WO2020179442A1 - マイクロ流体デバイスおよび試料分析方法 - Google Patents
マイクロ流体デバイスおよび試料分析方法 Download PDFInfo
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- WO2020179442A1 WO2020179442A1 PCT/JP2020/006402 JP2020006402W WO2020179442A1 WO 2020179442 A1 WO2020179442 A1 WO 2020179442A1 JP 2020006402 W JP2020006402 W JP 2020006402W WO 2020179442 A1 WO2020179442 A1 WO 2020179442A1
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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
- G01N33/54366—Apparatus specially adapted for solid-phase testing
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- 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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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- 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
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L7/00—Heating or cooling apparatus; Heat insulating devices
- B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/06—Fluid handling related problems
- B01L2200/0642—Filling fluids into wells by specific techniques
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/06—Fluid handling related problems
- B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/06—Fluid handling related problems
- B01L2200/0673—Handling of plugs of fluid surrounded by immiscible fluid
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/06—Fluid handling related problems
- B01L2200/0689—Sealing
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/12—Specific details about manufacturing devices
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0809—Geometry, shape and general structure rectangular shaped
- B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0848—Specific forms of parts of containers
- B01L2300/0851—Bottom walls
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
- B01L2300/0864—Configuration of multiple channels and/or chambers in a single devices comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
- B01L2300/0877—Flow chambers
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0893—Geometry, shape and general structure having a very large number of wells, microfabricated wells
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/16—Surface properties and coatings
- B01L2300/161—Control and use of surface tension forces, e.g. hydrophobic, hydrophilic
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/16—Surface properties and coatings
- B01L2300/161—Control and use of surface tension forces, e.g. hydrophobic, hydrophilic
- B01L2300/165—Specific details about hydrophobic, oleophobic surfaces
- B01L2300/166—Suprahydrophobic; Ultraphobic; Lotus-effect
Definitions
- the present invention relates to a microfluidic device and a sample analysis method.
- the present application claims priority with respect to Japanese Patent Application No. 2019-037543 filed in Japan on March 1, 2019, the contents of which are incorporated herein by reference.
- microwell arrays having various forms of fine flow path structures formed by using etching technology or photolithography technology used in semiconductor circuit manufacturing technology or a fine plastic molding method have been studied. ing.
- the wells of these microwell arrays are used as chemical reaction vessels for performing various biochemical or chemical reactions in a microvolume of fluid.
- Patent Documents 1 to 3 and Non-Patent Document 1 describe the use of such a microfluidic system as various microchips and biochips.
- digital measurement technology for example, as one of the new approaches in the detection and quantification of nucleic acid, there is a digital PCR (Digital Polymerase Reaction) technology.
- digital PCR technology a mixture of a reagent and nucleic acid is divided into innumerable microdroplets and PCR amplification is performed so that a signal such as fluorescence is detected from the droplet containing nucleic acid, and the signal is generated. This is a technique for quantifying by counting the detected droplets.
- a method for producing microdroplets a method of forming microdroplets by dividing a mixture of a reagent and a nucleic acid with a sealing solution, or a method of putting a mixture of a reagent and a nucleic acid in a pore formed on a substrate, followed by A method of forming fine droplets by adding a sealing liquid is being studied.
- a reagent when analyzing a sample with a microfluidic device, a reagent may adsorb nonspecifically to a lid member.
- fluorescent signals in micropores when fluorescent signals in micropores are detected, fluorescence emitted from non-specific adsorption of reagents, which is not a problem with methods other than digital measurement, is a fluorescent substance.
- An object of the present invention is to provide a microfluidic device capable of reducing nonspecific adsorption of a reagent to a lid member and improving detection efficiency.
- the present invention also provides a sample analysis method capable of correctly detecting a signal by suppressing the fluorescence generated by non-specific adsorption from becoming noise when detecting a signal generated from a microwell. The purpose is to
- the present invention includes the following aspects.
- a microwell array having a plurality of microwells, and a lid member facing the microwell array in a state of being separated from each other, and having a channel between the microwell array and the lid member,
- a sample analysis method comprising the operation and the detection of the signal.
- the present invention can provide a microfluidic device with reduced non-specific adsorption of reagents.
- the present invention also provides a sample analysis method capable of correctly detecting a signal by suppressing the fluorescence generated by non-specific adsorption from becoming noise when detecting a signal generated from a microwell. be able to.
- FIG. 2 is a sectional view taken along line bb of FIG. 1. It is a sectional view showing a microfluidic device concerning one embodiment of the present invention. It is a figure which shows the state at the time of use of the microfluidic device which concerns on one Embodiment of this invention. It is a figure which shows the state at the time of use of the microfluidic device which concerns on one Embodiment of this invention. It is a figure which shows the fluorescence image using the microfluidic device which concerns on one Embodiment of this invention. It is a figure which shows the fluorescence image using the microfluidic device which concerns on Comparative Example 1.
- FIGS. 1 to 5 An embodiment of the present invention will be described with reference to FIGS. 1 to 5.
- the dimensional ratio in each drawing is exaggerated for explanation and does not necessarily match the actual dimensional ratio.
- FIG. 1 is a perspective view showing a microfluidic device 1 according to this embodiment.
- FIG. 2 is a cross-sectional view taken along the line bb of FIG.
- the microfluidic device 1 includes a microwell array 30 having a plurality of wells and a lid member 20 facing the microwell array 30 in a state of being separated from the microwell array 30.
- the flow path 35 is provided between the cover member 20 and the cover member 20.
- the microwell array 30 may have only the substrate 10 or may have a bottom layer 31 and a wall layer 32 in addition to the substrate 10.
- a peripheral member 34 is located between the microwell array 30 and the lid member 20. The region sandwiched between the microwell array 30 and the lid member 20 and surrounded by the peripheral member 34 is the flow path 35.
- the peripheral edge member 34 may be formed integrally with the lid member 20.
- the substrate 10 may be capable of transmitting electromagnetic waves.
- examples of the electromagnetic waves include X-rays, ultraviolet rays, visible rays, infrared rays, and the like. Since the substrate 10 is capable of transmitting electromagnetic waves, fluorescence, phosphorescence, or the like generated by the reaction between the sample enclosed in the microfluidic device 1 and the reagent can be observed from the substrate 10 side.
- the substrate 10 may be capable of transmitting only electromagnetic waves in a predetermined wavelength range. For example, when the presence of a sample in a microwell is determined by detecting fluorescence having a peak in the wavelength range of 350 to 700 nm, which is a visible light region, a substrate capable of transmitting visible light in at least the above wavelength range is used as the substrate 10. Can be used as
- Examples of the material forming the substrate 10 include glass and resin.
- Examples of the material of the resin substrate include ABS resin, polycarbonate resin, COC (cycloolefin copolymer), COP (cycloolefin polymer), acrylic resin, polyvinyl chloride, polystyrene resin, polyethylene resin, polypropylene resin, polyvinyl acetate, PET. (Polyethylene terephthalate), PEN (polyethylene naphthalate) and the like can be mentioned.
- These resins may contain various additives. Examples of the additive include an antioxidant, an additive that imparts water repellency, an additive that imparts hydrophilicity, and the like.
- the resin substrate may contain only one of the above resins, or may be a mixture of a plurality of resins.
- substantially having no autofluorescence means that the substrate does not have any autofluorescence of the wavelength used for detecting the experimental result, or even if it has autofluorescence, it affects the detection of the experimental result. It means that it is too weak to give.
- autofluorescence that is 1 ⁇ 2 or less, preferably 1/10 or less of the fluorescence to be detected is weak enough not to affect the detection of the experimental result.
- the thickness of the substrate 10 can be appropriately determined, but is preferably 5 mm (mm) or less, more preferably 2 mm or less, and 1.6 mm or less, for example, in order to facilitate the transmission of fluorescence and phosphorescence emitted in sample analysis. More preferable. Further, in order to facilitate the processing, for example, 0.1 mm or more is preferable, and 0.2 mm or more is more preferable.
- the upper limit and the lower limit of the thickness of the substrate 10 can be arbitrarily combined.
- the thickness of the substrate 10 is preferably 0.1 mm or more and 5 mm or less, more preferably 0.2 mm or more and 2 mm or less, and further preferably 0.4 mm or more and 1.6 mm or less.
- the lid member 20 (may be simply referred to as the lid 20) is a plate-shaped or sheet-shaped member.
- the lid member 20 faces the microwell array 30 in a separated state. In other words, the lid member 20 covers the plurality of microwells 33.
- the flow path 35 is a region surrounded by the lid member 20, the microwell array 30, and the peripheral member 34. The flow path 35 is connected to the openings of the plurality of microwells 33 and is located above the plurality of microwells 33.
- the lid member 20 has a first hole 21 and a second hole 22 penetrating in the thickness direction.
- the first hole 21 and the second hole 22 are positioned so as to sandwich one or more of the liquid holding portions.
- the first hole 21 and the second hole 22 communicate with the internal space S including the microwell array 30 and the flow path 35 in the completed microfluidic device 1.
- the first hole 21 and the second hole 22 respectively function as an inlet for supplying a fluid to the internal space S and an outlet for discharging the fluid.
- the material forming the lid member 20 and the thickness of the lid member 20 can be the same as those of the substrate 10.
- the electromagnetic wave transparency can be appropriately set. For example, when the electromagnetic wave irradiation step described below is not performed from the lid member 20 side, the lid member 20 may not be able to transmit electromagnetic waves.
- the arithmetic mean roughness (Ra) of the surface 20a on the microwell array 30 side of the lid member 20 is 70 nm or less, preferably 60 nm or less, more preferably 50 nm or less, and 40 nm or less. Is more preferable, and 35 nm or less is particularly preferable. Further, the arithmetic mean roughness (Ra) of the surface 20a on the microwell array 30 side of the lid member 20 may be 30 nm or less, 25 nm or less, 20 nm or less, or 15 nm. It may be the following.
- the lower limit of the arithmetic mean roughness (Ra) of the surface 20a of the lid member 20 on the microwell array 30 side is not particularly limited, but is, for example, 5 nm.
- the upper and lower limits of the arithmetic average roughness (Ra) of the surface 20a of the lid member 20 on the microwell array 30 side can be arbitrarily combined.
- the arithmetic mean roughness (Ra) of the surface 20a of the lid member 20 on the microwell array 30 side is 5 nm or more and 70 nm or less, 5 nm or more and 60 nm or less, 6 nm or more and 50 nm or less, 7 nm or more and 40 nm or less, 7 nm or more and 35 nm or less. It may be 8 nm or more and 30 nm or less, 8 nm or more and 25 nm or less, 9 nm or more and 20 nm or less, or 10 nm or more and 15 nm or less.
- the arithmetic average roughness (Ra) can be measured, for example, according to the measurement method specified in JIS B 0601-2001.
- the measurement range may be the entire surface of the lid member or a typical area.
- the measurement range in this embodiment is defined as a straight line having the center of the lid member 20 as the center of the measurement range and having a length of 800 ⁇ m to 1.5 mm.
- the method of setting the arithmetic mean roughness (Ra) of the surface 20a of the lid member 20 on the microwell array 30 side to 50 nm or less is not particularly limited, and for example, the lid member 20 may be mirror-polished.
- the mold of the lid member 20 may be mirror-finished with diamond paste or the like.
- the contact angle of the surface 20a of the lid member 20 on the microwell array 30 side with water may be 70 degrees or more. Since the contact angle of the surface 20a of the lid member 20 on the microwell array 30 side with water is 180 degrees or less, the contact angle of the surface of the lid member 20 on the microwell array 30 side with water is, for example, It is 70 degrees or more and 180 degrees or less.
- the contact angle with water can be measured, for example, according to the sessile drop method defined in JIS R 3257-1999. Instead of the intravenous drip method specified in JIS R 3257-1999, the contact angle may be measured by a method according to ASTM D5725-1997.
- the surface roughness (ten-point average roughness) (Rz) of the surface 20a on the microwell array 30 side of the lid member 20 is preferably 350 nm or less, more preferably 300 nm or less, and more preferably 250 nm or less. Is particularly preferable. Further, the surface roughness (ten-point average roughness) (Rz) of the surface 20a on the microwell array 30 side of the lid member 20 may be 200 nm or less, 150 nm or less, or 120 nm or less. It may be 110 nm or less, 100 nm or less, or 90 nm or less.
- the surface roughness (Rz) of the surface 20a on the microwell array 30 side of the lid member 20 is 350 nm or less, the non-specific adsorption of the reagent on the surface 20a on the microwell array 30 side of the lid member 20 is further increased. Can be suppressed.
- the lower limit value of the surface roughness (ten-point average roughness) (Rz) of the surface 20a of the lid member 20 on the microwell array 30 side is not particularly limited, but is, for example, 50 nm.
- the upper limit value and the lower limit value of the surface roughness (ten-point average roughness) (Rz) of the surface 20a on the microwell array 30 side of the lid member 20 can be arbitrarily combined.
- the surface roughness (ten-point average roughness) (Rz) of the surface 20a of the lid member 20 on the microwell array 30 side is 50 nm or more and 350 nm or less, 55 nm or more and 300 nm or less, 60 nm or more and 250 nm or less, 60 nm or more and 200 nm or less. , 60 nm or more and 150 nm or less, 65 nm or more and 120 nm or less, 70 nm or more and 110 nm or less, 70 nm or more and 100 nm or less, or 80 nm or more and 90 nm or less.
- the surface roughness (Rz) can be measured according to the measuring method specified in JIS B 0601-2001.
- the measurement range may be the entire surface of the lid member or a typical area.
- the measurement range in this embodiment is defined as a straight line having the center of the lid member 20 as the center of the measurement range and having a length of 800 ⁇ m to 1.5 mm.
- the method of setting the surface roughness (Rz) of the surface 20a of the lid member 20 on the microwell array 30 side to 350 nm or less is not particularly limited, and examples thereof include performing a mirror polishing process on the lid member 20.
- the mold of the lid member 20 may be mirror-finished with diamond paste.
- the Ra / Rz of the surface 20a of the lid member 20 on the microwell array 30 side is preferably 0.10 or more.
- the Ra / Rz of the surface 20a of the lid member 20 on the microwell array 30 side is preferably 0.24 or less, more preferably 0.23 or less, and even more preferably 0.225 or less. ..
- Ra/Rz of the surface 20a of the lid member 20 on the microwell array 30 side may be 0.16 or less, 0.15 or less, or 0.14 or less. , 0.13 or less.
- Ra / Rz of the surface 20 a of the lid member 20 on the microwell array 30 side can be arbitrarily combined.
- Ra / Rz of the surface 20a of the lid member 20 on the microwell array 30 side is 0.10 or more and 0.24 or less, 0.10 or more and 0.23 or less, 0.10 or more and 0.225 or less, 0. It may be 10 or more and 0.16 or less, 0.10 or more and 0.15 or less, 0.10 or more and 0.14 or less, or 0.10 or more and 0.13 or less.
- Ra / Rz of the surface 20a of the lid member 20 on the microwell array 30 side is 0.10 or more and 0.24 or less, even if the material of the lid member 20 is hydrophilic, the microwell of the lid member 20 Non-specific adsorption of the reagent to the surface 20a on the array 30 side can be further suppressed. This is not only the relationship between the electrical and chemical interaction between the surface 20a of the lid member 20 on the microwell array 30 side and the reagent, but also the minute unevenness of the surface 20a of the lid member 20 on the microwell array 30 side. It is considered that the small size can suppress the physical interaction.
- Ra / Rz When Ra / Rz is smaller than 0.10, it means that there is a partially protruding portion on the surface 20a of the lid member 20 on the microwell array 30 side, and non-specific adsorption of the reagent is likely to occur in that portion. Presumed to be.
- Ra / Rz When Ra / Rz is larger than 0.24, it means that the surface 20a of the lid member 20 on the microwell array 30 side is rough as a whole, and it is presumed that non-specific adsorption of the reagent is likely to occur.
- the surface 20a of the lid member 20 on the microwell array 30 side is coated with a hydrophobic coating to replace the arithmetic mean roughness (Ra) within the above range.
- a hydrophobic coating agent is applied to the surface 20a of the lid member 20 on the microwell array 30 side.
- a method of drying, and the like examples include a fluorine-based coating agent, a fluorine-containing polymer, and a silicone resin.
- the coating method include a dry coating method and a wet coating method.
- the thickness of the coating layer is preferably 0.01 ⁇ m or more and 3 ⁇ m or less, and more preferably 0.05 ⁇ m or more and 1 ⁇ m or less. ..
- the microwell array 30 may include a bottom layer 31, a wall layer 32 (may be described as a partition wall 32), and a plurality of microwells 33.
- the bottom layer 31 is provided on the substrate 10.
- the wall layer 32 is formed on the bottom layer 31.
- the plurality of microwells 33 are composed of a bottom layer 31 and a plurality of through holes 32 a formed in the thickness direction of the wall layer 32.
- the plurality of microwells 33 are formed in an array in the wall layer 32.
- the bottom layer 31 constitutes the bottom surface of the microwell 33. Therefore, when it is desired to impart hydrophilicity to the bottom surface, the bottom layer 31 may be formed of a hydrophilic material. It is preferable that the bottom layer 31 is formed so that the bottom layer 31 can transmit an electromagnetic wave so as not to interfere with the observation of the sample in the microwell 33 from the substrate 10 side. When it is desired to impart hydrophobicity to the bottom surface, the bottom layer 31 may be formed of a hydrophobic material. It is preferable that the bottom layer 31 not interfere with the observation of the sample in the microwell 33 from the substrate 10 side. Further, it is preferable to use a material that does not substantially have autofluorescence for the bottom layer 31.
- the wall portion layer 32 may be formed directly on the substrate 10 without providing the bottom layer 31. Therefore, in that case, the surface of the substrate 10 and the through holes 32 a of the wall layer 32 form the microwell 33.
- the wall layer 32 has a plurality of through holes 32a provided in an array in a state viewed in the thickness direction.
- the inner surface of each through hole 32 a constitutes the inner wall surface of each microwell 33.
- the wall layer 32 may be integrally formed with the substrate 10. Therefore, in that case, the microwell 33 is formed on the surface of the substrate 10.
- the same resin or the like as the material for forming the substrate 10 can be used, but a material in which a colored component that absorbs electromagnetic waves of a predetermined wavelength is mixed with the resin may be used. ..
- a hydrophilic resin in which the molecules of the constituent components of the resin have a hydrophilic group and a hydrophobic resin in which the molecules of the constituent components of the resin have a hydrophobic group can be used.
- hydrophilic group examples include a hydroxyl group, a carboxyl group, a sulfone group, a sulfonyl group, an amino group, an amide group, an ether group, and an ester group.
- hydrophilic resins include siloxane polymers; epoxy resins; polyethylene resins; polyester resins; polyurethane resins; polyacrylamide resins; polyvinylpyrrolidone resins; acrylic resins such as polyacrylic acid copolymers; cationized polyvinyl alcohols and silanolated polyvinyls.
- Polyvinyl alcohol resins such as alcohol and sulfonated polyvinyl alcohol; polyvinyl acetal resins; polyvinyl butyral resins; polyethylene polyamide resins; polyamide polyamine resins; cellulose derivatives such as hydroxymethyl cellulose and methyl cellulose; polyethylene oxide, polyethylene oxide-polypropylene oxide copolymers, etc.
- a polyalkylene oxide derivative; a maleic anhydride copolymer; an ethylene-vinyl acetate copolymer; a styrene-butadiene copolymer; and a combination of the above resins can be appropriately selected and used.
- hydrophobic resin examples include, for example, novolac resin; acrylic resin; methacrylic resin; styrene resin; vinyl chloride resin; vinylidene chloride resin; polyolefin resin; polyamide resin; polyimide resin; polyacetal resin; polycarbonate resin; polyphenylene sulfide resin; Sulfone resin; Fluorine resin; Silicone resin; Urya resin; Melamine resin; Guanamin resin; Phenol resin; Cellulose resin; and combinations of the above resins according to the sessile drop method specified in JIS R3257-1999.
- a material having a contact angle of 70 degrees or more can be appropriately selected and used.
- the hydrophobicity in the present specification means that the contact angle measured according to the intravenous drip method defined in JIS R3257-1999 is 70 degrees or more.
- the contact angle may be measured by a method based on ASTM D5725-1997 instead of the intravenous drip method specified in JIS R3257-1999.
- Both the hydrophilic resin and the hydrophobic resin may be a thermoplastic resin or a thermosetting resin. Further, it may be a resin that is cured by an active energy ray such as an electron beam or UV light, or may be an elastomer.
- a photoresist is used as the resin material, a large number of fine through holes 32a can be accurately formed in the wall layer 32 by photolithography.
- photolithography known means can be appropriately selected for selecting the type of photoresist to be used, coating, and exposing (photosensitizing), and removing unnecessary photoresist.
- the wall layer 32 can be formed by, for example, injection molding.
- Examples of the colored component include organic or inorganic pigments.
- examples of the black pigment include carbon black, acetylene black, iron black and the like.
- Yellow pigments include chrome yellow, zinc yellow, loess, Hansa yellow, permanent yellow, and benzine yellow.
- Orange pigments include orange lake, molybdenum orange, and benzine orange.
- Red pigments include Bengara, Cadmium Red, Antimony Zhu, Permanent Red, Risole Red, Lake Red, Brilliant Scarlet, and Thioindigo Red.
- Blue pigments include ultramarine blue, cobalt blue, phthalocyanine blue, ferrocyanine blue, and indigo.
- examples of green pigments include chrome green, viridian naphthol green, and phthalocyanine green.
- the wall layer 32 is formed by injection molding or the like, not only the pigment dispersed in the resin but also various dyes dissolved in the resin can be used as colored components.
- the dye can be exemplified by various dye methods. Specific examples thereof include direct dyes, basic dyes, cationic dyes, acidic dyes, mordant dyes, acidic mordant dyes, sulfide dyes, construction dyes, naphthol dyes, disperse dyes, and reactive dyes. In particular, when dyeing a resin, a disperse dye is often selected.
- a microwell means a well having a volume of 10 nanoliters (nL) or less.
- enzymatic reactions such as PCR and ICA (Invasive Cleared Assay) reactions performed in a microspace can be suitably performed.
- ICA Intelligent Cleared Assay
- mutation detection of a gene can be performed.
- the volume of the microwell 33 is not particularly limited, but is preferably 10 femtoliter (fL) or more and 100 picoliter (pL) or less, more preferably 10 fL or more and 5 pL or less, and most preferably 10 fL or more and 2 pL or less. Setting the volume in such a range is suitable for accommodating one to several biomolecules or carriers in one microwell 33 at the time of sample analysis described later.
- the shape of the microwell 33 is not particularly limited as long as the volume is within the above range.
- a cylinder a polyhedron composed of multiple faces (eg, a rectangular parallelepiped, a hexagonal prism, an octagonal prism, etc.), an inverted cone, and an inverted pyramid (an inverted triangular pyramid, an inverted quadrangular pyramid, an inverted pentagonal pyramid).
- the shape of the plurality of macrowells 33 may be a shape obtained by combining two or more of the above-described shapes.
- a part of the plurality of macrowells 33 may be cylindrical and the rest may be inverted conical.
- the bottom surface of the cone or the pyramid serves as an opening for communicating the flow path 35 and the micro well 33.
- the bottom of the microwell 33 may be flattened by using a shape obtained by cutting a part from the top of the inverted cone or the inverted pyramid.
- the bottom portion of the macrowell 33 may have a curved surface shape protruding toward the opening portion, or the bottom portion of the macrowell 33 may have a recessed curved surface shape.
- the thickness of the wall layer 32 defines the depth of the microwell 33.
- the thickness of the wall layer 32 is, for example, 10 nm or more and 100 ⁇ m or less, preferably 100 nm or more and 50 ⁇ m or less, and more preferably, for the purpose of enclosing an aqueous liquid (sample) containing biomolecules.
- each part of the microwell 33 one or several biomolecules are included in one microwell in consideration of the amount of the aqueous liquid to be contained and the size of the carrier such as beads to which the biomolecule is attached. It may be determined as appropriate so that it will be accommodated.
- the number and density of the microwells 33 provided in the microwell array 30 can be appropriately set.
- the number of microwells 33 per cm 2 is, for example, 10,000 or more and 10 million or less, preferably 100,000 or more and 5 million or less, and more preferably 100,000 or more and 1 million or less.
- the number of microwells 33 per cm 2 may be referred to as the density of microwells.
- the density of the microwells is within this range, the operation of enclosing the aqueous liquid as a sample in a predetermined number of wells is easy. Further, when the density of the microwells is within this range, it is easy to observe the wells for analyzing the experimental results.
- FIG. 1 shows an example of a one-dimensional array in which a plurality of microwells 33 are arranged in a line.
- a two-dimensional array in which a plurality of microwells are two-dimensionally arranged may be used.
- a peripheral member 34 having a frame shape in a plan view is arranged around the microwell array.
- the dimension of the peripheral member 34 in the thickness direction of the microfluidic device 1 is larger than that of the wall layer 32.
- the peripheral edge member 34 secures a gap between the lid member 20 and the microwell array, and maintains the flow path 35. That is, the flow path 35 is a region sandwiched between the microwell array 30 and the lid member 20 and surrounded by the peripheral member 34.
- the material of the peripheral member 34 is not particularly limited, and examples thereof include a double-sided adhesive tape in which an acrylic adhesive is laminated on both sides of a core material film formed of silicone rubber or acrylic foam.
- the peripheral edge member 34 may be integrally molded with the lid member 20. In that case, the peripheral edge member 34 becomes a stepped portion of the lid member 20, and the stepped portion secures a gap between the lid member 20 and the microwell array to maintain the flow path 35.
- the microfluidic device 1 configured as described above can be manufactured by the following procedure, for example. First, the substrate 10 is prepared, and the wall portion resin layer to be the wall portion layer 32 is formed on the surface of the substrate 10. When the bottom layer 31 is provided, the bottom layer 31 is formed before the wall resin layer is formed. Even if the bottom layer 31 is not provided, an anchor layer or the like that enhances the adhesion between the substrate 10 and the resin layer for the wall portion may be provided on the surface of the substrate 10 as needed.
- the wall resin layer may be formed of a material in which a colored component is mixed with a resin material.
- the content ratio of the colored component with respect to the total mass of the resin material and the colored component can be, for example, 0.5 mass% (mass%) or more and 60 mass% or less.
- the content is preferably 5 mass% or more and 55 mass% or less, more preferably 20 mass% or more and 50 mass% or less.
- the content of the color component with respect to the total mass of the resin material and the color component can be appropriately set so that a desired pattern can be constructed in consideration of the ratio of the photosensitive component contained in the resist.
- the particle size of the pigment is set and prepared so that the above-described predetermined condition is satisfied for the microwell to be formed.
- a dispersant may be appropriately added to the resin material together with the pigment.
- a through hole 32a is formed in the formed wall resin layer.
- the through hole 32a can be formed easily and accurately.
- the wall resin layer is formed by injection molding or the like, the wall resin layer and the through holes can be formed by the same process.
- the through hole 32a can be formed by etching using a pattern mask.
- the peripheral member 34 is arranged around the microwell array 30, and then the lid member 20 is arranged on the peripheral member 34.
- the Ra of the surface 20a of the lid member 20 on the microwell array 30 side is arranged to be 50 nm or less.
- the substrate 10, the peripheral member 34, and the lid member 20 are integrally joined to complete the microfluidic device 1.
- the flow path is formed between the lid member 20 and the substrate 10 by the peripheral member 34.
- the joining method is not particularly limited, and examples thereof include joining with an adhesive, joining with a double-sided tape, and joining by laser welding.
- FIG. 3 shows a microfluidic device 2 in which the substrate 10 and the wall layer 32 are integrally molded, and the peripheral member 34 and the lid member 20 are integrally molded.
- the microfluidic device 2 is formed by arranging the substrate 10 integrally molded with the wall layer 32 on the lid member 20 integrally molded with the peripheral edge member 34, and integrally molding the peripheral edge member 34 and the lid member 20. It is possible to manufacture by joining the formed step portion to the substrate 10 integrally formed with the wall layer 32.
- the flow path 35 is formed between the lid member 20 and the substrate 10 by the step portion formed on the lid member 20.
- the configuration of the microfluidic device 2 is the same as that of the above-described microfluidic device 1 except that the substrate 10 and the wall layer 32 are integrally molded, and the peripheral member 34 and the lid member 20 are integrally molded.
- the microfluidic device may have the substrate 10 and the wall layer 32 as separate elements, and the peripheral member 34 and the lid member 20 are integrally formed. Also in this case, the configuration of the microfluidic device is the same as that of the above-described microfluidic device 1 except that the peripheral member 34 and the lid member 20 are integrally formed.
- the sample analysis method of the present embodiment is a sample analysis method using the microfluidic device 1 according to the present embodiment, Supplying an aqueous liquid containing a sample to the channel 35, A sealing liquid is introduced into the flow path 35 to replace the aqueous liquid existing in the flow path 35, and the aqueous liquid is sealed in the microwell 33. Heating the microfluidic device to cause a reaction in the microwell 33 and generate a signal for detection; Detecting the signal, And a sample analysis method.
- the aqueous liquid can include water, a buffer solution, a detection reaction reagent, and the like in addition to the sample.
- the aqueous liquid may contain an enzyme.
- the PCR method, the ICA method, the LAMP method (registered trademark, Loop-Mediated Isothermal Amplification), the TaqMan method (registered trademark), the fluorescent probe method, or the like can be used.
- the sample is a protein
- the ELISA method registered trademark
- the aqueous liquid may contain additives such as a surfactant.
- buffer solution examples include Tris-HCl buffer solution, acetate buffer solution, and phosphate buffer solution.
- the enzyme examples include DNA polymerase, RNA polymerase, reverse transcriptase, flap endonuclease and the like.
- the microfluidic device of the present embodiment can suitably retain the aqueous liquid in the well even when the temperature of the encapsulated aqueous liquid is changed in, for example, gene mutation detection.
- the range of the temperature to be changed that is, the range from the lower limit value to the upper limit value of the temperature change is, for example, 0 ° C. to 100 ° C., preferably 0 ° C. to 80 ° C., and more preferably 20 ° C. to 70 ° C.
- a reaction such as PCR or ICA reaction performed in a minute space.
- Samples to be analyzed using the microfluidic device 1 include DNA, RNA, miRNA, mRNA, protein, lipid, cell, bacterium and the like.
- the sample may be, for example, a sample collected from a living body such as blood.
- the detection target to be detected by the sample analysis may be a PCR product or the like using the DNA contained in the sample as a template, or an artificially synthesized compound (for example, artificially imitating the sample DNA). It may be a synthesized nucleic acid) or the like.
- the well may have a shape and size that can accommodate one molecule of DNA.
- an aqueous liquid containing a sample to be enclosed in microwells is prepared.
- the aqueous liquid containing the sample is a liquid in which water containing the detection target is the main solvent.
- examples thereof include ICA reaction solution containing FEN-1 and a fluorescent substrate.
- a surfactant may be added to make the sample more accessible to the microwells.
- beads that specifically recognize the detection target may be added to capture the detection target.
- the detection target may be suspended in an aqueous liquid without directly or indirectly binding to a carrier such as beads.
- the aqueous liquid 100 containing the prepared sample is supplied from the first hole 21 to the flow path 35 using a syringe or the like (also referred to as a sample supply step).
- the aqueous liquid 100 containing the supplied sample is filled in each microwell 33 and in the flow path 35 as shown in FIG.
- the gas in the flow path 35 is degassed in advance by a degassing operation before the sample supply step. This degassing operation may be performed by filling the flow path 35 with a buffer.
- the buffer include water, water containing a buffer solution, water containing a surfactant, and water containing a buffer solution and a surfactant.
- an encapsulation process of enclosing the aqueous liquid containing the sample 100 in the microwell 33 is performed.
- the detection target in the sample contained in the aqueous liquid may be labeled with fluorescence or the like.
- the fluorescent label treatment may be performed before the sample supply step, for example, when preparing the sample, or after the sample supply step, the fluorescent label may be introduced into the channel 35.
- the sealing liquid 110 is supplied from the first hole 21 to the flow path 35 using a syringe or the like. The supplied sealing liquid 110 flows in the channel, and pushes the aqueous liquid 100 containing the sample existing in the channel 35 toward the second hole 22 as shown in FIG.
- the sealing liquid 110 is replaced with the aqueous liquid 100 filled in the channel 35, and the channel 35 is filled with the sealing liquid 110.
- the aqueous liquid 100 containing the sample is placed only in each microwell 33 independently of each other, and the sealing of the sample is completed.
- the sealing liquid 110 means a liquid used for separating the aqueous liquids introduced into the microwells 33 of the microwell array 30 so as not to be mixed with each other, and examples thereof include oils and the like.
- oils for example, the product name "FC40” manufactured by Sigma, the product name “HFE-7500” manufactured by 3M, mineral oil used for PCR reaction and the like can be used.
- the sealing liquid 110 preferably has a contact angle with the material of the wall layer 32 of 5 degrees or more and 30 degrees or less. When the contact angle of the sealing liquid 110 is within this range, the sealing liquid 110 easily pushes the aqueous liquid 100, and the aqueous liquid 100 does not easily remain on the surface of the lid member 20.
- the contact angle of the sealing liquid may be measured, for example, by using the sealing liquid instead of water according to the intravenous drip method specified in JIS R3257-1999.
- the contact angle may be measured by a method according to ASTM D5725-1997 instead of the intravenous drop method specified in JIS R3257-1999.
- a reaction step of heating the microfluidic device 1 to cause a reaction in the microwell 33 and generating a signal for detection is performed.
- the signal for detection include fluorescence, chemiluminescence, color development, potential change, pH change and the like, but fluorescence is preferable.
- the microfluidic device 1 may be subjected to a thermal cycler, and an enzymatic reaction such as a PCR reaction or an ICA reaction may be performed as necessary.
- the reaction may be, for example, a biochemical reaction, more specifically an enzymatic reaction.
- the temperature for heating is appropriately determined depending on the reaction, but is, for example, 60° C. or higher and 100° C. or lower.
- the temperature for heating is not the actual temperature of the reagent solution in the microwell 33, but the heating temperature of the microfluidic device set by a thermal cycler, an incubator, or the like. Further, the temperature at the time of heating is, for example, 60 ° C. or higher and 100 ° C. or lower means that the maximum temperature reaches 60 ° C. or higher and 100 ° C. or lower, and it is not always necessary to be 60 ° C. or higher and 100 ° C. or lower. That is, the temperature of the microfluidic device 1 may change within the above-mentioned range of changing temperature.
- An example of the reaction is a signal amplification reaction.
- the microfluidic device 1 is placed in the microwell 33 under a constant temperature condition where the desired enzyme activity can be obtained, for example, at 60 ° C. or higher, in a state where the reagent solution containing the enzyme for signal amplification is contained in the microwell 33. It is an isothermal reaction which is maintained at 100° C. or lower for a predetermined time, for example, at least 10 minutes, preferably about 15 minutes.
- the signal generated from the microwell 33 by the above reaction is detected (detection step).
- the signal is fluorescence
- it is set in a fluorescence microscope and irradiated with excitation light (electromagnetic wave).
- the wavelength of the excitation light is appropriately set according to the fluorescent label used.
- the irradiation of the electromagnetic wave may be performed from the substrate 10 side of the microfluidic device 1, the lid member 20 side, that is, the upper side of the microwell 33, or from any other direction.
- the detection of fluorescence or phosphorescence generated as a result of irradiation with electromagnetic waves may be performed from the substrate side of the microwell array, the well side, or any other direction. When fluorescence or phosphorescence is detected using a microscope, it is convenient to perform it from the substrate 10 side of the microfluidic device 1.
- the measurement may be performed by capturing a fluorescence image of the microwell array 30 and using the fluorescence image. For example, by performing a PCR reaction in the microwell array 30 and detecting the fluorescence of SYBR Green in the microwells 33 in which PCR amplification was observed, the microwells 33 in which amplification was observed for all the numbers of the microwells 33 were detected. The percentage of numbers can be calculated.
- the detection target is, for example, a single nucleotide polymorphism (SNP: Single Nucleotide Polymorphism)
- the frequency of SNP expression can be analyzed by counting the number of microwells 33 that emit fluorescence.
- the aqueous liquid may contain proteins and enzymes as samples or enzymes as reagents.
- proteins and enzymes are not adsorbed on the beads but suspended in the aqueous liquid, adsorption of the proteins and enzymes on the surface of the lid member 20 is particularly likely to occur.
- proteins or enzymes are non-specifically adsorbed on the lid member 20 and these proteins or enzymes emit fluorescence or phosphorescence, the fluorescence or phosphorescence is detected as noise.
- the microfluidic device of the present invention the adsorption of proteins and enzymes contained in the sample and the reagent onto the lid member 20 is reduced, so that the generation of the noise can be suppressed.
- a microwell array having a plurality of microwells, and a lid member facing the microwell array in a state of being separated from each other, and having a channel between the microwell array and the lid member,
- a microfluidic device having an arithmetic average roughness Ra of 5 nm or more and 50 nm or less and a ten-point average roughness (Rz) of 50 nm or more and 250 nm or less on the surface of the lid member on the microwell array side.
- the microfluidic device according to [8] which is located between the microwell array and the lid member and further has a peripheral member surrounding the flow path.
- microfluidic device [10] The microfluidic device according to [9], wherein the peripheral member is a step portion formed integrally with the lid member. [11] The microfluidic device according to any one of [8] to [10], wherein a contact angle of the surface of the lid member on the microwell array side with water is 70 degrees or more. [12] The microfluidic device according to any one of [8] to [11], wherein Ra/Rz of the surface of the lid member on the microwell array side is 0.10 or more and 0.23 or less. [13] The microfluidic device according to any one of [8] to [12], wherein a surface of the lid member on the microwell array side is coated with a hydrophobic coating.
- a sample analysis method comprising the operation and the detection of the signal.
- Example 1 Two resin members were prepared: a rectangular substrate made of COP (ZEONOR1010R, manufactured by ZEON Corporation) formed by injection molding, and a rectangular lid member made of COP.
- a substrate made of COP a substrate formed by injection molding in which cylindrical micropores having a diameter of 10 ⁇ m and a depth of 15 ⁇ m were arranged on the entire surface of the substrate was used.
- the lid member was formed with a stepped portion having a height of 100 ⁇ m, that is, a peripheral member, an inlet and an outlet. The inlet and the outlet were arranged inside the peripheral member along the longitudinal direction of the lid member.
- the surface of the steel lid member corresponding to the lid member bottom surface (the surface on which the step portion is formed) of the die was polished with #3000 diamond paste, and the die was mirror-finished.
- the contact angle of the bottom surface of the molded lid member with water was measured using a contact angle measuring instrument SA-20 (manufactured by Kyowa Interface Science Co., Ltd.) according to the sessile drop method specified in JIS R 3257-1999.
- the contact angle of the bottom surface of the lid member of Example 1 with water was 85 degrees.
- the surface roughness of the molded lid member was measured using a contact type surface roughness measuring device (TALYSURF PGI1240, manufactured by Taylor Hobson). In this measurement, the height difference with respect to the starting point (the height is 0 nm) in the scanning range of 800 ⁇ m was acquired. Table 1 shows the measurement results of Ra and Rz on the bottom surface of the lid member.
- the COP substrate and the lid member were adhered to each other by applying mineral oil to the stepped portion of the lid member so that the mirror-finished surface of the lid member faces the substrate side, to fabricate a microfluidic device.
- 200 ⁇ L of a buffer having the composition shown in Table 2 below was fed into the flow path between the substrate and the lid member to fill each well of the micropore chip with the buffer.
- a fluorescent reagent (Fluorescein, manufactured by Tokyo Chemical Industry Co., Ltd.) having the composition shown in Table 3 below was sent to the above flow path, and the buffer was replaced with the fluorescent reagent. Further, 150 ⁇ l of fluorocarbon oil (FC40, manufactured by Sigma) was sent to individually seal each well of the micropore tip. In this example, the fluorescent reaction was not performed, but an enzyme was added to the fluorescent reagent in order to obtain the same state as when the fluorescent reaction was performed.
- FC40 fluorocarbon oil
- the above microfluidic device is set on a hot plate, heated at 66 ° C. for 15 minutes, and then the fluorescence image of the micropore chip is observed with a fluorescence microscope (BZ-710, manufactured by KEYENCE) using a 4x objective lens. did.
- the exposure time was set to 3000 msec with a bright field of 20 msec and a GFP (Green Fluorescent Protein) fluorescent filter.
- FIG. 6A shows the fluorescence observation result of the micropore chip after droplet formation in the microfluidic device of Example 1.
- the size of the observed image was 580 ⁇ m ⁇ 580 ⁇ m.
- the microfluidic device of Example 1 was able to correctly measure the number of droplets without nonspecifically adsorbing the reagent on the bottom surface of the lid member.
- Example 1 A microfluidic device was produced in the same manner as in Example 1 except that the surface corresponding to the bottom surface of the lid member in the mold was not mirror-finished. Table 1 shows the measurement results of Ra and Rz on the bottom surface of the lid member. The contact angle of the bottom surface of the lid member of Comparative Example 1 with water was 85 degrees.
- FIG. 6B shows the result of fluorescence observation of the micropore chip after the droplet formation in the microfluidic device of Comparative Example 1.
- a typical reagent attachment is shown in the arrow portion in the figure.
- many reagents adhered to the entire visual field.
- the reagent in the microfluidic device of Comparative Example 1 using the lid member not mirror-finished, the reagent nonspecifically adheres to the bottom surface of the lid member, and the correct number of droplets is caused by the adhered portion. could not be measured. From this result, it is clear that even if the contact angle of the bottom surface of the lid member with water is the same, non-specific adsorption of the reagent to the bottom surface of the lid member can be reduced because Ra is 50 nm or less. became.
- Example 2 A base material made of COP was produced by the same method as in Example 1.
- a lid member made of COP was manufactured by the same method as in Example 1 except that the time for mirror finishing the surface corresponding to the bottom surface of the lid member in the mold of the lid member was lengthened.
- the surface roughness of the molded lid member was measured using a surface roughness measuring instrument (SJ-210, manufactured by Mitutoyo). The scanning range of this measurement was set so that the center of the lid member was the center of the scanning range and was 1.5 mm in the direction perpendicular to the longitudinal direction of the lid member, and the height difference with respect to the starting point (the height was 0 nm) was acquired.
- Table 4 shows the measurement results of Ra and Rz on the bottom surface of the lid member of Example 2.
- 7: is a figure which shows the measurement data of the surface roughness of the lid member of the microfluidic device of Example 2.
- the base material and the lid member were adhered by applying mineral oil to the stepped portion of the lid member so that the mirror-finished surface of the lid member faced the substrate, to fabricate a microfluidic device.
- the buffer, the fluorescent reagent and the mineral oil were sent to the microfluidic device under the same conditions as in Example 1, and the fluorescent reagent was individually sealed in each well of the micropore chip.
- the fluorescence reaction was not performed as in Example 1, but an enzyme was added to the fluorescent reagent in order to bring the state to the same as when the fluorescence reaction was performed.
- the above microfluidic device is set on a hot plate, heated at 66 ° C. for 15 minutes, and then the fluorescence image of the micropore chip is observed with a fluorescence microscope (BZ-710, manufactured by KEYENCE) using a 4x objective lens. did.
- the exposure time was set to 3000 msec with a bright field of 20 msec and a GFP (Green Fluorescent Protein) fluorescent filter.
- the observation visual field was a region of 3.6 mm ⁇ 2.7 mm.
- the region emitting the fluorescence intensity higher than the reference is the part where the fluorescent reagent is adsorbed.
- the area of the part where the fluorescent reagent was adsorbed was calculated.
- the area of the portion where the fluorescent reagent was adsorbed with respect to the area of the entire fluorescent image was determined as the ratio (%) of the adsorbed area.
- Example 3 The ratio of the adsorption area of the reagent of Example 3 was obtained by the same method as in Example 2 except that the mirror-finishing time of the surface corresponding to the bottom surface of the lid member in the mold of the lid member was shortened. The results are shown in Table 4. 8: is a figure which shows the measurement data of the surface roughness of the lid member of the microfluidic device of Example 3. FIG.
- Comparative Examples 2 and 3 in which Ra of the surface of the lid member on the microwell array side is larger than 70 nm, the ratio of the adsorption area was 22.3% and 21.0%, respectively.
- Comparative Examples 2 and 3 since the surface corresponding to the bottom surface of the lid member in the mold of the lid member was not mirror-finished, the surface roughness of the bottom surface of the lid member could not be controlled and the surface roughness varied. ..
- the reason why the suppression of adsorption is evaluated to be good when the ratio of the adsorption area is less than 10% is as follows.
- the ratio of the adsorption area exceeds 10%, for example, when trying to measure a low-concentration sample such that only one target molecule enters all the wells of the microfluidic device, the reagent is adsorbed with a probability of 10% or more.
- the target molecule is encapsulated in the well that overlaps the portion where the target molecule is present. Even if the well of the portion where the reagent is adsorbed emits light due to the enzymatic reaction, the light emission cannot be detected, and it is erroneously determined (pseudo-negative) as negative.
- a microfluidic device and a sample analysis method capable of detecting fluorescence and phosphorescence of an aqueous liquid in a well as accurately as possible. For example, when a diagnosis is performed by detecting DNA or RNA derived from a living body, it becomes possible to put a nucleic acid together with a reagent into a minute space.
- Microfluidic Device 10 Substrate 20 Lid Member 30 Microwell Array 32 Wall Layer 33 Microwell 100 Aqueous Liquid 110 Sealing Liquid
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Abstract
Description
本願は、2019年3月1日に日本に出願された特願2019-037543号について優先権を主張し、その内容をここに援用する。
蓋部材にPDMS等の一般的に疎水性であるといわれる材料を使うことにより蓋部材への試薬の吸着を抑制することもできるが、用いることができる材料が制約されてしまう。
[1]複数のマイクロウェルを有するマイクロウェルアレイと、前記マイクロウェルアレイと離間した状態で対向する蓋部材と、を備え、前記マイクロウェルアレイと前記蓋部材との間に流路を有し、前記蓋部材のマイクロウェルアレイ側の面の算術平均粗さRaが70nm以下であるマイクロ流体デバイス。
[2]前記蓋部材のマイクロウェルアレイ側の面の水との接触角が70度以上である[1]に記載のマイクロ流体デバイス。
[3]前記蓋部材のマイクロウェルアレイ側の面の表面粗さRzが350nm以下である[1]または[2]に記載のマイクロ流体デバイス。
[4]前記蓋部材のマイクロウェルアレイ側の面のRa/Rzが0.10以上0.23以下である[1]~[3]のいずれか一項に記載のマイクロ流体デバイス。
[5]前記蓋部材のマイクロウェルアレイ側の面に疎水的なコーティングが施されている[1]~[4]のいずれか一項に記載のマイクロ流体デバイス。
[6][1]~[5]のいずれか一項に記載のマイクロ流体デバイスを用いた試料分析方法であって、試料を含有する水性液体を前記流路に供給することと、前記流路に封止液を導入して前記流路内に存在する前記水性液体と置換し、前記マイクロウェルに前記水性液体を封入することと、前記マイクロウェルにおいて反応を起こし、検出のためのシグナルを発生させることと、前記シグナルを検出することと、を備える試料分析方法。
[7]前記試料が、DNA、RNA、タンパク質、脂質、細胞、または細菌である[6]に記載の試料分析方法。
基板10は、所定の波長範囲の電磁波のみを透過可能であってもよい。例えば、マイクロウェル内の試料の存在を、可視光領域である350~700nmの波長範囲にピークを有する蛍光の検出により判定場合には、少なくとも上記波長範囲の可視光を透過可能な基板を基板10として用いればよい。
蓋部材20を形成する材料や蓋部材20の厚みについては、基板10と同様とすることができる。
蓋部材20が電磁波透過性を有する場合は、電磁波透過性については適宜設定できる。例えば、後述する電磁波照射工程を蓋部材20側から行わない場合は、蓋部材20は電磁波を透過可能でなくてもよい。
なお、蓋部材20のマイクロウェルアレイ30側の面20aの水との接触角は、180度以下であるので、蓋部材20のマイクロウェルアレイ30側の面の水との接触角は、例えば、70度以上180度以下である。
水との接触角は、例えば、JIS R 3257-1999に規定された静滴法に準じて測定することができる。JIS R 3257-1999に規定された静滴法に代えて、ASTM D5725-1997に準拠した方法で接触角を測定してもよい。
前記蓋部材20のマイクロウェルアレイ30側の面20aの表面粗さ(十点平均粗さ)(Rz)の上限値と下限値は、任意に組み合わせることができる。例えば、前記蓋部材20のマイクロウェルアレイ30側の面20aの表面粗さ(十点平均粗さ)(Rz)は、50nm以上350nm以下、55nm以上300nm以下、60nm以上250nm以下、60nm以上200nm以下、60nm以上150nm以下、65nm以上120nm以下、70nm以上110nm以下、70nm以上100nm以下または80nm以上90nm以下であってもよい。
前記コーティング剤としては、フッ素系コーティング剤及びフッ素含有ポリマー、シリコーン樹脂等が挙げられる。コーティング方法としては、ドライコーティング法及びウェットコーティング法等が挙げられる。
なお、壁部層32は、基板10と一体成形されていてもよい。従って、その場合、基板10の表面にマイクロウェル33が構成される。
樹脂材料としては、マイクロウェル33に求める特性等を考慮して、樹脂の構成成分の分子が親水性基を有する親水性樹脂と、樹脂の構成成分の分子が疎水性基を有する疎水性樹脂とのいずれも用いることができる。
疎水性樹脂の例としては、例えば、ノボラック樹脂;アクリル樹脂;メタクリル樹脂;スチレン樹脂;塩化ビニル樹脂;塩化ビニリデン樹脂;ポリオレフィン樹脂;ポリアミド樹脂;ポリイミド樹脂;ポリアセタール樹脂;ポリカーボネート樹脂;ポリフェニレンスルフィド樹脂;ポリスルフォン樹脂;フッ素樹脂;シリコーン樹脂;ユリヤ樹脂;メラミン樹脂;グアナミン樹脂;フェノール樹脂;セルロース樹脂;および上記の樹脂の組み合わせ等の中から、JIS R3257-1999に規定された静滴法に準じて測定した接触角が70度以上である材料を適宜選択して使用することができる。すなわち、本明細書における疎水性とは、JIS R3257-1999に規定された静滴法に準じて測定した接触角が70度以上であることを意味する。なお、JIS R3257-1999に規定された静滴法に代えて、ASTM D5725-1997に準拠した方法で接触角を測定してもよい。
樹脂材料としてフォトレジストを用いると、フォトリソグラフィにより壁部層32に多数の微細な貫通孔32aを精度良く形成することができる。
フォトリソグラフィを用いる場合、使用するフォトレジストの種類の選択、塗布、および露光(感光)、および不要なフォトレジストの除去の方法には公知の手段を適宜選択することができる。
レジストを用いない場合は、例えば射出成型等により壁部層32を形成することができる。
マイクロウェル33の容積は、特に限定されるものではないが、10フェムトリットル(fL)以上100ピコリットル(pL)以下が好ましく、10fL以上5pL以下がより好ましく、10fL以上2pL以下が最も好ましい。このような範囲に容積を設定すると、後述する試料分析の際に、一つのマイクロウェル33に、1から数個だけ生体分子または担体を収容するのに適している。
さらに複数のマクロウェル33の形状は、上述の形状を二つ以上組み合わせたような形状であってもよい。例えば、複数のマクロウェル33の一部が円筒形であり、残りが逆円錐形であってもよい。また、複数のマクロウェル33が逆円錐形または逆角錐形の場合、円錐または角錐の底面が流路35とマイクロウェル33とを連通する開口部となる。この場合、逆円錐形または逆角錐形の頂上から一部を切り取った形状を用いて、マイクロウェル33の底部を平坦にしてもよい。他の例として、マクロウェル33の底部が開口部に向かって突出した曲面形状であってもよく、マクロウェル33の底部が窪んだ曲面形状であってもよい。
マイクロウェル33の各部寸法は、収容する水性液体の量や、生体分子を付着させたビーズ等の担体の大きさ等を考慮して、1つのマイクロウェルに1つ、もしくは数個の生体分子が収容されるように適宜決定すればよい。
1cm2あたりのマイクロウェル33の数は、例えば1万以上1000万以下であり、好ましくは10万以上500万以下であり、更に好ましくは10万以上100万以下である。本明細書において1cm2あたりのマイクロウェル33の数をマイクロウェルの密度と呼ぶことがある。マイクロウェルの密度がこの範囲内であると、所定数のウェルに試料である水性液体を封入させる操作が容易である。また、マイクロウェルの密度がこの範囲内であると、実験結果を解析するためのウェルの観察も容易である。例えば、セルフリーDNAの変異を検出する場合において、野生型DNAに対する検出対象である変異DNAの存在割合が0.01%程度である場合、例えば、100万~200万個程度のマイクロウェルを使用することが好適である。
図1では、複数のマイクロウェル33が一列に並んだ一次元アレイの例を示している。しかしながら、上述のように多数のマイクロウェルを設ける場合、複数のマイクロウェルを二次元に配列した二次元アレイを用いてもよい。
周縁部材34の材質等に特に制限はないが、例えばシリコーンゴムまたはアクリル発泡体から形成される芯材フィルムの両面にアクリル系粘着剤が積層された両面粘着テープ等が挙げられる。
なお、周縁部材34は蓋部材20と一体成形されていてもよい。その場合、周縁部材34は、蓋部材20の段差部となり、前記段差部により蓋部材20とマイクロウェルアレイとの間に隙間を確保し、流路35を維持している。
まず、基板10を準備し、基板10の面上に壁部層32となる壁部用樹脂層を形成する。底部層31を設ける場合は、壁部用樹脂層の形成前に底部層31を形成する。底部層31を設けない場合でも、必要に応じて基板10の面上に基板10と壁部用樹脂層との密着性を高めるアンカー層等を設けてもよい。
形成された壁部樹脂層が樹脂材料に有色成分を混合した材料から形成されている場合、壁部樹脂層は、壁部樹脂層に含有される有色成分に基づいた色彩を有する。
貫通孔32aが形成されると、壁部樹脂層が壁部層32となり、マイクロウェルアレイ30が完成する。
基板10と壁部層32とが一体成形され、周縁部材34と蓋部材20とが一体成形されている以外のマイクロ流体デバイス2の構成は、上述のマイクロ流体デバイス1と同じである。
本実施形態の試料分析方法は、本実施形態に係るマイクロ流体デバイス1を用いた試料の分析方法であって、
試料を含む水性液体を前記流路35に供給することと、
前記流路35に封止液を導入して前記流路35内に存在する前記水性液体と置換し、前記マイクロウェル33に前記水性液体を封入することと、
前記マイクロ流体デバイスを加熱して前記マイクロウェル33において反応を起こし、検出のためのシグナルを発生させることと、
前記シグナルを検出することと、
を備える試料分析方法である。
酵素としては、例えばDNAポリメラーゼ、RNAポリメラーゼ、逆転写酵素、及びフラップエンドヌクレアーゼ等が挙げられる。
界面活性剤としては、例えばTween20(ポリオキシエチレンソルビタンモノラウラートともいう)、Triton-X100(ポリエチレングリコールモノ-4-オクチルフェニルエーテル(n=約10)ともいう)、グリセロール、オクチルフェノールエトキシレート、及びアルキルグリコシド等が挙げられる。
封入工程では、シリンジ等を用いて、封止液110を第一孔21から流路35に供給する。供給された封止液110は流路内を流れ、図5に示すように、流路35内に存在する試料を含有する水性液体100を第二孔22に向かって押す。そして、封止液110は、流路35内に充填されていた水性液体100と置換され、流路35は封止液110で充填される。その結果、試料を含有する水性液体100は、各マイクロウェル33内のみに、互いに独立した状態で配置され、試料の封入が完了する。
封止液110は、壁部層32の材質に対する接触角が5度以上30度以下であることが好ましい。封止液110の接触角がこの範囲であると、封止液110が水性液体100を押しやすく、蓋部材20の表面に水性液体100が残留しにくくなる。その結果、各マイクロウェル33に好適に試料を封入することができる。封止液の接触角は、例えば、JIS R3257-1999に規定された静滴法に準じて、水の代わりに封止液を用いて測定すればよい。JIS R3257-1999に規定された静滴法に代えて、ASTM D5725-1997に準拠した方法で接触角を測定してもよい。
検出のためのシグナルの例としては、蛍光、化学発光、発色、電位変化、またはpH変化等が挙げられるが、蛍光が好ましい。
反応工程の前に、マイクロ流体デバイス1をサーマルサイクラーにかけて、PCR反応やICA反応等の酵素反応等を必要に応じて行ってもよい。
前記反応は、例えば生化学反応、より具体的には酵素反応であってもよい。加熱する際の温度は、反応に応じて適宜決定されるが、例えば、60℃以上100℃以下である。加熱する際の温度とは、マイクロウェル33内の試薬液の実際の温度ではなく、サーマルサイクラーまたはインキュベーター等によって設定される、マイクロ流体デバイスの加熱温度のことである。また、加熱する際の温度が例えば60℃以上100℃以下であるとは、温度の最高温度が60℃以上100℃以下に達することをいい、常に60℃以上100℃以下である必要はない。すなわち、上述した変化させる温度の範囲内において、マイクロ流体デバイス1の温度が変化しても構わない。反応の一例として、シグナル増幅反応が挙げられる。シグナル増幅反応は、シグナル増幅のための酵素を含んだ試薬液がマイクロウェル33内に収容された状態で、マイクロ流体デバイス1を、所望の酵素活性が得られる一定温度条件下、例えば60℃以上100℃以下で、所定時間、例えば少なくとも10分間、好ましくは約15分間、維持する等温反応である。
電磁波の照射は、マイクロ流体デバイス1の基板10側から行ってもよく、蓋部材20側、つまりマイクロウェル33の上側から行ってもよく、その他の任意の方向から行ってもよい。また、電磁波の照射の結果発生する蛍光または燐光の検出は、マイクロウェルアレイの基板側から行ってもよく、ウェル側から行ってもよく、その他の任意の方向から行ってもよいが、例えば蛍光顕微鏡を用いて蛍光または燐光を検出する場合には、マイクロ流体デバイス1の基板10側から行うことが簡便である。
例えば、マイクロウェルアレイ30内でPCR反応を行い、PCR増幅が見られたマイクロウェル33におけるSYBR Greenの蛍光を検出することにより、全てのマイクロウェル33の数に対する増幅が見られたマイクロウェル33の数の割合を算出することができる。検出対象が例えば一塩基多型(SNP:Single Nucleotide Polymorphism)の場合、蛍光を発するマイクロウェル33の数を数えることで、SNPの発現頻度等を分析することができる。
[8]複数のマイクロウェルを有するマイクロウェルアレイと、前記マイクロウェルアレイと離間した状態で対向する蓋部材と、を備え、前記マイクロウェルアレイと前記蓋部材との間に流路を有し、前記蓋部材のマイクロウェルアレイ側の面の算術平均粗さRaが5nm以上50nm以下であり、十点平均粗さ(Rz)が50nm以上250nm以下であるマイクロ流体デバイス。
[9]前記マイクロウェルアレイと前記蓋部材との間に位置し、且つ前記流路を囲む周縁部材をさらに有する[8]に記載のマイクロ流体デバイス。
[10]前記周縁部材が前記蓋部材と一体に形成された段差部である、[9]に記載のマイクロ流体デバイス。
[11]前記蓋部材のマイクロウェルアレイ側の面の水との接触角が70度以上である[8]~[10]のいずれか一項に記載のマイクロ流体デバイス。
[12]前記蓋部材のマイクロウェルアレイ側の面のRa/Rzが0.10以上0.23以下である[8]~[11]のいずれか一項に記載のマイクロ流体デバイス。
[13]前記蓋部材のマイクロウェルアレイ側の面に疎水的なコーティングが施されていることを特徴とする[8]~[12]のいずれか一項に記載のマイクロ流体デバイス。
[14]前記疎水的なコーティングは、フッ素系コーティング剤及びフッ素含有ポリマー、シリコーン樹脂のうちの1つである、[8]~[13]のいずれか一項に記載のマイクロ流体デバイス。
[15][8]~[14]のいずれか一項に記載のマイクロ流体デバイスを用いた試料分析方法であって、試料を含有する水性液体を前記流路に供給することと、前記流路に封止液を導入して前記流路内に存在する前記水性液体と置換し、前記マイクロウェルに前記水性液体を封入することと、前記マイクロウェルにおいて反応を起こし、検出のためのシグナルを発生させることと、前記シグナルを検出することと、を備える試料分析方法。
[16]前記試料が、DNA、RNA、タンパク質、脂質、細胞、または細菌である[15]に記載の試料分析方法。
射出成形で形成したCOP製(ZEONOR1010R、日本ゼオン社製)の矩形状の基板とCOP製の矩形状の蓋部材の2つの樹脂製の部材を準備した。COP製の基板は、基板全面に径10μm、深さ15μmの円筒状の微小孔が配置され、射出成形により成形された基板を用いた。蓋部材には、高さが100μmとなる段差部、つまり周縁部材と注入口と排出口とを形成した。注入口と排出口は、周縁部材の内側に蓋部材の長手方向に沿って配置させた。
スチール製の蓋部材の金型における蓋部材底面(段差部が形成されている面)に相当する面を、#3000ダイヤモンドペーストで磨き、金型の鏡面加工を実施した。
成形した蓋部材底面の水との接触角を、接触角測定器SA-20(協和界面科学社製)を用いて、JIS R 3257-1999に規定される静滴法に準じて測定した。実施例1の蓋部材底面の水との接触角は、85度であった。
また、成形した蓋部材の表面粗さを、接触式表面粗さ測定器(TALYSURF PGI1240、Taylor Hobson社製)を使用して測定した。本測定では、走査範囲800μmにおける、開始点(高さ0nmとする)に対する高低差を取得した。
表1に、上記蓋部材底面のRaおよびRzの測定結果を示す。
基板と蓋部材の間の流路に下記表2に示す組成のバッファを、200μL送液し、微小孔チップの各ウェルにバッファを充填した。
金型における蓋部材底面に相当する面に鏡面加工を行わない以外は、実施例1と同様にして、マイクロ流体デバイスを作製した。上記蓋部材底面のRaおよびRzの測定結果を表1に示す。比較例1の蓋部材底面の水との接触角は、85度であった。
図6Bに比較例1のマイクロ流体デバイスにおける、液滴形成後の微小孔チップの蛍光観察の結果を示す。図中の矢印の部分に代表的な試薬付着を示した。矢印部分以外にも、視野全体に、試薬付着が多数見られた。
図6Bに示したように、鏡面加工していない蓋部材を用いた比較例1のマイクロ流体デバイスは、蓋部材底面に試薬が非特異的に付着し、付着した箇所が原因で正しい液滴数が計測できなかった。
この結果から、蓋部材底面の水との接触角が同じであっても、Raが50nm以下であることにより、試薬の蓋部材底面への非特異的な吸着を低減させることができることが明らかとなった。
実施例1と同じ方法でCOP製の基材を製造した。蓋部材の金型における蓋部材底面に相当する面の鏡面加工の時間を長くした以外は、実施例1と同じ方法でCOP製の蓋部材を製造した。成形した蓋部材の表面粗さを、表面粗さ測定器(SJ-210、ミツトヨ製)を使用して測定した。本測定の走査範囲は、蓋部材の中心を走査範囲の中心とし、かつ蓋部材の長手方向と垂直な方向に1.5mmとし、開始点(高さ0nmとする)に対する高低差を取得した。表4に、実施例2の蓋部材底面のRaおよびRzの測定結果を示す。図7は、実施例2のマイクロ流体デバイスの蓋部材の表面粗さの測定データを示す図である。
実施例1と同じ条件でバッファ、蛍光試薬およびミネラルオイルをマイクロ流体デバイスに送液し、微小孔チップの各ウェルに蛍光試薬を個別に封止した。なお、本実施例でも、実施例1と同様に蛍光反応は行っていないが、蛍光反応を行った場合と同様の状態とするために、蛍光試薬に酵素を加えている。
蛍光画像全体の面積に対する、蛍光試薬が吸着している部分の面積を、吸着面積の比率(%)として求めた。吸着面積の比率が10%未満の場合を吸着の抑制が良好であると評価し、10%以上の場合を吸着の抑制が悪いと評価した。その結果を表4に示す。
蓋部材の金型における蓋部材底面に相当する面の鏡面加工の時間を短くした以外は、実施例2と同じ方法により、実施例3の試薬の吸着面積の比率を求めた。その結果を表4に示す。図8は、実施例3のマイクロ流体デバイスの蓋部材の表面粗さの測定データを示す図である。
蓋部材の金型における蓋部材底面に相当する面の鏡面加工を行わなかった以外は、実施例2と同じ方法により、比較例2および3の試薬の吸着面積の比率を求めた。その結果を表4に示す。図9および図10は、それぞれ比較例2および比較例3のマイクロ流体デバイスの蓋部材の表面粗さの測定データを示す図である。
吸着面積の比率が10%を超える場合、例えばマイクロ流体デバイスの全ウェル中に1個しか標的分子が入らないような低濃度のサンプルを測定しようとすると、10%以上の確率で試薬が吸着している部分と重なるウェルに標的分子が封入される。試薬が吸着している部分のウェルが酵素反応によって発光したとしても、その発光を検出することができず、陰性であると誤って判断(擬陰性)されてしまう。同じ測定を2回行ったとしても、1%以上の確率(100人に1人以上)で擬陰性になってしまう。
一方、吸着面積の比率が10%未満の場合、例えば実施例2および3のように吸着面積の比率が5%以下の場合、擬陰性は5%以下の確率となり、同じ測定を2回行うことで、偽陰性の確率を0.25%以下まで下げることができる。
10 基板
20 蓋部材
30 マイクロウェルアレイ
32 壁部層
33 マイクロウェル
100 水性液体
110 封止液
Claims (7)
- 複数のマイクロウェルを有するマイクロウェルアレイと、
前記マイクロウェルアレイと離間した状態で対向する蓋部材と、
を備え、
前記マイクロウェルアレイと前記蓋部材との間に流路を有し、
前記蓋部材のマイクロウェルアレイ側の面の算術平均粗さRaが70nm以下である、マイクロ流体デバイス。 - 前記蓋部材のマイクロウェルアレイ側の面の水との接触角が70度以上である、請求項1に記載のマイクロ流体デバイス。
- 前記蓋部材のマイクロウェルアレイ側の面の表面粗さRzが350nm以下である、請求項1または2に記載のマイクロ流体デバイス。
- 前記蓋部材のマイクロウェルアレイ側の面のRa/Rzが0.10以上0.24以下である、請求項1~3のいずれか一項に記載のマイクロ流体デバイス。
- 前記蓋部材のマイクロウェルアレイ側の面に疎水的なコーティングが施されている、請求項1~4のいずれか一項に記載のマイクロ流体デバイス。
- 請求項1~5のいずれか一項に記載のマイクロ流体デバイスを用いた試料分析方法であって、
試料を含有する水性液体を前記流路に供給することと、
前記流路に封止液を導入して前記流路内に存在する前記水性液体と置換し、前記マイクロウェルに前記水性液体を封入することと、
前記マイクロ流体デバイスを加熱して前記マイクロウェルにおいて反応を起こし、検出のためのシグナルを発生させることと、
前記シグナルを検出することと、
を備える試料分析方法。 - 前記試料が、DNA、RNA、タンパク質、脂質、細胞、または細菌であることを特徴とする請求項6に記載の試料分析方法。
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