WO2014021060A1 - 分析システム及び分析方法 - Google Patents
分析システム及び分析方法 Download PDFInfo
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- WO2014021060A1 WO2014021060A1 PCT/JP2013/068586 JP2013068586W WO2014021060A1 WO 2014021060 A1 WO2014021060 A1 WO 2014021060A1 JP 2013068586 W JP2013068586 W JP 2013068586W WO 2014021060 A1 WO2014021060 A1 WO 2014021060A1
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- analysis system
- microfluidic device
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- flow path
- pipe
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
- G01N35/08—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a stream of discrete samples flowing along a tube system, e.g. flow injection analysis
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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
- B01L3/502715—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 interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
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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
- B01L3/502769—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 multiphase flow arrangements
- B01L3/502784—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 multiphase flow arrangements specially adapted for droplet or plug flow, e.g. digital microfluidics
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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
- B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
- B01L2300/0874—Three dimensional network
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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
- B01L2300/0883—Serpentine channels
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
- G01N2035/00178—Special arrangements of analysers
- G01N2035/00237—Handling microquantities of analyte, e.g. microvalves, capillary networks
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
- G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
- G01N2035/1027—General features of the devices
- G01N2035/1034—Transferring microquantities of liquid
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- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
- H01J49/00—Particle spectrometers or separator tubes
- H01J49/02—Details
- H01J49/04—Arrangements for introducing or extracting samples to be analysed, e.g. vacuum locks; Arrangements for external adjustment of electron- or ion-optical components
- H01J49/0404—Capillaries used for transferring samples or ions
Definitions
- This invention relates to an analysis system and an analysis method for analyzing minute droplets.
- Non-Patent Document 1 discloses a technique using a mass spectrometer as a technique for detecting droplets generated in a microfluidic device.
- the generated droplets are carried in the flow path of the microfluidic device by a continuous phase of fluoro oil surrounding the droplets.
- this continuous phase Prior to the droplet components being analyzed by a mass spectrometer, this continuous phase is removed by an emulsion separation mechanism in the device. This is realized as follows. In the flow path of the emulsion separation mechanism, the flow of fluorine oil containing droplets flows adjacent to and parallel to the aqueous side flow.
- Electrodes are provided on both sides of the flow path, and when a voltage is applied, the droplets merge with the aqueous side stream, and the components of the droplets are extracted into the aqueous side stream.
- Non-Patent Document 1 in order to detect the components of the droplets with a mass spectrometer, it is necessary to separate the continuous phase carrying the droplets using an emulsion separation mechanism in the microfluidic device. It was. Such a mechanism complicates the structure of the microfluidic device and complicates the design, manufacturing, and operation processes. Furthermore, in this technique, the components are diluted to extract the components of the droplets into an aqueous side stream. If the flow path after the emulsion separation mechanism or the fused silica capillary is too long, the components of the droplets diffuse in the aqueous side stream, and the information may further diminish and become difficult to detect.
- the problem to be solved by the present invention is to deliver the droplets in the microfluidic device to the analyzer with a simple structure without separating the continuous phase.
- the system of the present invention is an analysis system including a microdevice having a microchannel and an analysis apparatus, and the microdevice has a first inlet and a second inlet, and these inlets The fluid channels injected from the respective inlets are discharged to the analyzer.
- the liquid is injected from the first inlet and the fluid from the second inlet is alternately arranged, that is, by dividing one fluid by the other fluid, Can be orderly removed from the microfluidic device.
- This allows the droplets to be delivered to the analyzer with a simple structure and method and analyzed.
- the system of the present invention includes one or more microfluidic devices, one or more pipes, and one or more analyzers. Further, the system of the present invention includes one or more connection units.
- the “microfluidic device” includes a flow path and has at least one of functions of generating, holding, and transporting droplets.
- the “analyzer” of the present invention provides a function of analyzing the characteristics of a droplet, is constituted by a structure and software, and does not necessarily take the form of an independent device.
- the “pipe” of the present invention has a fluid path inside and provides a function of transporting droplets from the microfluidic device to the analyzer.
- the “connecting portion” of the present invention fluidly communicates the flow path of the microfluidic device and the piping, and can flow droplets in an orderly manner.
- FIG. 1 is a schematic diagram of an embodiment of the analysis system of the present invention.
- the analysis system includes a microfluidic device 101, a pipe 102, an analysis apparatus 103, and a connection unit 104.
- the microfluidic device 101 includes a flow path 105 and has at least one of a function for generating, holding, or transporting the droplet 106.
- the analysis device 103 provides a function of analyzing the characteristics of the droplet, and is constituted by a structure and software, and does not necessarily have to be in the form of an independent device.
- the pipe 102 has a fluid path inside, and transports the droplet 106 from the microfluidic device 101 to the analyzer 103.
- the connection unit 104 is used for fluid communication between the flow path 105 of the microfluidic device 101 and the pipe 102, and can flow the droplets 106 in an orderly manner.
- each part will be described in detail.
- the microfluidic device 101 may be a so-called microfluidic device, a microfluidic chip, a microchip, or a so-called MEMS (Micro Electro Mechanical Systems) device, and is typically a plate-shaped chip.
- the thickness is in the range of about 1 ⁇ m to about 50 mm, and one side (width, depth, diameter) is about 10 ⁇ m to about 500 mm.
- the microfluidic device 101 includes a flow path, for example, a micro flow path.
- the microchannel has a part of the dimension of the channel, for example, a part of the cross-sectional dimension, for example, the channel width or diameter, at least 1 mm or less, preferably 500 ⁇ m or less, or 300 ⁇ m or less, or 100 ⁇ m or less, or 100 ⁇ m or less, Alternatively, the flow path is 10 ⁇ m or less, or 1 ⁇ m or less.
- the microfluidic device 101 includes an inlet and an outlet for injecting and / or discharging a fluid handled inside the device.
- the inlet and the outlet are referred to for convenience on the basis of their main applications, but it is generally possible to discharge the fluid from the inlet or the fluid from the outlet.
- the inlet and the outlet are openings opened to the outside of the microfluidic device provided at the end or in the middle of the flow path.
- the microfluidic device 101 can be manufactured using a so-called microfluidic device, a microfluidic chip, a microchip, or a manufacturing method of a MEMS (Micro Electro Mechanical Systems) device.
- a MEMS Micro Electro Mechanical Systems
- the components of the microfluidic device 101 can be formed of a solid material, in which case the flow path is formed by lithography technology such as photolithography or electron beam lithography, imprint technology such as nanoimprint, spin Coating, chemical vapor deposition, physical vapor deposition, film formation technology such as sputtering, various wet etching using hydrofluoric acid or potassium hydroxide, reactive ion etching, various dry etching such as Bosch process, ion milling, etc.
- lithography technology such as photolithography or electron beam lithography
- imprint technology such as nanoimprint
- spin Coating chemical vapor deposition
- physical vapor deposition film formation technology
- film formation technology such as sputtering
- the constituent elements of the microfluidic device 101 include various materials, for example, semiconductors such as silicon, glass, metals, polymer materials (natural polymer materials such as paper, thermoplastic resins, thermosetting resins, elastomers, and more specifically. Can be formed using silicone resin, various Teflon (registered trademark), acrylic, polycarbonate, polystyrene, and the like. Different components of the microfluidic device may be formed from different materials, and structures that form the channel 105, for example, structures corresponding to the walls, bottom, and ceiling of the channel 105 are also formed from a plurality of materials. May be.
- the flow channel 105 can be formed as a groove-like structure on the surface of a solid member that is a component of the microfluidic device 101, with most or part of the flow channel 105 being a component.
- a groove is formed in advance in the joining interface of one or more members, so that each member is surrounded on all sides.
- the flow path 105 can be formed.
- most or part of the flow path 105 can be arranged on a plane parallel to the largest plane of the microfluidic device 101.
- the microfluidic device 101 is formed using a flat plate member, for example, a silicon and glass wafer.
- the channel 105 can be formed as a groove on one or more surfaces of the wafer.
- known techniques such as etching, film formation, and lithography can be easily used, and reliability, cost, types of techniques that can be used, etc. You can enjoy many advantages in terms.
- One of the preferable flow paths is formed as a groove having a depth and a width of 100 ⁇ m on the silicon wafer surface by deep reactive ion etching, for example.
- the surfaces having grooves can be bonded by using anodic bonding.
- the microfluidic device 101 having the flow path 105 inside can be obtained.
- the inlet and outlet can be formed as openings such as through holes in contact with the flow path in the silicon wafer, the glass wafer, or both prior to bonding. Or you may form after joining and dicing.
- various etching methods, powder blasting, cutting and other processing methods may be used.
- the inner surface of the flow path 105 may be coated or covered with a different material.
- coatings or coating materials include physicochemical properties (wetting or affinity or water repellency with various liquids), chemical properties (reactive, non-reactive, passivating, catalytic ability), machinery That affect the physical characteristics (strength, elasticity, wear resistance, etc.) and optical characteristics (transparency, scattering intensity, and various wavelength characteristics affected by optical matching with surrounding members, surface roughness, etc.) It's okay.
- the pipe 102 provided in the analysis system may be a capillary, a tube, a pipe, a union, an adapter, a fitting, or the like.
- the material may be various solid materials, preferably fused quartz or PDMS (polydimethylsiloxane). Silicone resin containing, fluororesin containing various Teflon (registered trademark), PEEK (polyetheretherketone), PC (polycarbonate), other resin containing PS (polystyrene), metal containing stainless steel may be used.
- the pipe 102 may be coated or coated with a different material on the surface (inner surface) in contact with the internal space or on the outer surface.
- These coatings or coating materials include physicochemical properties (wetting or affinity or water repellency with various liquids), chemical properties (reactive, non-reactive, passivating, catalytic ability), machinery That affect the physical characteristics (strength, elasticity, wear resistance, etc.) and optical characteristics (transparency, scattering intensity, and various wavelength characteristics affected by optical matching with surrounding members, surface roughness, etc.) It's okay.
- one of the cross-sectional dimensions, for example, the inner diameter, of the internal space of the pipe 102 is in the range of about 0.1 ⁇ m to 1 mm, preferably 500 ⁇ m or less, or 300 ⁇ m or less, or 100 ⁇ m or less, or 50 ⁇ m or less, It is.
- a fused silica capillary (outer diameter: about 360 ⁇ m, inner diameter: about 50 ⁇ m to 100 ⁇ m) can be used.
- fused quartz is chemically stable, has sufficient physical strength for handling, and various surface modifications (coating) using chlorosilane agents and the like are possible.
- the above-described fused silica capillary may be coated with a film formed of perfluorooctyltrichlorosilane (1H, 1H, 2H, 2H, -Perfluorooctyl-trichlorosilane).
- the inner surface becomes water-repellent with respect to the aqueous solution, and nonspecific adsorption of various substances, particularly hydrophilic or lipophilic (solvent-soluble) substances, to the inner surface can be reduced.
- the outer surface may be coated with a polyimide film, thereby increasing the mechanical strength and facilitating handling.
- the fluid path is a passage of fluid, and is a space that can hold and / or transport the fluid.
- it includes a space inside a hollow tube and a flow path of a so-called microfluidic device.
- the member provided with a fluid path may provide a function as a fluid path alone, or may provide a function as a fluid path in cooperation with different members.
- a plate-like member having a groove on the surface is sandwiched between the groove and the smooth surface by being combined with another flat member having a smooth surface by pressing or joining each other.
- a fluid such as water can flow or be held in the space.
- the space is referred to as a fluid path
- the groove before combination is also referred to as a fluid path since it is a factor that determines the position of the path through which fluid flows during combination.
- the fluid path may have merging or branching.
- fluid communication means that when two or more structures having fluid paths such as flow paths and pipes are in contact with each other, those fluid paths are in contact with each other and pass through the respective structures. It means that the structure is in close contact with the fluid in such a way that the fluid can move between the structures in one direction or in multiple directions, and the fluid does not leak at that time. At this time, it can be considered that these fluid paths form one large fluid path.
- the droplets flow in an orderly manner means that the droplets or portions of the droplets do not merge with each other and do not break up, along with the fluid flowing around the droplets, along the fluid path. It means flowing.
- the droplets flow without disturbing the permutation intended by the system design or the user's operation, that is, without changing the order between the droplets, and generally at the intended timing.
- connection unit 104 that realizes fluid communication capable of orderly flowing the droplet 106 is provided.
- the connection unit 104 included in the analysis system is shown in FIG.
- the connection part 104 connects the fluid path 201 in the fused silica capillary 200 as the pipe 102 and the flow path 105 in the microfluidic device 101 in fluid communication.
- the microfluidic device 101 has a structure in which a silicon layer 209 and a glass layer 210 are joined, and a flow path 105 and a connection hole 204 are formed in the silicon layer 209 using reactive ion etching.
- the joint 203 is inserted into the hole 204, and the capillary 200 is inserted into the opening 211 on the piping side of the joint 203.
- the capillary 200 is held by a holding ferrule 202 made of fluoro rubber, and is fixed so that the opening 205 of the capillary is at an appropriate position.
- the joint 203 has a shape as shown in FIG. 3, and a groove 206 as a fluid path is provided on the flow path 105 side of the joint 203.
- the joint 203 is brought into close contact with the glass layer 210 of the microfluidic device 101 by the holding ferrule 203.
- the space formed by the groove 206 and the glass layer 210 functions as a fluid path
- the end of the groove 206 functions as the opening 208 on the flow path side.
- the opening 208 on the flow path side of the joint 203 is aligned so as to face and coincide with the opening 207 at the end of the flow path, and is also fixed by being pressed against the glass layer 210 of the microfluidic device 101 by the holding ferrule 202.
- the joint 203 is formed of an elastic material such as PDMS, and is pressed against the wall of the hole 204 of the glass layer 210 or the silicon layer 209 and the outer surface of the capillary 200 when pressed, so that the flow path 105 is formed.
- the flowing liquid will not leak.
- Fluid communication between the fluid path 201 and the flow path 105 in the capillary is realized. Fluorine oil flows in the flow path 105, and the droplet 106 flows toward the connecting portion 104 in the oil.
- the droplet 106 passes through the opening 207 of the flow path and the opening 208 on the flow path side of the joint 203, passes through the fluid path of the joint 203, passes through the pipe-side opening 211 of the joint 203 and the opening 205 of the capillary, and enters the capillary.
- the fluid path 201 Into the fluid path 201.
- the flow path 105 and the opening 205 of the capillary have substantially the same cross-sectional size, and the cross-sectional size of the fluid path in the joint 203 connecting them is almost the same, the fluid path Substantially forms one continuous fluid path and flows substantially laminarly as it does in channel 105 or pipe 102. For this reason, the droplet 106 flowing through the inside can flow in an orderly manner.
- the shortest straight line is referred to herein as an ideal fluid path.
- the size of the cross section being substantially the same means that either the cross sectional area or the length in the direction in which the cross sectional diameter is minimum is substantially equal. More preferably, both the cross-sectional area and the length in the direction in which the cross-sectional diameter is minimum are substantially equal.
- the ideal fluid path 413 is formed inside the connection hole 404.
- a large dead volume 412 is added to the fluid path.
- This additional dead volume 412 may cause irregularities in the shape of the fluid path or stagnation of the flow.
- the droplet 106 is sometimes trapped by the unevenness or flows into the stagnation of the dead volume 412 and stagnates. Then, it may merge with another droplet that flows next.
- the droplet 106 may break up by flowing into the capillary 200 while the remaining portion 106 remains stagnant.
- connection part 104 illustrated here can flow the droplet 106 orderly remarkably.
- the connection portion 104 included in the analysis system preferably includes at least a fluid communication opening 207 provided in one of the flow paths 105 of the microfluidic device 101, a fluid communication opening 205 provided in the pipe 102, and at least And a joint 203 having a fluid path having two openings 208 and 211 capable of fluid communication.
- the opening 207 in the flow path is in fluid communication with one of the openings in the joint, and the opening 205 in the piping is in fluid communication with a different one of the openings 208, 211 in the joint. This provides fluid communication that allows the droplets 106 to flow in an orderly manner.
- the joint 203 used for the connecting portion 104 has a plurality of openings.
- One example of a preferred joint is that the face with one opening and at least another different face with an opening are not parallel. Thereby, even when the directions of the flow path 105 and the pipe 102 of the microfluidic device 101 are different, desirable fluid communication can be realized.
- the “surface” is one continuous surface, and includes a flat surface or a curved surface. Therefore, being parallel to the surface having the opening means being substantially parallel to the plane in contact with the opening portion.
- the two openings 208 and 211 of the joint 203 are substantially perpendicular to each other.
- the connection portion 104 can be easily made by using a typical microfluidic device manufacturing method. For example, when a channel 204 and a hole 204 for a connecting portion are formed on a flat substrate (for example, a silicon or glass wafer) using reactive ion etching, the hole 204 is formed between the substrate and the channel as shown in FIG. It is formed perpendicular to.
- the shape of the connection hole 204 may be, for example, a cylindrical shape as shown in FIG. 2 or a shape having corners such as a polygon as shown in FIG. 5a.
- the joint 503a does not rotate freely like a circle, it becomes easy to match the opening direction of the joint 503a with the opening of the flow path 507a.
- an asymmetric polygon as shown in FIG. since the joint 503b only fits in the connection hole 504b, the orientation is further facilitated.
- the shape of the connection holes 504a and 504b may be formed in accordance with the joints 503a and 503b.
- the joint used for the connecting portion 104 forms a non-linear fluid path in the structure. More preferably, the fluid path provided in the joint is bent substantially vertically once in the middle thereof. Thereby, even when the directions of the flow path 105 and the pipe 102 of the microfluidic device 101 are different, desirable fluid communication can be realized. Examples of such joints and connections 104 are illustrated in FIGS.
- the fluid path of the joint 203 is in contact with another structure (here, the glass layer 210 positioned on the bottom surface of the connection hole 204 of the microfluidic device 101), thereby forming a fluid path.
- the fluid path of the joint 603 may be formed only by the structure of the joint 603.
- the shape of the connection hole 204 may be formed in accordance with the joint 603. For example, by digging down the bottom surface of the hole 204, the opening 608 on the side of the joint 603 in contact with the microfluidic device 101 can be aligned with the opening 207 of the flow path of the microfluidic device 101.
- a preferred example of fluid communication between the joint opening 208 and the opening 207 of the microfluidic device 101 is easily and reversibly removable.
- liquid-tightness is achieved by self-adsorption or pressing of surfaces with moderate elasticity at least one of the surfaces, or surfaces that have a sufficiently smooth surface and complementary mating or flat surfaces
- Such pressing may be performed by a screw, a spring, a fluid pressure such as hydraulic pressure or pneumatic pressure, or the like.
- a preferable example of fluid communication between the opening 208 of the joint and the opening 207 of the microfluidic device 101 is realized by pressing with a screw or a spring without using an adhesive. Since no adhesive is used, there is an advantage that there is no concern about the melting of the adhesive component due to the fluid flowing through the fluid path coming into contact with the adhesive.
- Such pressing necessary for detachable fluid communication can be realized by a structure using a holder shown in FIG. 7, for example.
- the holder consists of a holder upper part 714 and a holder lower part 715, both of which are sandwiched by screws 717.
- the holding ferrule 202 is pressed down.
- the holding ferrule 202 is deformed by being pressed against the silicon layer 209 of the microfluidic device 101 from above with a nut 716, and is brought into close contact with the capillary 200 and the joint 203 to fix them.
- the fluid communication opening provided in the flow path of the microfluidic device 101 can be provided at one end or in the middle of the flow path.
- the fluid communication opening provided in the pipe 102 can be provided at one end of the pipe 102.
- the connecting portion 104 can be provided in the middle of a series of flow paths that continue from the flow path 805 to the flow path 818.
- the fluid may flow from both the flow path 805 and the flow path 818 and be discharged to the capillary 200.
- a part of the fluid flowing from the flow path 805 may be discharged to the capillary 200 and the rest may flow to the flow path 818.
- any of the above operations may be switched each time by providing an adjusting mechanism such as a valve in the middle of one of the capillary 200 and the flow paths 805 and 818. It is clear from the description so far that the connection unit 104 of FIG. 8 can function in this way. With such a structure, a confluence is provided in the flow path 105 through which the droplet 106 flows, so that a plurality of flow paths 805 and 818 are used in parallel to increase the throughput or process under a plurality of conditions at the same time. It becomes possible.
- FIG. 9 An example of another preferred joint structure is shown in FIG.
- the basic structure is the same as that shown in FIG. 2, but in this example, the additional dead volume slightly existing under the opening of the capillary 200 in FIG. 2 is filled with the structure of the joint 903.
- the tip 221 of the capillary is held in a state where it is pressed against a part of the step 922 of the joint, thereby realizing fluid communication.
- the fluid path of the joint 903 has almost no dead volume, and substantially realizes the ideal fluid path in FIG. 2, thereby contributing to more reliable flow of the droplets 106.
- the following items may be used as a mode of fluid communication between two structures each having a fluid path having an opening in the connecting portion 104.
- a surface having an opening having a structure having an opening is in liquid-tight contact with a surface having an opening having another structure having an opening at least part of each surface.
- the surface including the opening and the combined surface of one or more surfaces surrounding the surface are in liquid-tight contact with the surface having the opening of the other structure including the opening in at least a part of each surface. .
- Any of the above can be used to realize fluid communication.
- the modes (1) and (2) may be mixed and used. For example, in the structure shown in FIGS.
- the fluid communication between the flow path 105 of the microfluidic device 101 and the joint 903 is in the manner of (1), and the fluid communication between the joint 903 and the capillary 200 is performed. Is realized by the form (2). Moreover, in the connection part 104 shown in FIG. 11, both fluid communication is implement
- the structure of the joint 1103 used in FIG. 11 is almost the same as the joint 203 in FIG. 3 as shown in FIG. However, the diameter of the opening 1111 on the pipe side corresponds to the outer diameter of the pipe 102 in FIG. 3, whereas in the example of FIG. 12, it is made to correspond to the inner diameter of the pipe 102 or the diameter of the opening 205 of the pipe. Yes.
- the mode (1) is simple because the surfaces having two openings need only be in liquid-tight contact with each other.
- the connecting portion 104 in FIGS. 9 and 11 forms a fluid path having substantially the same shape.
- the joint 903 in FIG. The joint 1103 has a simpler structure.
- the mode (2) has an advantage that positioning for facing the openings becomes easy. For example, in FIG. 11, in order to make the capillaries and joint openings 205 and 1111 coincide with each other, it is necessary to accurately align the capillaries 200. For example, the microfluidic device 101 is observed with a microscope from the transparent glass layer 210 side.
- FIG. 1 An example of another joint in which the fluid communication between the channel 105 and the joint of the microfluidic device 101 is realized in the manner (1) and the fluid communication between the joint and the capillary 200 is realized in the manner (2). Is shown in FIG. As in the connection part 104 of FIG. 9, in order to realize a fluid path almost free of dead volume, it contributes to the flow of the droplets 106 more reliably and orderly. At the same time, the joint 1303 and the opening 205 of the capillary 200 are easily positioned to face each other.
- connection hole 1404 is formed according to the outer diameter of the capillary 200, and the size of the joint 1403 is also formed according to this. Since the outer surface of the capillary 200 is in contact with the wall surface of the hole 1404, the position of the capillary 200 is guided by the wall surface of the hole 1404. This facilitates the alignment of the opening 205 of the capillary 200 and the opening 1411 of the joint 1403. Further, as in the example of FIG. 11, in order to realize a fluid path having almost no dead volume, it contributes to the flow of the droplets 106 more reliably and orderly.
- the two openings connected in fluid communication in the above-described manner have substantially the same cross-sectional area.
- the channel opening 207 and the joint opening 208 of the microfluidic device 101 have approximately the same cross-sectional area.
- a fluid especially a fluid that is hardly compressed, such as water
- the flow rate is constant between points in the fluid path. Therefore, the flow velocity at each point is generally inversely proportional to the cross-sectional area of the fluid path. Therefore, a constant cross-sectional area leads to a constant flow velocity. If the flow velocity is too small, the droplet 106 may stagnate as described above.
- the average flow velocity is set to the representative speed, and the minimum value of the diameter of the fluid path cross section (thickness for a flat flow path) is selected. It can be said that if the Reynolds number is large, it becomes turbulent, and if it is small, it tends to be laminar.
- the joint material may be various solid materials, preferably fused quartz, silicone resin containing PDMS (polydimethylsiloxane), fluororesin containing various Teflon (registered trademark), PEEK (polyetheretherketone), PC (polycarbonate). ), Other resins including PS (polystyrene), and metals including stainless steel may be used. More preferably, a material having an appropriate elasticity and rigidity that is convenient for forming a liquid-tight contact surface with another structure can be used. As such a material, various resin materials can be used. In addition, the joint may be coated or coated with a different material on the surface (inner surface) in contact with the internal space or the outer surface.
- coatings or coating materials include physicochemical properties (wetting or affinity or water repellency with various liquids), chemical properties (reactive, non-reactive, passivating, catalytic ability), machinery That affect the mechanical properties (strength, elasticity, wear resistance, etc.) may be used.
- physicochemical properties wetting or affinity or water repellency with various liquids
- chemical properties reactive, non-reactive, passivating, catalytic ability
- machinery That affect the mechanical properties may be used.
- the wettability with the fluid to be used is matched with the wettability between the fluid path wall surface in the flow path 105 or the pipe 102 and the fluid, the orderly flow of the liquid droplets 106 is not hindered. Can help.
- the joints as described above may use various known processing methods.
- the joint 203 shown in FIG. 3 can be formed by a soft lithography method using PDMS. Specifically, a first layer of negative photoresist SU-8 is spin-coated on a silicon wafer, and a pattern corresponding to the groove 206 forming the fluid path of the joint 203 is exposed and cured. Subsequently, a second layer of SU-8 is coated, and a doughnut-shaped pattern is masked and exposed to form a structure that defines the outer periphery of the pipe-side opening 211 and the joint 203.
- the thickness of the groove 206 forming the fluid path of the joint 203 can be controlled by the thickness of the first layer, and the thickness of the entire joint 203 can be controlled by the sum of the first layer and the second layer and the last cutting step.
- the outer diameter of the joint 203, the size of the opening 211 of the joint, and the width of the groove 206 forming the fluid path can be controlled by the pattern of the photomask used for lithography.
- it can be manufactured using various processing methods such as injection molding, cutting, and 3D printing.
- connection unit 104 An example of the connection unit 104 included in the analysis system is shown in FIG.
- the connecting portion 104 connects the fluid path 201 in the fused silica capillary 200 as the pipe 102 and the flow path 1605 in the microfluidic device 1601 in fluid communication.
- the end portion 1603 of the flow channel 1605 is perpendicular to the surface of the microfluidic device 1601, and the opening 1607 of the flow channel is located on the surface of the microfluidic device 1601.
- the surface around the opening 1607 of the flow channel 1605 and the surface around the opening 205 of the capillary, that is, the end surface 221 of the capillary are sufficiently smooth, the openings 1607 and 205 are in contact with each other, and the holding ferrule 202 or the like is used.
- the smoothness of the surface around the opening 1607 of the flow path can be realized by an ordinary known technique such as a combination of a silicon wafer and etching, and the smoothness of the end face 205 of the capillary can also be realized by a known technique using a diamond cutter or the like.
- the structure shown in FIG. 15 can be fixed and held using a holder or the like already shown in FIG. Even with this configuration, by realizing fluid communication with almost no dead volume, the droplet 106 can flow more orderly.
- connection unit 104 is shown in FIGS. 16 and 17.
- the configuration is almost the same as that of FIG. 15, the microfluidic devices 1701 and 1801 are provided with connection holes 1704 and 1804, respectively.
- the capillary 200 is inserted into the hole 1704 together with the holding ferrule 1702.
- the end face 221 of the capillary and the bottom face of the hole are sufficiently smooth, and fluid communication is realized by contacting each other.
- the shape of the holding ferrule 1702 is formed so as to fit in the hole 1704, and facilitates alignment of the capillary opening 205 and the flow path opening 1707.
- connection hole 1804 is formed in accordance with the outer diameter of the capillary 200, and only the capillary 200 is inserted into the hole 1804. This also facilitates alignment of the capillary opening 205 and the flow path opening 1807. 16 and FIG. 17 also enables fluid droplets 106 to flow more orderly by realizing fluid communication with almost no dead volume.
- connection unit 104 An example of the connection unit 104 included in the analysis system is shown in FIG.
- the connecting portion 104 connects the fluid path 201 in the fused silica capillary 200 as the pipe 102 and the flow path 1905 in the microfluidic device 1901 in fluid communication.
- the tip of the channel 1905 is formed as a groove 1919 in the glass layer 1910, and the channel opening 1907 is located on the bottom surface of the connection hole 1904.
- the connection hole 1904 is formed as a through hole in the silicon layer 1909. Such a through hole can be formed by, for example, deep reactive ion etching before the glass layer and the silicon layer are bonded.
- the capillary 200 is inserted into the hole 1904 together with the holding ferrule 1902, fixed by the holding ferrule 1902, and pressed against the bottom surface of the hole 1904. By this pressing, the holding ferrule 1902 and the capillary 200 are in liquid-tight contact with the glass layer 1910 forming the bottom surface of the hole 1904.
- the tip of the channel 1905 functions as a fluid path by combining the groove 1919 in the glass layer with the holding ferrule 1902 and the end face 221 of the capillary. With this configuration, the connection unit 104 can achieve fluid communication with almost no dead volume, thereby allowing the droplets 106 to flow more orderly.
- connection unit 104 is shown in FIGS. 19 and 20.
- the holding ferrule 1902 in the connection portion of FIG. 18 is replaced with a combination of the holding ferrule 2002 and the grooved ferrule 2003.
- the fluid path at the tip of the flow path 1905 is formed by a combination of the groove 1919 in the glass layer, the grooved ferrule 2003 and the end face 221 of the capillary.
- the groove 2006 of the grooved ferrule is arranged so as to face the groove 1919 of the glass layer.
- the connecting portion 104 can achieve fluid communication with almost no dead volume, thereby allowing the droplets 106 to flow more orderly.
- the analysis system can be used to control and execute a target reaction in a solution containing a sample and measure the result of the reaction for various types of analysis of the sample.
- the analysis system can be used for scientific analysis (enzyme reaction kinetics, measurement of DNA sequence and number of DNA, etc.), clinical analysis, synthesis, monitoring for production, and the like.
- the analysis system as its function, (1) generates a minute droplet 106 (reaction droplet) that causes a target reaction in the microfluidic device 101, and (2) uses the droplet 106 as a reaction vessel. (3) The droplet 106 is taken out of the microfluidic device 101 and (4) the characteristics of the droplet 106 are measured to provide a means for analyzing the result of the target reaction.
- reaction droplet The reaction includes, for example, a chemical, physical or biological reaction.
- the reaction can be started by adding, applying, and mixing reaction elements, for example.
- the reaction element may be a main element that causes a reaction, for example, a substance such as an enzyme, a substrate, an antibody, an antigen, etc., or a small individual or animal or plant cell or group of cells, tissue piece, bacteria, fungus, virus, etc. Or a biological sample.
- the reaction element also includes a reaction sub-element.
- the sub-elements are substances that promote, inhibit, assist, or “start” the reaction, and prevent the deactivation due to aggregation, coagulation, precipitation, modification, adsorption, etc. of the reaction element, and the environment that affects the reaction.
- Substances that can be provided such as catalysts, promoters, agonists, inhibitors, antagonists, pH buffers, redox agents, various metal ions and salts in general, surfactants, anti-denaturing agents, various high molecular or low molecular drugs, pharmaceuticals And drug candidates and precursors, media, inducers, etc.
- sub-elements may be changes in physical and chemical quantities such as temperature, pressure, velocity, light (electromagnetic wave) reaction, sound wave, electric field, magnetic field, pH, etc. It is only necessary to be able to control the start of. Further, it may be brought into contact with a solid phase such as a catalyst. Moreover, these combinations may be sufficient and reaction may be started by the free combination of said main element and subelement.
- reaction elements for example, in a PCR reaction using a hot start enzyme (AmpliTaq Gold (registered trademark) DNA Polymerase), an enzyme, primer DNA, template DNA, and buffer (pH buffer) are included as reaction elements.
- the reaction may be started by mixing the enzyme after mixing, or may be started by incubating at a suitable temperature (for example, 95 ° C. for 5 minutes) after mixing all of the enzyme. Good. This method of adjusting the temperature appropriately is known to those skilled in the art.
- reaction can be terminated by a reaction termination step.
- reaction ending step treatment for changing or removing reaction elements (main elements, subelements), substance addition, and the like can be used. Further, it may be changes in physical and chemical quantities such as temperature, pressure, speed, light (electromagnetic wave) reaction, sound wave, electric field, magnetic field, pH, and the like, as long as the end of the reaction can be controlled by these changes.
- the completion of the reaction can be controlled by adding a droplet containing an inhibitor that can bind to the activation site of the enzyme and lose the activity of the enzyme to the reaction droplet.
- the pH of the reaction droplet is removed from the optimum pH of the enzyme, and further, the enzyme is denatured and deactivated.
- the end of the reaction can be controlled.
- a droplet having an enzyme as a reaction element at an optimum temperature of 36 ° C. is caused to flow along the flow path, the enzyme is denatured and deactivated by being held at 80 ° C. for 1 minute or longer. It is also possible to control the end of the reaction by keeping a part of the temperature at 100 ° C. and allowing the droplets to pass through the range over 1 minute or longer.
- reaction end step is from the start to the end of each treatment, from the start of the addition of the substance until the substance is mixed into the entire reaction droplet until substantially all the reaction is stopped, or each physical quantity It refers to the period from the start of changes such as the chemical amount to the stop of substantially all reactions.
- the droplet 106 may be generated by various methods known to those skilled in the art, and may be performed by a passive droplet generation method, an active droplet generation method, or a combination thereof.
- a passive droplet generation method a flow focus described in JP 2010-506136, or a T junction described in Lab ⁇ on a Chip (2006), 2006vol.6, pp.437-446 is used.
- an active droplet generation method the method described in Lab on a Chip2010 (2010), vol.10, pp.816-818 may be used.
- the opening / closing time of the valve provided inside or outside the fluidic device 101 and the pressure difference before and after the valve the volume of the minute droplet 106 can be controlled and discharged from various nozzles.
- Both droplet generation methods use at least two fluids that are immiscible with each other.
- immiscible fluid combinations include polar molecules such as water, liquids such as oils and ionic liquids, and these three groups can all be immiscible with each other.
- polar molecules such as water
- liquids such as oils and ionic liquids
- these three groups can all be immiscible with each other.
- organic oils ordinary organic molecules containing various types of hydrocarbons and fluorocarbons (fluorocarbons) may be immiscible with each other by selecting an appropriate combination.
- a gas immiscible with the liquid may be used. Either of these is used as either a continuous phase or a discontinuous phase.
- the continuous phase and the discontinuous phase are merged, and the discontinuous phase is divided into the continuous phase to generate droplets 106.
- the structure used for these merges is typically provided in the microfluidic device 101.
- a fluid that forms a continuous phase and a discontinuous phase is introduced from the inlets 11 and 12 of the microfluidic device 101.
- the introduction timing may be used in advance, for example, during analysis or in advance.
- two or more fluids may each be assigned a dedicated inlet.
- the microfluidic device 101 has a valve-like function, which is switched each time it is introduced, so that different fluids are stored in different reservoirs, and different immiscible fluids are sent out from the respective reservoirs at the time of use. But you can.
- the reaction elements in the droplet 106 are quickly mixed with the addition and become uniform in the droplet 106.
- the reaction can be started by adding and mixing the reaction elements, but the added reaction elements are uniformly mixed in the droplets 106, thereby reducing the reaction conditions in the droplets 106. This makes it possible to obtain a uniform measurement result regardless of the characteristics of any part in the droplet 106. This also minimizes the difference between the actual reaction time and various reaction conditions and the intended reaction time and reaction conditions.
- the above mixing may be based only on the properties of the minute droplets 106 themselves.
- the minute droplet 106 has an effect of promoting the mixing of the reaction elements. This is because the droplet 106 has a small spatial size as compared with a general reaction vessel, and thus mixing can be completed in a very short time even in the case of mixing only by diffusion.
- the spiral flow in the droplet 106 has an effect of promoting mixing.
- it can be passed through a meandering channel or a channel with irregularities on the wall. In this case, the vortex flow in the droplet 106 becomes more prominent and the liquid is stirred, so that the mixing can be completed in a shorter time.
- mixing may be performed in a state (continuous flow) before droplet generation.
- a flow path having a structure that promotes various types of mixing which is already known, may be used.
- the fluids may be mixed prior to the generation of the droplets, or may be mixed by fusing the droplets after the generation of a plurality of droplets including the reaction elements. Also, by combining these, some reaction elements are mixed first before the droplets are generated, and after the mixed liquid droplets and the remaining element droplets are generated, the droplets are fused and mixed. Also good. In this case, for example, a reaction element that has a relatively small diffusion coefficient and takes a long time to mix is mixed in advance, and a reaction element that has a relatively large diffusion coefficient and can be mixed immediately is mixed at the end. Time can be shortened.
- reaction time is defined by the time from the reaction start time to the reaction end time of each reaction droplet 106. By controlling these times, the reaction time can be controlled.
- the time when the reaction droplet 106 is generated or the time when the reaction is started may be considered as the reaction start time. More specifically, the time at which the reaction elements start to be mixed, the time at which mixing is completed, and the representative time between them may be considered as the reaction start time. Alternatively, the time when the reaction droplet 106 is generated may be used. Similarly, the start time or end time of the reaction end process, the time representative of the period of the process, or the analysis time may be used as the reaction end time.
- the analysis time is the time of measurement of the characteristics of the reaction droplet 106 necessary for analysis of the reaction droplet.
- the analysis time can be used as the reaction end time.
- the analysis end time as the reaction end time, a measurement start time, a measurement end time, or a time representative of a period from the start to the end may be used. Which of the above examples is adopted as the reaction start time and reaction end time may be selected in consideration of the reaction type, analysis type, purpose, etc. A correct time may be used.
- water which is a discontinuous phase
- oil which is a continuous phase
- An aqueous solution containing an enzyme and a buffer is injected from the first inlet
- an aqueous solution containing a substrate and a buffer is injected from the second inlet.
- a droplet 106 is generated at the T junction, and the substantial reaction start is completed in this example when the contents of the droplet 106 are completely mixed while the droplet 106 flows in the flow path. .
- the junction point and the T junction to be very short, the time from the junction to the generation of the droplet 106 can be made extremely short in comparison with the flow velocity, the size of the droplet, the reaction rate, and the like. Then, since the liquids are mixed faster after the droplet 106 is generated, as a result, from the start of the reaction (confluence) to the completion of the reaction start (completion of the mixing) can be regarded as substantially simultaneous. It is also possible to execute in a short time.
- any of the candidates for the reaction start time listed above will not be greatly affected.
- the reaction used is very fast, or when mixing in the droplet 106 does not proceed sufficiently fast due to the characteristics of the solution used, the volume of the droplet 106, the flow path 105, and the volume flow rate, the start of the reaction starts. Since the time difference from (confluence) to the completion of the reaction start (mixing completion) affects the reaction time, the time at which the reaction start is completed may be adopted. Alternatively, the time point when the reaction start is completed by about half may be used as an intermediate point between the start and the completion of the reaction start or a predicted value obtained by calculation when the mixing is completed by about half.
- reaction product as a time integral of the reaction rate, for example, by substituting the time change of the reaction rate during the start of the reaction, for example by substituting the time change of the degree of mixing by calculation based on experiments or theory. You can also. In this case, when it is assumed that the reaction rate is always equal to the reaction rate after completion of the reaction immediately after the start of the reaction, a virtual reaction start time that gives the same amount of reaction product as the actual reaction is obtained, and the reaction starts. It can also be used as time.
- the absorbance for the light having a specific wavelength of the droplet 106 flowing in the flow path 105 can be measured as an analysis without providing a reaction completion step.
- the reaction time is substantially equal to the movement time of the droplet 106 from the reaction start point to the analysis start point.
- the moving time of the droplet 106 can be obtained by calculation, experiment, or measurement of the actual moving time. In the case of calculation, for example, it can be estimated from the volume flow rate and the flow path volume.
- the volume flow rate can provide any volume flow rate. For example, by using a syringe and a syringe pump, a liquid can be poured into the microfluidic device 101 at an arbitrary constant volume flow rate.
- the channel volume may be calculated from a known size (length, inner diameter, height and width of the cross section, stroke volume, dead volume, etc.) of the channel 105, or may be experimentally measured. Experimentally, for example, the time taken for a fluid of a certain volume flow rate to pass through the volume may be measured by visual observation or an image taken with a camera, and may be calculated from these. After filling the volume, another second immiscible second liquid is injected, with the volume of the second liquid injected from the beginning of the second liquid passing through the volume to the end of the passage, It is good also as the said volume.
- the experimentally measured time may be used as the droplet moving time, and used to determine the reaction time.
- the moving time of the droplet 106 may be measured by measuring the time taken for a discontinuous phase having a certain volume flow rate to pass through the volume. More preferably, the continuous phase may be flowed at a volume flow rate actually used in the actually used flow channel 105 or an equivalent flow channel, and the moving time of the droplet 106 flowing through the continuous phase may be measured and used. .
- the wall wettability, the volume flow rate of the continuous phase and the discontinuous phase, and the like may be set over a value or a range of values that are actually used, and an experimentally measured moving time may be used. This is because it is known that these parameters may affect the moving speed of the droplet 106.
- the moving speed of the droplet 106 is such that the relative length l / w of the droplet 106 in the channel 105 (w is the flow rate).
- the width of the channel, l is the droplet length), the ratio of the viscosity between the continuous phase and the discontinuous phase, the number of capillaries Ca, and the like.
- the present invention provides means for transporting droplets 106 generated in microfluidic device 101 to analyzer 103.
- the microfluidic device 101 is connected to the pipe 102 which has already been described through the connection unit 104 which has already been described.
- the droplet 106 is transported from the microfluidic device 101 to the analyzer 103 via the connection unit 104 and the pipe 102.
- the flow path 105 of the microfluidic device 101 is connected in fluid communication with a fluid path in the pipe 102, and both form an integrated fluid path.
- the droplet 106 is a continuous stream of oil or the like flowing through this integrated fluid path. Flowing through the phases.
- the transportation route may be a single road, or may be branched or merged. If there is a branch or merge, programmed control or a stochastic control method can be used.
- the analysis includes measuring various characteristic (s) of the droplet 106. It also includes obtaining a set of absolute values and relative values of a plurality of features by measurement.
- Non-limiting examples of the characteristics of the droplet 106 include fluorescence, absorption, spectrum (eg, absorption or emission in optical, visible light, infrared light, ultraviolet light, terahertz wave, etc., various scattering, resonance spectrum). , Or nuclear magnetic resonance spectrum, etc.), radioactivity, mass, volume, density, temperature, viscosity, electromagnetic properties (conductivity, dielectric constant, permeability), pH, chemical and biological substances (eg protein Concentration of substances such as nucleic acids, etc.). Further, when measuring each feature, the feature of one droplet 106 may be measured once. After measuring a plurality of times, a representative value such as an average value is calculated, or the feature in the droplet 106 is measured. The distribution may be evaluated.
- fluorescence eg, absorption or emission in optical, visible light, infrared light, ultraviolet light, terahertz wave, etc., various scattering, resonance spectrum). , Or nuclear magnetic resonance spectrum, etc.
- radioactivity mass, volume, density, temperature,
- photometers and spectrometers such as atomic absorption, absorptiometer, and fluorometer, mass spectrometer (MS), NMR (nuclear magnetic resonance apparatus), emission spectroscopic analysis (ICP), HPLC, various types A microscope or the like may be used.
- the continuous phase separating the droplets 106 shows a value different from the characteristic value of the droplets, especially when the feature is hardly detected
- the continuous phase temporally measures the measured values of the characteristics indicated by the plurality of droplets. Acts as a spacer that divides into two. Specifically, if the characteristic values indicated by the plurality of liquid droplets 106 are plotted against time, a shape such as a peak or a pulse is shown. Thus, each droplet 106 can be easily associated with the measured feature value.
- the flow may include a laminar flow and a turbulent flow in part or all of the flow, and includes an electroosmotic flow, a pressure driven flow, and the like.
- the pressure-driven flow may be driven by a syringe and a syringe pump, or may be driven by a pressure source configured by an air cylinder or a combination of a pump and a valve.
- oils and oils in general for example, vegetable oil, mineral oil, hydrocarbon (linear, aromatic), fluorine oil (fluorocarbon, etc.) can be used, More preferably, among fluoro oils, perfluorodecalin and Fluorinert (registered trademark) (such as FC-40 and FC-3283, 3M) may be used. Alternatively, a mixture of these various oils and a surfactant may be used.
- the surfactant various surfactants can be used according to the constituent materials of the droplets such as the oil, the analysis object, and the solvent.
- Nonionic surfactants such as Tween 20 and NP-40, and fluorine-based surfactants such as 1H, 1H, 2H, 2H-Perfluoro-1-octanol, and EA Surfactant (Randance) may be used.
- water includes pure water and various aqueous solutions containing water as a component.
- the continuous phase is a fluid phase that occupies a substantially continuous space that occupies most of the flow path.
- a droplet is a mass of a fluid having a substantially constant mass surrounded by a structure such as a wall constituting a flow path or a continuous phase.
- the shape may be spherical, elliptical, bullet-shaped, cylindrical, or the like.
- the cross-sectional area of the flow path is small with respect to the volume of the droplet, it can be deformed into an arbitrary shape according to the shape of the flow path.
- the discontinuous phase is a phase of a fluid constituting a droplet.
- droplet generation means that a fluid flowing in a substantially spatially continuous state from an inlet of a discontinuous phase is separated by a continuous phase or a wall of a flow path and is substantially independent. Say to produce a lump with a constant mass.
- the continuous flow means a fluid flow that does not contain droplets and uniformly contains only a continuous phase or a discontinuous phase, or only oil or water. It refers to the flow of fluid that contains it.
- the present invention provides a system for preparing a reaction droplet containing an enzyme, controlling the substrate concentration in the droplet and the reaction time, and measuring the concentration of the substrate and reaction product in the droplet. Furthermore, the present invention provides a method for analyzing enzyme reaction kinetics in a droplet.
- a buffer (NH 4 HCO 3 , pH 8) was used as a solvent for the enzyme and substrate, and as a diluent for adjusting the concentration. Since trypsin cleaves peptides at specific sites, the reaction product is a peptide having the amino acid sequence VYPNGAEDESAEAFPLEF in this system.
- An equivalent peptide (ACTH22-39) was purchased from Sigma-Aldrich.
- LeuEnk leucine enkephalin
- FIG. 21 shows an outline of the system of this embodiment.
- the microfluidic device 2201 includes a microchannel 2205 formed as a groove by deep etching in a silicon wafer, and inlets 2211 to 2214 and outlet 2215 formed as through holes.
- a silicon wafer was anodically bonded to a glass wafer and then diced to obtain a microfluidic device.
- Syringes 2221 to 2224 are connected to the injection ports 2211 to 2214 via capillaries 2225 to 2228, respectively.
- the syringe 2221 has a substrate solution of 784 ⁇ M ACTH18-39, 196 ⁇ M LeuEnk, a 22.5 mM NH 4 HCO 3 aqueous solution, the syringe 2222 has a diluted 22.5 mM NH 4 HCO 3 aqueous solution, and the syringe 2223 has an enzyme solution of .43 ⁇ M trypsin, 100 ⁇ M.
- the aqueous HCl solution and the syringe 2224 contain oil.
- a fused silica capillary 2202 is connected to the discharge port 2215 through a connection portion 2204 in fluid communication.
- the other end of the capillary 2202 is connected to a stainless steel capillary, which is an ion source 2230, via a union 2229 provided in a mass spectrometer 2233 (Waters, Synchron HDMS).
- the sample flowing into the ion source 2230 is ionized by electrospray to become ions 2231, which are introduced into the mass analyzer 2232, and the mass thereof is measured as m / z.
- the wall surface of the flow path 2205 of the microfluidic device 2201 and the inner surface of the capillary 2202 were coated with fluorine.
- FIG. 22 shows details of the microfluidic device 2201.
- the substrate solution and the diluted solution are respectively injected from the injection ports 2211 and 2122, and merged at the T junction 2216 and mixed.
- the mixed solution of the substrate solution and the diluent is merged with the enzyme solution flowing in from the inlet 2213 at the T junction 2217 to become a reaction mixed solution.
- the reaction mixture immediately thereafter merges with the oil flowing in from the inlet 2214 at the T junction 2218 and is divided by the oil to generate droplets (reaction droplets) 2219.
- the droplet 2219 flows through the flow path 2205, and regularly flows to the capillary 2202 via the connection portion 2204 at the discharge port 2215.
- the droplet 2219 is ionized by the ion source 2230 and analyzed by the mass analysis unit 2232.
- the composition at the time when the droplet 2219 is generated is controlled by the flow rate of each liquid. For example, if the flow rate ratio of the substrate solution, diluent, and enzyme solution is 4: 4: 1, the composition is 382 nM trypsin, 348 ⁇ M ACTH18-39, 174 ⁇ M LeuEnk, and 20 mM NH 4 HCO 3 .
- the reaction time is defined by the time from the generation of mixed droplets to ionization at the ion source 2230, and can be controlled by the total flow rate including oil.
- the reaction time was changed in the range of 2.6 to 8.6 minutes by changing the total flow rate in the range of 3 to 10 ⁇ L / min while maintaining the flow ratio of the reaction mixture and oil at 9:10. .
- the data obtained as described above is shown in FIG.
- the signal intensity corresponding to ACTH18-39 is indicated by a circle
- the signal intensity corresponding to LeuEnk is indicated by a triangle.
- Each signal shows a pulse shape because a signal is obtained only while the droplet 2219 flows into the ion source and is ionized, and no signal is obtained while the oil is flowing, and the oil acts as a spacer. is there.
- one pulse corresponds to one reaction droplet 2219, whereby the composition of each droplet 2219 can be analyzed.
- the average value of the intensity ratio of ACTH18-39 and LeuEnk is calculated, and the concentration of the substrate and reaction product can be obtained by comparing with the calibration curve.
- Inlet 1 12 Inlet 2 15 outlet 101 microfluidic device 102 piping 103 Analyzer 104 connections 105 flow path 106 droplets 200 capillaries 201 Fluid path in capillary 202 holding ferrule 203 Fitting 204 Connection hole 205 Capillary opening 206 Groove as fluid path 207 Flow path opening 208 Opening on the flow path side of the joint 209 Silicon layer 210 glass layer 211 Opening on the joint piping side 213 Ideal fluid path 221 End face of capillary 401 Fluid path in capillary 402 holding ferrule 404 connection hole 405 Capillary opening 407 Flow path opening 408 Joint opening on channel side 409 Silicon layer 409 glass layer 411 Joint piping side opening 412 Additional dead volume 413 Ideal fluid path 503a, 503b Fitting 504a, 504b Connection hole 505a, 505b Capillary opening 506a, 506b Groove as fluid path 507a, 507b Microfluidic device flow path 508a, 508b joint channel side opening 509a
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Abstract
Description
マイクロ流体デバイス101は、いわゆるマイクロ流体デバイス、マイクロ流体チップ、マイクロチップ、またはMEMS(メムス、Micro Electro Mechanical Systems)デバイスと呼ばれるものを用いてよく、典型的には平板形のチップ状であり、その場合、厚みが約1μmから約50mm、側方の一辺(幅、奥行、直径)が約10μmから約500mmの範囲である。
本明細書において流体経路とは、流体の通り道のことで、流体を保持または輸送またはその両方を可能とする空間である。例えば、中空な管の内部の空間や、いわゆるマイクロ流体デバイスの流路などを含む。また、流体経路を備える部材は、単独で流体経路としての機能を提供してもよく、異なる部材と協同して流体経路としての機能を提供してもよい。例えば、表面に溝を持つ平板状の部材は、もう1つの平滑な面を持つ平板状の部材と互いに押しつけや接合により組み合わされることで、前記の溝と前記の平滑な面とに挟まれた空間に水などの流体を流したり、保持したりすることができる。この時、前記の空間を流体経路と呼び、また、組み合わせ前の前記の溝も、組み合せに際して流体の流れる経路の位置を決定する要素であることから、流体経路と呼ぶ。また、流体経路は、合流や枝分かれを持ってよい。
本実施形態では、液滴106を整然と流すことが可能な流体連通を実現する接続部104を提供する。分析システムが備える接続部104は、図2に示される。ここで接続部104は、配管102としての溶融石英製のキャピラリ200中の流体経路201と、マイクロ流体デバイス101中の流路105を流体連通に接続する。
Re = ρVL/μ
で定義される無次元数で、ここでρ[kg/m3]は流体の密度、μ[N・s/m2]は流体の粘性係数、V[m/s]は流体の代表速さ、L[m]は流体経路の代表長さである。代表速さはや代表長さは、系を特徴づける値を選び、例えば平均流速を代表速さに、流体経路断面の径の最小値(扁平な流路なら厚み)を選ぶ。レイノルズ数が大きければ乱流に、小さければ層流になりやすいと言える。
分析システムが備える接続部104の一例は、図15に示される。ここで接続部104は、配管102としての溶融石英製のキャピラリ200中の流体経路201と、マイクロ流体デバイス1601中の流路1605を流体連通に接続する。流路1605の終端部1603はマイクロ流体デバイス1601の表面に垂直で、流路の開口1607はマイクロ流体デバイス1601の表面に位置する。流路1605の開口1607の周囲の面とキャピラリの開口205の周りの面、すなわちキャピラリの端面221は十分に平滑であり、両開口1607、205を互いに向き合わせて接し、保持フェラル202などを用いて固定することで、液密を保ち、流体連通を実現できる。流路の開口1607の周囲の面の平滑は、シリコンウエハとエッチングの組合せ等、通常の公知の技術で実現でき、キャピラリの端面205の平滑もダイヤモンドカッター等を用いた公知の技術で実現できる。また、図15に示した構造は、すでに図7に示したようなホルダ等を用いて固定、保持できる。この構成によってもデッドボリュームのほぼない流体連通を実現することにより、液滴106をより整然と流すことが可能となる。
分析システムが備える接続部104の一例は、図18に示される。ここで接続部104は、配管102としての溶融石英製のキャピラリ200中の流体経路201と、マイクロ流体デバイス1901中の流路1905を流体連通に接続する。流路1905の先端部は、ガラス層1910の中に溝1919として形成され、流路の開口1907は、接続用の孔1904の底面に位置する。接続用の孔1904は、シリコン層1909に貫通孔として形成されている。このような貫通孔はガラス層とシリコン層の接合前に、例えば深堀反応イオン性エッチング等で形成することができる。貫通孔であるため、孔1904の形成時に、孔の深さを制御しなくて済み、加工が容易になる利点がある。キャピラリ200は、保持フェラル1902と共に孔1904に挿入され、保持フェラル1902によって固定され、孔1904の底面に押しつけられる。この押しつけにより、保持フェラル1902とキャピラリ200は、孔1904の底面をなすガラス層1910と液密に接する。流路1905の先端部は、ガラス層中の溝1919と、保持フェラル1902およびキャピラリの端面221とが組み合わされることで、流体経路として機能している。この構成によって、接続部104はデッドボリュームのほぼない流体連通を実現することにより、液滴106をより整然と流すことが可能となる。
これ以降は、以上で説明した構造を用いた分析システムの機能と、その動作についてより詳細に説明する。
反応には、例えば、化学的、物理的または生物学的な反応を含む。
反応時間は、各々の反応液滴106の、反応開始時刻から、反応終了時刻までの時間で定義される。これらの時刻を制御することで、反応時間を制御することができる。
Ca = μV/γ
で定義される無次元数で、ここでμ[N・s/m2]は連続相の粘性係数、V[m/s]は不連続相の代表速さ、γ[N/m]は連続相と不連続相の間の表面張力である。以上に述べた方法により、任意の体積流量を与えた際の液滴106の移動時間を求めることができる。これに基づいて、移動時間を制御することで、反応時間を制御することができる。
本発明は、マイクロ流体デバイス101中で生成された液滴106を、分析装置103まで輸送する手段を提供する。マイクロ流体デバイス101は、すでに説明した接続部104を介して、同じくすでに説明した配管102と接続される。これにより、液滴106はマイクロ流体デバイス101から接続部104、配管102を経由して、分析装置103まで輸送される。マイクロ流体デバイス101の流路105は配管102内の流体経路と流体連通に接続され、両者は一体化した流体経路を形成する、液滴106は、この一体化した流体経路を流れるオイルなどの連続相の中を流れる。上で述べたように、連続相の流れを制御することで、液滴106の輸送速度や、分析装置103に到達するタイミングまたは分析されるタイミングを制御することができる。輸送の経路は、***でもよく、分岐、合流があってもよい。分岐や合流がある場合には、プログラムされた制御や、確率的な制御方法を用いることができる。
分析には、液滴106の各種特徴(1つ、または複数)を測定することを含む。また、測定により複数の特徴の絶対値や相対値の組を得ることを含む。
本明細書において、流れとは、その一部または全部に層流、乱流を含んでよく、電気浸透流、圧力駆動流(pressure driven flow)、などを含む。圧力駆動流は、シリンジとシリンジポンプによって駆動されてよく、また、空気ボンベやポンプとバルブの組合せなどで構成される圧力源によって駆動されてもよい。
12 注入口2
15 排出口
101 マイクロ流体デバイス
102 配管
103 分析装置
104 接続部
105 流路
106 液滴
200 キャピラリ
201 キャピラリ中の流体経路
202 保持フェラル
203 継ぎ手
204 接続用孔
205 キャピラリの開口
206 流体経路としての溝
207 流路の開口
208 継ぎ手の流路側の開口
209 シリコン層
210 ガラス層
211 継ぎ手配管側の開口
213 理想的な流体経路
221 キャピラリの端面
401 キャピラリ中の流体経路
402 保持フェラル
404 接続用孔
405 キャピラリの開口
407 流路の開口
408 継ぎ手の流路側の開口
409 シリコン層
409 ガラス層
411 継ぎ手配管側の開口
412 追加のデッドボリューム
413 理想的な流体経路
503a,503b 継ぎ手
504a,504b 接続用孔
505a,505b キャピラリの開口
506a,506b 流体経路としての溝
507a,507b マイクロ流体デバイスの流路
508a,508b継ぎ手の流路側の開口
509a,509b マイクロ流体デバイスのシリコン層
603 継ぎ手
606 継ぎ手の流体経路
608継ぎ手の流路側の開口
611 配管側の開口
714 ホルダ上部
715 ホルダ下部
716 ナット
717 ねじ
804 接続用孔
805 流路
806 流体経路としての溝
809 シリコン層
810 ガラス層
811 継ぎ手配管側の開口
818 流路
819 流体経路としての溝
903 継ぎ手
906 流体経路としての溝
908 継ぎ手の流路側の開口
911 継ぎ手配管側の開口
922 継ぎ手の段差部分
1103 継ぎ手
1106 流体経路としての溝
1108 継ぎ手の流路側の開口
1111 継ぎ手配管側の開口
1303 継ぎ手
1304 接続用孔
1306 流体経路としての溝
1308 継ぎ手の流路側の開口
1311 継ぎ手配管側の開口
1403 継ぎ手
1404 接続用孔
1406 流体経路としての溝
1408 継ぎ手の流路側の開口
1411 継ぎ手配管側の開口
1601 マイクロ流体デバイス
1603 流路の終端部
1605 流路
1607 流路の開口
1609 シリコン層
1610 ガラス層
1701 マイクロ流体デバイス
1702 保持フェラル
1703 流路の終端部
1704 接続用孔
1705 流路
1707 流路の開口
1709 シリコン層
1710 ガラス層
1801 マイクロ流体デバイス
1802 保持フェラル
1803 流路の終端部
1804 接続用孔
1805 流路
1807 流路の開口
1809 シリコン層
1810 ガラス層
1901 マイクロ流体デバイス
1902 保持フェラル
1904 接続用孔
1905 流路
1907 流路の開口
1909 シリコン層
1910 ガラス層
1919 流路の先端部となるガラス層の溝
2002 保持フェラル
2003 溝付きフェラル
2006 流体経路としての溝
2102 溝付き保持フェラル
2106 流体経路としての溝
2201 マイクロ流体デバイス
2202 キャピラリ
2204 接続部
2205 マイクロ流路
2211 注入口1
2212 注入口2
2213 注入口3
2214 注入口4
2215 排出口
2216 Tジャンクション1
2217 Tジャンクション2
2218 Tジャンクション3
2219 液滴
2221 シリンジ1
2222 シリンジ2
2223 シリンジ3
2224 シリンジ4
2225 キャピラリ1
2226 キャピラリ2
2227 キャピラリ3
2228 キャピラリ4
2229 ユニオン
2230 イオン源
2231 イオン
2232 質量分析部
2233 質量分析計
2234 ホルダ
Claims (42)
- マイクロ流路を有するマイクロ流体デバイスと、分析装置とを備えた分析システムであって、
マイクロ流体デバイスは、第1の注入口と第2の注入口を有し、これらの注入口からの流路は内部で合流し、それぞれの注入口ら注入された流体は、分析装置へ排出されることを特徴とする分析システム。 - 請求項1において、
マイクロ流体デバイスから排出した流体を分析装置へ送液する配管を有し、
マイクロ流体デバイスの排出口付近において配管の周囲を覆う第1の接続部材を有することを特徴とする分析システム。 - 請求項2において、
第1の接続部材は、中心に貫通孔を有しており、
貫通孔の内側に密着して配管が挿入されており、
第1の接続部材は、貫通孔と異なる方向に伸び、貫通孔と連通した切り欠き部を有し、切り欠き部と流路とが連通していることを特徴とする分析システム。 - 請求項2において、
第1の接続部材は、中心に貫通孔を有しており、
貫通孔の内側に密着して配管が挿入されており、
第1の接続部材は、貫通孔と異なる方向に伸び、貫通孔と連通すると共に第1の接続部材の側面まで繋がった開口部を有し、開口部と流路とが連通していることを特徴とする分析システム。 - 請求項2において、
配管とマイクロ流体デバイスの流路の下面との間には隙間を有することを特徴とする分析システム。 - 請求項2において、
配管をマイクロ流体デバイスに固定するフェラルを有する特徴とする分析システム。 - 請求項6において、
フェラルが第1の接続部材をマイクロ流体デバイスに押し付けていることを特徴とする分析システム。 - 請求項2において、
配管を保持するホルダ上部と、マイクロ流体デバイスを保持するホルダ下部と、両者を挟みつけるネジと、を備えていることを特徴とする分析システム。 - 請求項8において、
配管をマイクロ流体デバイスに固定するフェラルと、ホルダ上部をフェラルに押さえつけるナットと、を有することを特徴とする分析システム。 - 請求項3において、
切り欠き部を複数有し、それぞれは互いに離間していることを特徴とする分析システム。 - 請求項4において、
開口部を複数有し、それらは互いに離間していることを特徴とする分析システム。 - 請求項3において、
貫通孔は、段差部分を有しており、段差部分に配管の端面が位置することを特徴とする分析システム。 - 請求項1において、
マイクロ流体デバイスは、第1の層状部材と第2の層状部材が接合されて構成されており、
マイクロ流体デバイスから排出した流体を分析装置へ送液する配管を有し、
マイクロ流体デバイスの出口付近において該配管と第2の層状部材との間に位置し、流路から配管へ流体を送液する第2の接続部材を有し、
第2の接続部材における少なくとも配管と同方向の流路の内径は配管の内径と略同一であることを特徴とする分析システム。 - 請求項13において、
第2の接続部材の一部は、配管の外周まで延びていることを特徴とする分析システム。 - 請求項13において、
第2の接続部材の外径が配管の外径と同一であることを特徴とする分析システム。 - 請求項13において、
第2の接続部材は、中心に貫通孔を有しており、
貫通孔の内側に密着して配管が挿入されており、
第2の接続部材は、貫通孔と異なる方向に伸び、貫通孔と連通した切り欠き部を有し、切り欠き部と流路とが連通していることを特徴とする分析システム。 - 請求項13において、
第2の接続部材は、中心に貫通孔を有しており、
貫通孔の内側に密着して配管が挿入されており、
第2の接続部材は、貫通孔と異なる方向に伸び、貫通孔と連通すると共に第1の接続部材の側面まで繋がった開口部を有し、開口部と流路とが連通していることを特徴とする分析システム。 - 請求項2において、
マイクロ流体デバイスは、第1の層状部材と第2の層状部材が接合されて構成されていることを特徴とする分析システム。 - 請求項18において、
マイクロ流体デバイスを構成する2つの層状部材は、シリコン層とガラス層のいずれかであることを特徴とする分析システム。 - 請求項18において、
第1の層状部材には貫通孔が形成されていることを特徴とする分析システム。 - 請求項20において、
第1の接続部材が第2の層状部材に密着していることを特徴とする分析システム。 - 請求項1において、
マイクロ流体デバイスは、第1の層状部材と第2の層状部材が接合されて構成されており、
配管と繋がった内径と同一径の流路、および、この流路と連通した第2の層状部材上の流路を形成するように、第1の層状部材には断面L字型の流路が形成されていることを特徴とする分析システム。 - 請求項22において、
第1の層状部材は、配管との接合部分が配管の外径よりも大きく、切り欠かれていることを特徴とする分析システム。 - 請求項22において、
第1の層状部材は、配管との接合部分が配管の外径と略同一の内径を有するような切り欠きを有することを特徴とする分析システム。 - 請求項1において、
マイクロ流体デバイスから流出した流体を分析装置へ送液する配管を有し、
マイクロ流体デバイスは、第1の層状部材と第2の層状部材が接合されて構成されており、
第1の層状部材は、貫通孔を有しており、
貫通孔の内側に配管が挿入されており、
第2の層状部材には、配管付近に設けられた溝を有する
ことを特徴とする分析システム。 - 請求項1において、
マイクロ流体デバイスから流出した流体を分析装置へ送液する配管を有し、
マイクロ流体デバイスは、第1の層状部材と第2の層状部材が接合されて構成されており、
第1の層状部材は、貫通孔と流路を有しており、
貫通孔の内側に配管が挿入されており、
第2の層状部材には、配管付近に設けられた溝を有し、
溝は流路と連通していることを特徴とする分析システム。 - 請求項25において、
配管とマイクロ流体デバイスの間には、配管をマイクロ流体デバイスに固定するためのフェラルを備えており、該フェラルには溝が設けられていることを特徴とする分析システム。 - 請求項27において、
フェラルは2層構造になっていることを特徴とする分析システム。 - 請求項1において、
分析装置は、質量分析装置であることを特徴とする分析システム。 - 請求項3において、
貫通孔の貫通方向と、流路の方向が直交しており、
切り欠き部と流路とが、直線的に接続されていることを特徴とする分析システム。 - 請求項4において、
貫通孔の貫通方向と、流路の方向が直交しており、
開口部と流路とが、直線的に接続されていることを特徴とする分析システム。 - 請求項3または4において、
第1の接続部材は、弾性材料で形成される
ことを特徴とする分析システム。 - 請求項16または17において、
第2の接続部材は、弾性材料で形成されていることを特徴とする分析システム。 - 請求項32または33において、
弾性材料はPDMSまたはフッ素ゴムである
ことを特徴とする分析システム。 - 請求項3または4において、
第1の接続部材は、ソフトリソグラフィ法、射出成型、3Dプリンティングのいずれかによって作製されることを特徴とする分析システム。 - 請求項16または17において、
第2の接続部材は、ソフトリソグラフィ法、射出成型、3Dプリンティングのいずれかによって作製されることを特徴とする分析システム。 - 請求項3または4において、
第1の接続部材は、マイクロ流体デバイスから着脱可能であることを特徴とする分析システム。 - 請求項16または17において、
第2の接続部材は、マイクロ流体デバイスから着脱可能であることを特徴とする分析システム。 - 流路を有するマイクロ流体デバイスと、分析装置とを備えた分析システムを用いた分析方法であって、
マイクロ流体デバイスは、第1の注入口と第2の注入口を有し、これらの注入口からの流路は内部で合流し、第1の注入口から注入した第1の流体が、第2の注入口から注入した第2の流体により分断され、分断された状態でこれらの流体が分析装置へ排出されることを特徴とする分析方法。 - 請求項39において、
第2の流体がフッ素オイル、またはフッ素オイルとフッ素系界面活性剤の混合液であることを特徴とする分析方法。 - 請求項39において、
第1の流体が試料であり、第2の流体がスペーサであることを特徴とする分析方法。 - 請求項39において、
分析装置は、間欠的に導入されてくる第1の流体を検出することを特徴とする分析方法。
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