薬が単一の光学系によって検出可能である分析キット。
(7)前記(1)~(4)のいずれか1記載のマイクロ流体デバイスの分析に用いるシステムであって、
測定を実行するための測定開始手段と、
マイクロ流体デバイス上をスキャンしながら蛍光強度を測定する単一の蛍光を検出するための検出手段と、
蛍光強度を数値として表示する表示手段とを備えるマイクロ流体デバイス用分析システム。
(8)マイクロ流体デバイスを製造する方法であって、
流路を少なくとも1つ含む基板を準備する基板準備工程、
一種類の特異的結合試薬又は一種類の検体と親水性光硬化性樹脂を混合した溶液を前記流路内に充填する充填工程、
前記流路内に充填された前記親水性光硬化性樹脂の一部に対してフォトマスクを用いて露光を行い樹脂を光硬化させる露光工程、
未硬化の樹脂を前記流路から洗浄除去する洗浄工程、
を含むマイクロ流体デバイス製造方法。
(9)前記(8)記載のマイクロ流体デバイスを製造する方法であって、
前記洗浄工程の後に、
前記特異的結合試薬又は検体とは異なる特異的結合試薬又は検体を親水性光硬化性樹脂と混合した溶液を前記流路内に充填する再充填工程、
前記流路内に充填された前記親水性光硬化性樹脂に対して、未硬化の光硬化性樹脂の存在する部位を露光可能なフォトマスクを用いて露光を行う再露光工程、
未硬化の樹脂を前記流路から洗浄除去する再洗浄工程、
再充填工程から再洗浄工程を繰り返し、複数の異なる特異的結合試薬及び/又は検体を流路内に固定するマイクロ流体デバイス製造方法。
(1)分子量400~6000のポリエチレングリコール1モルの両末端水酸基を(メタ)アクリル酸2モルでエステル化したポリエチレングリコールジ(メタ)アクリレート類
(2)分子量200~4000のポリプロピレングリコール1モルの両末端水酸基を(メタ)アクリル酸2モルでエステル化したポリプロピレングリコールジ(メタ)アクリレート類
(3)分子量400~6000のポリエチレングリコール1モルの両末端水酸基をトリレンジイソシアネート、キシリレンジイソシアネート、イソホロンジイソシアネートなどのジイソシアネート化合物2モルでウレタン化し、次いで(メタ)アクリル酸2-ヒドロキシエチルなどの不飽和モノヒドロキシエチル化合物2モルを付加した不飽和ポリエチレングリコールウレタン化物
(4)分子量200~4000のポリプロピレングリコール1モルの両末端水酸基をトリレンジイソシアネート、キシリレンジイソシアネート、イソホロンジイソシアネートなどのジイソシアネート化合物2モルでウレタン化し、次いで(メタ)アクリル酸2-ヒドロキシエチルなどの不飽和モノヒドロキシエチル化合物2モルを付加した不飽和ポリプロピレングリコールウレタン化物。
環状オレフィンポリマー(BS-X2194、住友ベークライト社製)のマイクロチップ基板(70mm×30mm×1.25mm)を用いた(図2C、写真参照。)。基板には直方体(1000μm×6500μm×50μm)のマイクロ流路が40か所設けられている。マイクロ流路の入り口、及び出口の直径は1.0mmである。
一次抗体: 抗PSA抗体(アブカム社製、ab10189、2mg/ml)
抗原: ヒトPSA(Acris Antibodies社製、P117-7)
二次抗体: 抗PSA抗体(Cell Signaling Technology社製、5365)
三次抗体: DyLight650(商標)標識ヤギ抗ウサギIgG(アブカム社製、ab96902)
一次抗体:抗CRP抗体(アブカム社製、ab136176,2mg/ml)
抗原:CRP(Acris Antibodies社製、P100-0)
二次抗体:抗CRP抗体(アブカム社製、ab31156)
三次抗体:DyLight650(商標)標識ヤギ抗ウサギIgG(アブカム社製、ab96902)
一次抗体:抗CEA抗体(アブカム社製、ab4451、2mg/ml)
抗原:ヒトCEA(R&D社製、4128-CM―050)
二次抗体:抗CEACAM5抗体(ウサギ)(アブカム社製、ab131070)
三次抗体:DyLight650(商標)標識ヤギ抗ウサギIgG(アブカム社製、ab96902)
次に、複数項目を同時に検出するデバイスを作製し検討を行った。抗CEA抗体、抗CRP抗体を1つの流路内に配置したマイクロ流体デバイスを作製した。固相化に用いた抗体、検出に用いた抗体、抗原は実施例1と同じものを用いている。
Claims (9)
- 基板には少なくとも1つ以上の流路が設けられ、
個々の流路内には、
光硬化した親水性樹脂中に混合された一種類の特異的結合試薬又は一種類の検体が架橋により保持された微小構造物が少なくとも一つ以上配置されているマイクロ流体デバイス。 - 請求項1記載のマイクロ流体デバイスであって、
前記微小構造物には夫々異なる特異的結合試薬及び/又は検体が架橋により保持されているマイクロ流体デバイス。 - 請求項1又は2記載のマイクロ流体デバイスであって、
前記特異的結合試薬が、抗体、抗原、アビジン、ストレプトアビジン、ビオチンのいずれか1つ以上であるマイクロ流体デバイス。 - 請求項1~3のいずれか1項記載のマイクロ流体デバイスであって、
前記検体が、細胞、細胞塊、細胞膜、オルガネラ、エクソソームを含むものであるマイクロ流体デバイス。 - 請求項1~4のいずれか1項記載のマイクロ流体デバイスと
検出対象に特異的に結合する標識された試薬を含む分析キット。 - 請求項5記載の分析キットであって、
前記標識された試薬が単一の光学系によって検出可能である分析キット。 - 請求項1~4のいずれか1項記載のマイクロ流体デバイスの分析に用いるシステムであって、
測定を実行するための測定開始手段と、
マイクロ流体デバイス上をスキャンしながら蛍光強度を測定する単一の蛍光を検出するための検出手段と、
蛍光強度を数値として表示する表示手段とを備えるマイクロ流体デバイス用分析システム。 - マイクロ流体デバイスを製造する方法であって、
流路を少なくとも1つ含む基板を準備する基板準備工程、
一種類の特異的結合試薬又は一種類の検体と親水性光硬化性樹脂を混合した溶液を前記流路内に充填する充填工程、
前記流路内に充填された前記親水性光硬化性樹脂の一部に対してフォトマスクを用いて露光を行い樹脂を光硬化させる露光工程、
未硬化の樹脂を前記流路から洗浄除去する洗浄工程、
を含むマイクロ流体デバイス製造方法。 - 請求項8記載のマイクロ流体デバイスを製造する方法であって、
前記洗浄工程の後に、
前記特異的結合試薬又は検体とは異なる特異的結合試薬又は検体を親水性光硬化性樹脂と混合した溶液を前記流路内に充填する再充填工程、
前記流路内に充填された前記親水性光硬化性樹脂に対して、未硬化の光硬化性樹脂の存在する部位を露光可能なフォトマスクを用いて露光を行う再露光工程、
未硬化の樹脂を前記流路から洗浄除去する再洗浄工程、
再充填工程から再洗浄工程を繰り返し、複数の異なる特異的結合試薬及び/又は検体を流路内に固定するマイクロ流体デバイス製造方法。
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JP2019100714A (ja) * | 2017-11-28 | 2019-06-24 | 東洋製罐グループホールディングス株式会社 | 免疫学的測定デバイス |
JP2021018177A (ja) * | 2019-07-22 | 2021-02-15 | 国立大学法人東海国立大学機構 | 分析デバイス |
WO2022176897A1 (ja) * | 2021-02-19 | 2022-08-25 | デンカ株式会社 | デバイス、チップおよび基板 |
JP7503817B2 (ja) | 2019-07-22 | 2024-06-21 | 国立大学法人東海国立大学機構 | 分析デバイス |
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JPWO2016152702A1 (ja) | 2018-01-18 |
US20180080929A1 (en) | 2018-03-22 |
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