WO2009145022A1 - Micropointe comportant un microcanal - Google Patents

Micropointe comportant un microcanal Download PDF

Info

Publication number
WO2009145022A1
WO2009145022A1 PCT/JP2009/058069 JP2009058069W WO2009145022A1 WO 2009145022 A1 WO2009145022 A1 WO 2009145022A1 JP 2009058069 W JP2009058069 W JP 2009058069W WO 2009145022 A1 WO2009145022 A1 WO 2009145022A1
Authority
WO
WIPO (PCT)
Prior art keywords
microchip
channel
fine
flow path
liquid
Prior art date
Application number
PCT/JP2009/058069
Other languages
English (en)
Japanese (ja)
Inventor
洋一 青木
彰久 中島
康博 山東
楠 東野
直樹 日影
Original Assignee
コニカミノルタエムジー株式会社
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by コニカミノルタエムジー株式会社 filed Critical コニカミノルタエムジー株式会社
Priority to JP2010514419A priority Critical patent/JPWO2009145022A1/ja
Publication of WO2009145022A1 publication Critical patent/WO2009145022A1/fr

Links

Images

Classifications

    • 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
    • G01N35/1009—Characterised by arrangements for controlling the aspiration or dispense of liquids
    • G01N35/1016—Control of the volume dispensed or introduced
    • 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
    • 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
    • B01L3/502746—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the means for controlling flow resistance, e.g. flow controllers, baffles
    • 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
    • B01L2200/02—Adapting objects or devices to another
    • B01L2200/026—Fluid interfacing between devices or objects, e.g. connectors, inlet details
    • B01L2200/027—Fluid interfacing between devices or objects, e.g. connectors, inlet details for microfluidic devices
    • 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
    • B01L2200/02—Adapting objects or devices to another
    • B01L2200/028—Modular arrangements
    • 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
    • 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
    • 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
    • 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
    • B01L2300/18—Means for temperature control
    • B01L2300/1805—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
    • B01L2300/1822—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks using Peltier elements
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
    • B01L2400/0487—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/08—Regulating or influencing the flow resistance
    • B01L2400/084—Passive control of flow resistance
    • 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
    • G01N35/1009—Characterised by arrangements for controlling the aspiration or dispense of liquids
    • G01N35/1016—Control of the volume dispensed or introduced
    • G01N2035/102—Preventing or detecting loss of fluid by dripping
    • 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
    • G01N2035/1039—Micropipettes, e.g. microcapillary tubes

Definitions

  • the present invention relates to a microchip having a fine channel whose surface is chemically modified.
  • micromachine technology In recent years, by making full use of micromachine technology and ultrafine processing technology, devices and means (for example, pumps, valves, flow paths, sensors, etc.) for performing conventional sample preparation, chemical analysis, chemical synthesis, etc. have been miniaturized. Systems integrated on a chip have been developed. This is also called ⁇ -TAS (Micro Total Analysis System), and a sample (for example, a DNA-treated extraction solution obtained by processing urine, saliva, blood, etc. of a subject to be examined) is placed on a member called a microchip. And the reagent are mixed, and the characteristics of the specimen are examined by detecting the reaction.
  • ⁇ -TAS Micro Total Analysis System
  • Microchips use a photolithographic process (a method of creating a groove by etching a pattern image with a chemical) or a groove using a laser beam on a substrate made of a resin material or glass material, and flow a reagent or specimen.
  • a fine flow path that can be used and a reservoir for storing the reagent are provided, and various patterns have been proposed.
  • the reagent and the specimen are separated by feeding a liquid such as a reagent or specimen contained in the microchip with a micropump.
  • the reaction is led to the detected part and detected.
  • the target substance is detected by, for example, an optical detection method.
  • Reaction in a microchip with a fine channel has advantages such as increased reaction efficiency by diffusion due to the effect of the minute volume, and shortening of the reaction time. If the amount of water is very small and adsorption or liquid residue occurs on the surface of the flow channel during liquid feeding, the specific signal of the target substance is lowered and the reaction efficiency is lowered.
  • non-specific adsorption of proteins in the micro flow path causes hydrophilicity of the flow path surface, resulting in a remaining liquid of the flowing reagent, and a problem that the liquid is not sent to the reaction section.
  • a liquid film is formed in the liquid containing protein due to the effect of non-specific adsorption, and the slit part where air is originally removed is blocked. Therefore, there is a problem that the target liquid feeding is hindered.
  • the surface is hydrophilized, so that there is a problem that a liquid residue is generated, or that it is difficult to control the fluid by running on the wall surface of the channel with capillary force.
  • coating with a biopolymer such as BSA is not a liquid film prevention measure.
  • silane coupling agents having a trifluoromethyl group and fluoroalkyl resin membranes were not able to adequately suppress protein adsorption, and liquid residue on the channel wall surface and slit channel closure were observed. .
  • the present invention has been made in view of the above problems and situations, and a solution to the problem is a microchip including a fine channel, and prevents adsorption of a sample such as a protein in the fine channel.
  • An object of the present invention is to provide a microchip that prevents adverse effects such as sample loss and channel closing caused by adsorption.
  • a microchip having a fine channel which is a composite compound of a hydrophobic fine silica compound modified with hexamethyldisilazane and a silane compound having a fluoroalkyl group or a fluoroalkyl ether group on the surface of the fine channel 2.
  • microchip having the fine flow path according to 4 above, wherein the slit structure is a side road structure.
  • microchip having the fine flow path according to 4 or 5, wherein the slit structure has fine irregularities.
  • a microchip having a micro flow channel is used to prevent the adsorption of a sample such as a protein in the micro flow channel, and the sample loss due to the adsorption, the channel closing, etc.
  • a microchip that prevents adverse effects can be provided.
  • FIG. 4A is a top view around the intermediate reservoir 139
  • FIG. 4B is a cross-sectional view around the intermediate reservoir 139
  • FIG. 4C is an AA cross-sectional view in FIG. 4A
  • 5A is a top view around the downstream outlet of the intermediate reservoir 139
  • FIG. 5B is a cross-sectional view taken along the line BB in FIG. 5A.
  • Perspective view of the central section around the intermediate reservoir 139 Conceptual diagram of a specific example of a fine flow path with a slit structure
  • the microchip of the present invention is a microchip provided with a fine channel, and a hydrophobic fine silica compound, a silane compound having a fluoroalkyl group or a fluoroalkyl ether group on the surface of the fine channel, or a composite thereof.
  • the compound is coated.
  • An embodiment of the present invention is a microchip provided with a fine channel, and on the surface of the fine channel, a hydrophobic fine silica compound modified with hexamethyldisilazane and a fluoroalkyl group or a fluoroalkyl ether group
  • a composite compound with a silane compound having a silane compound is coated is also preferable.
  • the silane compound having a fluoroalkyl group preferably has a chemical structure represented by the general formula (A).
  • the fine flow path is a mode in which a fine flow path having a slit structure is included as a flow path constituting the fine flow path.
  • the said slit structure is a side road structure (structure which has the side flow path 139s shown in FIG.4 and FIG.6).
  • an aspect in which the slit structure has fine irregularities is also preferable.
  • Hydrophobic fine silica compounds according to the present invention include various conventionally known hydrophobic fine silica compounds containing essentially any type of silicon-containing compound, including silica, diatomite, diatomaceous earth and other types of diatomaceous earth. Silica compounds can be used. In the present invention, a particularly preferred example is a modified product obtained by contact reaction of hexamethyldisilazane with OH groups on the surface of fine silica.
  • the average particle diameter of primary particles of the hydrophobic fine silica compound is preferably 5 to 50 nm from the viewpoint of hydrophobicity and film properties.
  • the amount of carbon in the hydrophobic fine silica compound is preferably 2 to 5% by mass, and particularly preferably 2.2 to 4% by mass, from the same viewpoint as described above.
  • hydrophobic fine silica compound used in the present invention when modifying by contact reaction of hexamethyldisilazane, first, an alkyl halogeno such as methyltrichlorosilane or dimethyldichlorosilane is added to the OH group on the surface of the fine silica. It is also preferable to use a hydrophobic fine silica compound modified by further contact reaction of hexamethyldisilazane after contact reaction of silane. Examples of the method for producing the hydrophobic fine silica compound used in the present invention include the methods disclosed in Japanese Patent No. 2886037 and Japanese Patent No. 2886105. The hydrophobic fine silica compound used in the present invention can also be obtained as a commercial product.
  • HM-20L modified by contact reaction of hexamethyldisilazane with OH groups on the surface of fine silica.
  • HM-30S manufactured by Tokuyama Co., Ltd.
  • Rheolocyl ZD-30ST modified by contact reaction of alkylhalogenosilane with OH groups on the surface of fine silica and further contact reaction with hexamethyldisilazane (( Etc.) manufactured by Tokuyama Corporation.
  • silane compound having fluoroalkyl group or fluoroalkyl ether group As the “silane compound having a fluoroalkyl group or fluoroalkyl ether group” according to the present invention, heptadecatrifluorodecyltrimethoxysilane, trifluoropropyltrimethoxysilane, compounds described later, and the like can be used. In addition, it can also obtain as a commercial item, for example, Shin-Etsu Chemical Co., Ltd. KBM7803 (heptadecatrifluorodecyltrimethoxysilane), KBM7103 (trifluoropropyltrimethoxysilane), etc. can be mentioned.
  • the surface of the fine channel is preferably treated with a silane compound solution having a fluoroalkyl group or a fluoroalkyl ether group diluted to 0.01 to 10% by mass with an organic solvent not containing fluorine.
  • the fluoroalkyl group in the silane compound is bonded to Si atoms at a ratio of 1 or less to one Si atom, and the rest Is preferably a silane compound which is a hydrolyzable group or a siloxane bond group.
  • the hydrolyzable group here is, for example, a group such as an alkoxy group, and becomes a hydroxyl group by hydrolysis, whereby the silane compound forms a polycondensate.
  • the silane compound is reacted with water (in the presence of an acid catalyst if necessary), usually in the range of room temperature to 100 ° C. while distilling off the by-produced alcohol.
  • the alkoxysilane is (partially) hydrolyzed to cause a partial condensation reaction, and can be obtained as a hydrolyzate having a hydroxyl group.
  • the degree of hydrolysis and condensation can be appropriately adjusted depending on the amount of water to be reacted.
  • water is not actively added to the silane compound solution. It is preferable to dilute and use the solid content concentration of the solution in order to cause a hydrolysis reaction with water.
  • the silane compound having a fluoroalkyl group is represented by the following general formula (A) and used as a solution in which the concentration of the silane compound is diluted to 0.01 to 5% by mass (surface treatment). )It is to be.
  • m is an integer of 1 to 10
  • n is an integer of 0 to 10
  • Ra represents the same or different alkyl group.
  • Ra is an alkyl group having 3 or less carbon atoms and consisting only of carbon and hydrogen, for example, a group such as methyl, ethyl, isopropyl and the like.
  • Examples of the silane compound having a fluoroalkyl group or a fluoroalkyl ether group preferably used in the present invention include CF 3 (CH 2 ) 2 Si (OCH 3 ) 3 , CF 3 (CH 2 ) 2 Si (OC 2 H 5 ) 3.
  • CF 3 (CH 2) 2 Si (OC 3 H 7) 3, CF 3 (CH 2) 2 Si (OC 4 H 9) 3, CF 3 (CF 2) 5 (CH 2) 2 Si (OCH 3) 3 , CF 3 (CF 2 ) 5 (CH 2 ) 2 Si (OC 2 H 5 ) 3 , CF 3 (CF 2 ) 5 (CH 2 ) 2 Si (OC 3 H 7 ) 3 , CF 3 (CF 2 ) 7 (CH 2 ) 2 Si (OCH 3 ) 3 , CF 3 (CF 2 ) 7 (CH 2 ) 2 Si (OC 2 H 5 ) 3 , CF 3 (CF 2 ) 7 (CH 2 ) 2 Si (OC 3 ) H 7) , CF 3 (CF 2) 7 (CH 2) 2 Si (OCH 3) (OC 3 H 7) 2, CF 3 (CF 2) 7 (CH 2) 2 Si (OCH 3) 2 OC 3 H 7, CF 3 (CF 2 ) 7 (CH 2 ) 2 SiCH 3 (OCH 3 ) 2 ,
  • silane compounds When these silane compounds are used, they are diluted to 0.01 to 10% by mass, preferably 0.03 to 5% by mass, more preferably 0.05 to 2% by mass with an organic solvent not containing fluorine. Used in.
  • Solvents for the coating composition that can be used in the present invention include propylene glycol mono (C1-C4) alkyl ether and / or propylene glycol mono (C1-C4) alkyl ether ester, propylene glycol mono (C1-C4) alkyl.
  • Specific examples of the ether include propylene glycol monomethyl ether (PGME), propylene glycol monoethyl ether, propylene glycol mono-n-propyl ether, propylene glycol monoisopropyl ether and propylene glycol monobutyl ether.
  • propylene glycol mono (C1 to C4) alkyl ether ester examples include propylene glycol monoalkyl ether acetate, specifically, propylene glycol monomethyl ether acetate, propylene glycol monoethyl ether acetate and the like.
  • the solvent to be mixed is not particularly limited to these.
  • solvents particularly preferable solvents are one or more organic solvents selected from ethanol, isopropyl alcohol, propylene glycol, and propylene glycol monomethyl ether.
  • solvents those having a boiling point of less than 100 ° C. (low boiling solvent) such as methanol, ethanol and isopropyl alcohol, and boiling points of 100 ° C. or more such as propylene glycol monomethyl ether and n-butyl alcohol. It is preferable to use those having a boiling point of 60 to 98 ° C. and those having a boiling point of 100 to 160 ° C. in particular.
  • the ratio of the low boiling point solvent to the high boiling point solvent when used in combination is preferably 98.0% by mass or more of the low boiling point solvent and 0.5 to 2% by mass of the high boiling point solvent.
  • composition for coating which concerns on this invention, it is also preferable to add acid and to adjust pH to 5.0 or less. Since the acid promotes hydrolysis of the silane compound and acts as a catalyst for the polycondensation reaction, formation of a polycondensation film of the silane compound on the substrate surface can be facilitated.
  • the pH is in the range of 1.5 to 5.0, preferably in the range of 2.0 to 4.0.
  • the microchip of the present invention is a microchip having a fine channel.
  • a typical example of the configuration of the microchip will be described in detail, but is not limited to the following exemplary embodiments.
  • FIG. 1 is a schematic perspective view of a microchip analysis system 8 using a microchip according to the present invention
  • FIG. 2 is a configuration diagram.
  • the microchip analysis system 8 includes a driving liquid tank 70 that stores a driving liquid L0 for feeding a sample and a reagent previously injected into the microchip 1, and a micropump 5 for supplying the driving liquid L0 to the microchip 1.
  • the pump connection part 6 that connects the micropump 5 and the microchip 1 so that the driving liquid L0 does not leak, the temperature control unit 3 that controls the temperature of the necessary part of the microchip 1, and the temperature control so that the microchip 1 does not deviate.
  • a chip pressing plate 2 for bringing the chip pressing plate 2 into close contact with the unit 3 and the pump connection unit 6; a pressing plate driving unit 21 for moving the chip pressing plate 2 up and down; a regulating member 22 for positioning the microchip 1 with respect to the micro pump 5 with high accuracy;
  • a light detection unit 4 (4a and 4b) for detecting a reaction state between the specimen and the reagent in the microchip 1 is provided.
  • the chip pressing plate 2 is retracted upward from the position shown in FIG. 2 in the initial state. Thereby, the microchip 1 can be inserted / removed in the direction of the arrow X, and the person inspecting inserts the microchip 1 from the insertion port until it comes into contact with the regulating member 22. Thereafter, the chip pressing plate 2 is lowered by the pressing plate driving unit 21 and comes into contact with the microchip 1, and the lower surface of the microchip 1 is in close contact with the temperature adjustment unit 3 and the pump connection unit 6.
  • the temperature control unit 3 includes a Peltier element 31 and a heater 32 on the surface facing the microchip 1.
  • the Peltier element 31 and the heater 32 are attached to the microchip 1. It comes to adhere closely.
  • the portion containing the reagent is cooled by the Peltier element 31 so that the reagent is not denatured, or the intermediate reservoir 139 that causes the sample and the reagent to join and react is heated by the heater 32 to promote the reaction. .
  • the light from the light emitting unit 4a made of, for example, a mercury lamp is supplied to the microchip 1 as excitation light through an excitation filter that allows light of a wavelength in a specific region to pass. Irradiation is performed, and fluorescence emitted from the fluorescent material present in the detection unit 148 of the microchip 1 is transmitted, and the transmitted light is detected by the light receiving unit 4b.
  • the light receiving portion 4b is integrally provided inside the chip pressing plate 2.
  • the light emitting unit 4a and the light receiving unit 4b are provided to face the detecting unit 148 of the microchip 1 shown in FIG.
  • the micropump 5 is located on the pump chamber 52, the piezoelectric element 51 that changes the volume of the pump chamber 52, the first throttle channel 53 located on the microchip 1 side of the pump chamber 52, and the driving fluid tank 70 side of the pump chamber.
  • the second throttle channel 54 is formed.
  • the first throttle channel 53 and the second throttle channel 54 are narrow and narrow channels, and the first throttle channel 53 is longer than the second throttle channel 54.
  • the piezoelectric element 51 is driven so as to rapidly reduce the volume of the pump chamber 52. Then, a turbulent flow is generated in the second throttle channel 54 that is a short throttle channel, and the channel resistance in the second throttle channel 54 is relatively larger than that of the first throttle channel 53 that is a throttle channel. growing. As a result, the driving liquid L0 in the pump chamber 52 is predominantly pushed toward the first throttle channel 53 and fed. Next, the piezoelectric element 51 is driven so that the volume of the pump chamber 52 is gradually increased. Then, the driving liquid L0 flows from the first throttle channel 53 and the second throttle channel 54 as the volume in the pump chamber 52 increases.
  • the driving liquid L 0 flows into the pump chamber 52 predominantly from the second throttle channel 54.
  • the piezoelectric element 51 repeats the above operation, the driving liquid L0 is fed in the forward direction.
  • the piezoelectric element 51 is driven so as to gently reduce the volume of the pump chamber 52. Then, since the length of the second throttle channel 54 is shorter than that of the first throttle channel 53, the channel resistance of the second throttle channel 54 is smaller than that of the first throttle channel 53. . As a result, the driving liquid L0 in the pump chamber 52 is predominantly pushed out toward the second throttle channel 54 and fed. Next, the piezoelectric element 51 is driven so that the volume of the pump chamber 52 is rapidly increased. Then, the driving liquid L0 flows from the first throttle channel 53 and the second throttle channel 54 as the volume in the pump chamber 52 increases.
  • the pump connection portion 6 is formed of a contact surface made of a resin having flexibility (elasticity, shape followability) such as polytetrafluoroethylene or silicon resin in order to ensure necessary sealing performance and prevent leakage of driving liquid. It is preferred that Such a close contact surface having flexibility may be, for example, due to the constituent substrate of the microchip itself, or may be a separate additional having flexibility attached around the flow path opening in the pump connection portion 6. It may be due to a member.
  • a resin having flexibility such as polytetrafluoroethylene or silicon resin
  • FIG. 3 shows an example of the microchip 1 according to the present embodiment.
  • middle storage part 139 in the state from which the sheet-like coating substrate was removed is shown typically.
  • the microchip 1 uses a water repellent (also referred to as hydrophobic) base material, and a microchannel r and a flow for mixing and reacting a liquid specimen (sample) on the microchip 1 as well as a liquid reagent.
  • a road element is disposed.
  • the material of the base material include resins such as polystyrene, polyethylene, polypropylene, polyethylene terephthalate, polyethylene naphthalate, polyethylene vinyl alcohol, polycarbonate, polymethylpentene, fluorocarbon, and saturated cyclic polyolefin.
  • polystyrene is excellent as transparency, mechanical properties, and moldability, and can be easily finely processed. Therefore, polystyrene is preferable as a material for forming a groove forming substrate.
  • a water-repellent substrate is used for a sheet-like coated substrate (not shown).
  • the fine channel r is formed in the order of micrometers, for example, the width w is several tens to several hundreds ⁇ m, preferably 50 to 300 ⁇ m, and the height h is about 25 to 1000 ⁇ m, preferably 50 to 300 ⁇ m.
  • G is an upstream opening released from one surface of the microchip 1 to the outside.
  • the plurality of upstream openings g are aligned with the channel openings provided on the connection surface of the micropump 5 when the microchip 1 is overlapped and connected to the micropump 5 via the pump connection 6. It communicates with the micropump 5. Then, a driving liquid L0 for feeding liquid is injected from the upstream opening.
  • I are injection holes for injecting a liquid such as a reagent or a specimen (hereinafter also simply referred to as a liquid), and are openings opened from the upper surface of the microchip 1 to the outside.
  • a liquid is injected into each injection hole i with the upstream opening g opened.
  • the injected liquid is sent through the fine channel toward the nearby upstream opening g.
  • the liquid is sequentially injected from the downstream injection hole i5 to the upstream injection hole i1.
  • the injected liquid is stored in the fine flow path r1 upstream of the intermediate storage part 139.
  • the upstream opening g and the injection hole i are opened, and only the injection hole i is sealed after the reagent injection. Then, the liquid supply pressure is applied to the liquid such as the reagent or the sample through the gas such as air or inert gas by the driving liquid L0 sent from the micropump 5 communicating with the upstream opening g, so that the liquid Is fed through the fine channel r.
  • FIG. 4 is an enlarged view of the periphery of the intermediate storage portion 139 of the microchip shown in FIG. 3, FIG. 4 (a) is a top view, FIG. 4 (b) is a sectional view, and FIG. 4 (c) is AA. It is sectional drawing.
  • FIG. 5 is an enlarged view of the vicinity of the downstream outlet of the intermediate reservoir 139, FIG. 5 (a) is a top view, and FIG. 5 (b) is a BB cross-sectional view.
  • FIG. 6 is a perspective view of the central cross section of the intermediate reservoir 139.
  • the intermediate storage part 139 is composed of a main part 139m and a side flow path 139s.
  • the side flow path 139s communicates with the upstream fine flow path r1 and the downstream fine flow path r2 through the communication portion CN.
  • one side flow path 139s is provided on each side of the main portion 139m of the intermediate storage section 139.
  • the present invention is not limited to this, and the side flow path 139s may be provided only on one side. Good.
  • the side flow path 139s and the fine flow path r have the same upper surface position, but their height (depth) and width are different.
  • the side flow path 139s has a rectangular shape with a width of 250 ⁇ m and a height (depth) of 100 ⁇ m, and its cross-sectional area is 25 ⁇ 10 ⁇ 9 m 2 .
  • the microchannels r1 and r2 have a rectangular shape with a width of 300 ⁇ m and a height of 250 ⁇ m, and the cross-sectional area is 75 ⁇ 10 ⁇ 9 m 2 . That is, the cross-sectional area of the side channel 139s is formed so that the cross-sectional area is smaller than that of the fine channel r1 on the upstream side. Note that the cross-sectional area of the side flow path 139s may be further narrowed to have a width of 250 ⁇ m and a height of 50 ⁇ m.
  • the surface of the covering substrate to be attached to the upper surface (not shown) and the inner wall surfaces s1 and s2 of the side channel 139s are formed of a water-repellent material.
  • the cross-sectional area is smaller than that of the upstream fine flow channel r1 and is formed of a water-repellent material, the flow resistance of the side flow channel 139s is very large compared to the fine flow channel r1. That is, in the side flow path 139s, it is difficult for liquid to pass through, but gas can easily pass through.
  • the fine flow path which concerns on this invention is an aspect containing the fine flow path of a slit structure as a flow path which comprises it.
  • the said slit structure is a side road structure.
  • the slit structure has a fine unevenness.
  • Fig. 7 and Fig. 8 show conceptual diagrams of specific examples of a fine flow path having a slit structure.
  • pillars columnumns
  • the shape of the pillar 140 is not limited to this shape, and preferably, the height is 10 to 500 ⁇ m, the diameter is 10 to 500 ⁇ m, and the pillar interval is 5 to 100 ⁇ m.
  • the “slit structure” in this case refers to a space between pillars.
  • fine unevenness” in the structure refers to an uneven shape including a gap portion between pillars corresponding to concave portions and a pillar portion corresponding to convex portions.
  • the air between the reagent 141 and the reagent 142 passes the reagent 141 from the slit structure part between the pillars, and then the reagent 141 and the reagent 142 joins.
  • a groove 143 having a depth of 100 ⁇ m and a width of 50 ⁇ m is provided in the intermediate storage portion 139.
  • the number of grooves is not limited, and a plurality of grooves may be provided.
  • the depth is 10 to 500 ⁇ m
  • the width is 10 to 500 ⁇ m
  • the groove interval is 5 to 100 ⁇ m.
  • the “slit structure” in this case refers to the groove 143.
  • fine irregularities” in the structure refers to an irregular shape composed of a groove and a non-groove portion.
  • the air between the reagent 141 and the reagent 142 passes the reagent 141 from the slit structure part, and then the reagent 141 and the reagent 142 are Join.
  • the intermediate reservoir 139 has an overall length of 4000 ⁇ m.
  • the main portion 139m has a flat bottom surface portion having the largest cross-sectional area (see FIG. 4C), has a width of 1500 ⁇ m and a height of 1500 ⁇ m, and both the width and height are larger than those of the fine channel r. Yes.
  • the length of the flat portion is 2000 ⁇ m. Because of this shape, the volume (capacity) of the main portion 139m is 7.3 ⁇ l.
  • the height of the main portion 139m of the intermediate storage portion 139 may be 250 ⁇ m, which is the same height as the fine flow path r1, and the shape may be widened only in the width direction. By doing so, the microchip can have a simple shape.
  • the microchip used in this example has a channel formed by pasting a cover such as an adhesive sheet on a glass or resin in which a groove is dug.
  • a hydrophobic fine silica compound 5% solution (contact angle of 100 ° or more) is poured into a microchip channel as shown in FIGS. 1 to 6, removed, and then dried, so that hydrophobic silica is formed on the channel surface.
  • the compound was applied (coating).
  • silane compound having a fluoroalkyl ether group was poured, removed, and dried to coat the compound having a fluoroalkyl ether group on the surface of the flow path.
  • hydrophobic fine silica compound for example, Adesso MR-1 manufactured by Nikka Chemical Co., Ltd. was diluted to 5% with 3M perfluoroether HFE-7100.
  • silane compound having a fluoroalkyl ether group for example, an OPTOOL DSX manufactured by Daikin was diluted to 0.5% with perfluoroether HFE-7100 manufactured by 3M.
  • Air is applied to the coated channel with 1% (10 mg / ml) BSA solution (A solution in FIG. 3) and 0.2% fluorescent dye fluorescein solution (FITC) (B solution in FIG. 3). Then, 3 ⁇ L of BSA solution 1% (10 mg / ml), 4 ⁇ L of air, and 5 ⁇ L of FITC 0.2% were sent in this order.
  • the drive method was driven by suction from a syringe pump from the downstream.
  • Example 2 Flow through a microchip channel as shown in Figs. 1 to 6 with a mixture of a 5% hydrophobic fine silica compound solution (contact angle of 100 ° or more) and a silane compound having a fluoroalkyl ether group in advance. Then, by drying, a mixture of a hydrophobic fine silica compound and a compound having a fluoroalkyl ether group was applied to the channel surface.
  • hydrophobic fine silica compound As the hydrophobic fine silica compound, Adesso MR-1 manufactured by Nikka Chemical Co., Ltd. was diluted to 5% with 3M perfluoroether HFE-7100.
  • the silane compound having a fluoroalkyl ether group was obtained by diluting Daikin Optool DSX to 0.5% with 3M Perfluoroether HFE-7100.
  • Air is sandwiched between 1% (10 mg / ml) BSA solution (A solution in FIG. 3) and 0.2% (fluorescent dye fluorescein solution (FITC) solution (B solution in FIG. 3)) to the coated channel. Then, BSA solution 1% (10 mg / ml) 3 ⁇ L-air 4 ⁇ L-FITC 0.2% 5 ⁇ L in this order.
  • the drive method was driven by suction from a syringe pump from the downstream.
  • Example 1 it was confirmed that the stability of the air vent channel by suppressing the liquid film generation was more stable than those in Example 3 and Example 4 described later.
  • Example 1 In order to compare with Examples 1 and 2, the same liquid feeding as Example 1 and 2 was performed without a coating agent.
  • the drive method was driven by suction from a syringe pump from the downstream.
  • Example 3 A hydrophobic fine silica compound 5% solution (contact angle 100 °) is poured into the flow path of the microchip as shown in FIGS. 1 to 6, removed, and then dried, so that the hydrophobic silica compound is formed on the flow path surface. Was applied.
  • hydrophobic fine silica compound for example, Adesso MR-1 manufactured by Nikka Chemical Co., Ltd. was diluted to 5% with 3M perfluoroether HFE-7100.
  • Air is applied to the coated channel with 1% (10 mg / ml) BSA solution (A solution in FIG. 3) and 0.2% fluorescent dye fluorescein solution (FITC) (B solution in FIG. 3). Then, 3 ⁇ L of BSA solution 1% (10 mg / ml), 4 ⁇ L of air, and 5 ⁇ L of FITC 0.2% were sent in this order.
  • the drive method was driven by suction from a syringe pump from the downstream.
  • Example 4 A silane compound having a fluoroalkyl ether group is poured into the flow path of a microchip as shown in FIGS. 1 to 6 and then removed, followed by drying, whereby a compound having a fluoroalkyl ether group is applied to the surface of the flow path. did.
  • Optool DSX manufactured by Daikin was diluted to 0.5% with perfluoroether HFE-7100 manufactured by 3M.
  • Air is sandwiched between 1% (10 mg / ml) BSA solution (A solution in FIG. 3) and 0.2% of fluorescent dye fluorescein solution (FITC) (B solution in FIG. 3) against the coated channel.
  • the drive method was driven by suction from a syringe pump from the downstream.
  • Example 4 As in Example 4, 1% (10 mg / ml) BSA solution (A solution in FIG. 3) and 0.2% of fluorochrome fluorescein solution (FITC) (FI solution B in FIG. 3) were applied to the coated channel. ), Air was sandwiched, and BSA solution 1% (10 mg / ml) 3 ⁇ L-air 4 ⁇ L-FITC 0.2% 5 ⁇ L was fed in this order.
  • BSA solution 1% (10 mg / ml) 3 ⁇ L-air 4 ⁇ L-FITC 0.2% 5 ⁇ L was fed in this order.
  • the drive method was driven by suction from a syringe pump from the downstream.
  • Example 5 A mixture of 5% hydrophobic fine silica compound solution (contact angle 100 °) and fluorine-based coating agent is poured into the microchip channel as shown in FIGS. 1 to 6 and removed, and then dried. Thus, a mixture of a hydrophobic fine silica compound and a silane compound having a fluoroalkyl ether group was applied to the flow path surface.
  • hydrophobic silica compound for example, Adesso MR-1 manufactured by Nikka Chemical Co., Ltd. was diluted to 5% with 3M perfluoroether HFE-7100.
  • silane compound having a fluoroalkyl ether group for example, an OPTOOL DSX manufactured by Daikin was diluted to 0.5% with perfluoroether HFE-7100 manufactured by 3M.
  • the liquid was fed in the order of 0.05% 5 ⁇ L.
  • the drive method was driven by suction from a syringe pump from the downstream.
  • tween20 0.05% (both A and B liquids in FIG. 3) is sandwiched with respect to the coated channel, and tween20 0.05% 3 ⁇ L-air 4 ⁇ L.
  • -Tween20 Liquid was fed in the order of 0.05% 5 ⁇ L.
  • the drive method was driven by suction from a syringe pump from the downstream.
  • the top tween 20 0.05% passes through the flow path, and then air does not escape to the air flow path (139s in FIG. 4), and a large amount of fine bubbles are generated, affecting the liquid feed. It was.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Analytical Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Clinical Laboratory Science (AREA)
  • Dispersion Chemistry (AREA)
  • Hematology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biochemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Automatic Analysis And Handling Materials Therefor (AREA)
  • Physical Or Chemical Processes And Apparatus (AREA)

Abstract

La présente invention concerne une micropointe comportant un microcanal empêchant l'adsorption d'un échantillon, par exemple une protéine, dans le microcanal, tout en empêchant également toute perte d'échantillon provoquée par l'adsorption et des problèmes tels que l'engorgement du canal. La micropointe est une micropointe comportant un microcanal à la surface duquel sont déposés un fin composé de silice hydrophobe, un composé silane comportant des groupes fluoroalkyle ou fluoroalkyléther ou un composé composite associant les composés précédents.
PCT/JP2009/058069 2008-05-27 2009-04-23 Micropointe comportant un microcanal WO2009145022A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2010514419A JPWO2009145022A1 (ja) 2008-05-27 2009-04-23 微細流路を備えたマイクロチップ

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2008-137757 2008-05-27
JP2008137757 2008-05-27

Publications (1)

Publication Number Publication Date
WO2009145022A1 true WO2009145022A1 (fr) 2009-12-03

Family

ID=41376908

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2009/058069 WO2009145022A1 (fr) 2008-05-27 2009-04-23 Micropointe comportant un microcanal

Country Status (2)

Country Link
JP (1) JPWO2009145022A1 (fr)
WO (1) WO2009145022A1 (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2011214996A (ja) * 2010-03-31 2011-10-27 Enplas Corp マイクロ流路チップ及びマイクロ分析システム
WO2018125901A1 (fr) 2016-12-28 2018-07-05 Bio-Rad Laboratories, Inc. Modification des propriétés de surface de dispositifs microfluidiques
US10850273B2 (en) 2014-11-28 2020-12-01 Dexerials Corporation Master for micro flow path creation, transfer copy, and method for producing master for micro flow path creation
JP2021522363A (ja) * 2018-05-18 2021-08-30 ザ ユニバーシティ オブ ノース カロライナ アット チャペル ヒルＴｈｅ Ｕｎｉｖｅｒｓｉｔｙ Ｏｆ Ｎｏｒｔｈ Ｃａｒｏｌｉｎａ Ａｔ Ｃｈａｐｅｌ Ｈｉｌｌ 基板の表面特性を改善する為の組成物、デバイス、及び方法
WO2023127759A1 (fr) * 2021-12-28 2023-07-06 凸版印刷株式会社 Puce microfluidique et procédé de fabrication d'une puce microfluidique
US11802826B2 (en) 2018-10-29 2023-10-31 Kyocera Corporation Measurement apparatus

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005274368A (ja) * 2004-03-25 2005-10-06 Nippon Sheet Glass Co Ltd バイオチップ用基板
JP2006105813A (ja) * 2004-10-06 2006-04-20 Alps Electric Co Ltd 検査用プレート及びその検査用プレートを用いた検査方法
JP2007238931A (ja) * 2006-02-08 2007-09-20 Nicca Chemical Co Ltd 疎水性コーティング膜形成組成物、疎水性コーティング膜、その形成方法及びそれを備えた機能性材料
JP2008064748A (ja) * 2006-08-08 2008-03-21 Sekisui Chem Co Ltd 微量液体秤取装置、それを有するマイクロチップ及び微量な液体の秤取方法
JP2008111798A (ja) * 2006-10-31 2008-05-15 Shimadzu Corp マイクロ反応装置

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005274368A (ja) * 2004-03-25 2005-10-06 Nippon Sheet Glass Co Ltd バイオチップ用基板
JP2006105813A (ja) * 2004-10-06 2006-04-20 Alps Electric Co Ltd 検査用プレート及びその検査用プレートを用いた検査方法
JP2007238931A (ja) * 2006-02-08 2007-09-20 Nicca Chemical Co Ltd 疎水性コーティング膜形成組成物、疎水性コーティング膜、その形成方法及びそれを備えた機能性材料
JP2008064748A (ja) * 2006-08-08 2008-03-21 Sekisui Chem Co Ltd 微量液体秤取装置、それを有するマイクロチップ及び微量な液体の秤取方法
JP2008111798A (ja) * 2006-10-31 2008-05-15 Shimadzu Corp マイクロ反応装置

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2011214996A (ja) * 2010-03-31 2011-10-27 Enplas Corp マイクロ流路チップ及びマイクロ分析システム
US10850273B2 (en) 2014-11-28 2020-12-01 Dexerials Corporation Master for micro flow path creation, transfer copy, and method for producing master for micro flow path creation
WO2018125901A1 (fr) 2016-12-28 2018-07-05 Bio-Rad Laboratories, Inc. Modification des propriétés de surface de dispositifs microfluidiques
CN110099968A (zh) * 2016-12-28 2019-08-06 生物辐射实验室股份有限公司 微流体装置的表面性质修饰
EP3562893A4 (fr) * 2016-12-28 2020-07-29 Bio-Rad Laboratories, Inc. Modification des propriétés de surface de dispositifs microfluidiques
US10919038B2 (en) 2016-12-28 2021-02-16 Bio-Rad Laboratories, Inc. Modification of surface properties of microfluidic devices
US11759780B2 (en) 2016-12-28 2023-09-19 Bio-Rad Laboratories, Inc. Modification of surface properties of microfluidic devices
JP2021522363A (ja) * 2018-05-18 2021-08-30 ザ ユニバーシティ オブ ノース カロライナ アット チャペル ヒルＴｈｅ Ｕｎｉｖｅｒｓｉｔｙ Ｏｆ Ｎｏｒｔｈ Ｃａｒｏｌｉｎａ Ａｔ Ｃｈａｐｅｌ Ｈｉｌｌ 基板の表面特性を改善する為の組成物、デバイス、及び方法
US11802826B2 (en) 2018-10-29 2023-10-31 Kyocera Corporation Measurement apparatus
WO2023127759A1 (fr) * 2021-12-28 2023-07-06 凸版印刷株式会社 Puce microfluidique et procédé de fabrication d'une puce microfluidique
JP2023098037A (ja) * 2021-12-28 2023-07-10 凸版印刷株式会社 マイクロ流路チップ及びマイクロ流路チップの製造方法
JP7310875B2 (ja) 2021-12-28 2023-07-19 凸版印刷株式会社 マイクロ流路チップ及びマイクロ流路チップの製造方法

Also Published As

Publication number Publication date
JPWO2009145022A1 (ja) 2011-10-06

Similar Documents

Publication Publication Date Title
WO2009145022A1 (fr) Micropointe comportant un microcanal
JP6279654B2 (ja) ガラス様表面を有する高分子化合物基板、および前記高分子化合物基板で作られたチップ
JP4199609B2 (ja) 分析用チップ、分析用チップユニット及び分析装置ならびに分析用チップの作製方法
US9604210B2 (en) Controlled fluid delivery in a microelectronic package
US7790109B2 (en) Micro-fluid reaction vessel, method for manufacturing the same, and micro-fluid reaction method using the vessel
WO2004008142A1 (fr) Puce d'analyse, unite de puce d'analyse, appareil d'analyse, methode d'analyse effectuee avec l'appareil et procede de production de la puce d'analyse
WO2009081722A1 (fr) Micropuce et procédé d'envoi de liquide pour micropuce
JP2006292472A (ja) マイクロ総合分析システム
JPWO2008117651A1 (ja) マイクロチップ
US20210394185A1 (en) Microfluidic device and sample analysis method
US20070034269A1 (en) Method of controlling fluid
CN204710358U (zh) 一种微流控芯片
US7767437B2 (en) System for detection of a component in a liquid
JP4590542B2 (ja) マイクロ液滴輸送デバイス
JP4579867B2 (ja) 化学物質検出用チップ
US20220362774A1 (en) Microfluidic chips with one or more vias
JP2005140511A (ja) 微量流体制御機構及び該機構を有するマイクロチップ
WO2010122720A1 (fr) Dispositif de trajet d'écoulement
JP2022175985A (ja) マイクロ流路チップにおいてエマルジョンを生成する方法及び装置
JP2018171660A (ja) 流体デバイス用シリコーン部材およびその製造方法
US20150191772A1 (en) Method of charging a test carrier and a test carrier
KR101533425B1 (ko) 유리비드 혼합몰드를 이용한 미세채널 제조방법 및 이 미세채널을 이용한 면역센서
JP7475050B2 (ja) アッセイ用カートリッジデバイス
WO2022044505A1 (fr) Dispositif à microcanaux, procédé de production de gouttelettes d'huile, procédé de production de bulles, procédé de production de microcapsules, procédé de production d'émulsion multiple, procédé de production de gouttelette d'encapsulation de bulle et procédé de production de dispositif à microcanaux
TWI696834B (zh) 微流體檢測單元及流體檢測方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 09754530

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 2010514419

Country of ref document: JP

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 09754530

Country of ref document: EP

Kind code of ref document: A1