WO2018025899A1 - Solution for filling plastic microfluidic device - Google Patents

Solution for filling plastic microfluidic device Download PDF

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Publication number
WO2018025899A1
WO2018025899A1 PCT/JP2017/027994 JP2017027994W WO2018025899A1 WO 2018025899 A1 WO2018025899 A1 WO 2018025899A1 JP 2017027994 W JP2017027994 W JP 2017027994W WO 2018025899 A1 WO2018025899 A1 WO 2018025899A1
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group
general formula
microfluidic device
filling
solution
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PCT/JP2017/027994
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French (fr)
Japanese (ja)
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和宏 寺嶌
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和光純薬工業株式会社
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Publication of WO2018025899A1 publication Critical patent/WO2018025899A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N37/00—Details not covered by any other group of this subclass

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  • the present invention relates to a plastic microfluidic device filling solution, a method for filling a plastic microfluidic device using the solution, and a PCR method using the device.
  • a microfluidic device is filled with an aqueous solution and performs various reactions and analyzes therein.
  • Many microfluidic devices are made of plastic materials from the viewpoint of component compatibility and disposal.
  • the plastic microfluidic device has non-uniform surface properties, formation of bubbles is promoted when filling with an aqueous solution, and reaction and analysis using the device may be hindered. Therefore, there is a demand for development of a method for uniformly filling a plastic microfluidic device with an aqueous solution without leaving bubbles or the like.
  • a chemical reaction involving a temperature change such as an enzyme reaction
  • a heat source such as a Peltier element
  • the temperature in the device is uneven due to the difference in specific heat between the aqueous solution and air, so that accurate temperature control can be achieved. As a result, an accurate enzyme reaction or the like may not be performed.
  • the plastic surface (chamber inner wall surface) of the plastic microfluidic device is made uniform hydrophilic by plasma treatment of the plastic surface, and the aqueous solution is put into the chamber.
  • a method of introducing Patent Document 1, Non-Patent Document 1.
  • the effect of hydrophilizing the plastic surface by plasma treatment decreases with time. Therefore, a plastic microfluidic device stored for a long time after manufacture has a problem that it cannot be uniformly filled with an aqueous solution.
  • the present inventors have conceived a method of filling a plastic microfluidic device with an aqueous solution containing a compound (additive) such as a specific surfactant. It has been found that according to the filling method, the aqueous solution can be uniformly filled in the plastic microfluidic device without leaving air (bubbles). However, among the specific surfactants, the surfactant in the filled aqueous solution causes a chemical attack on the plastic surface (inner wall surface of the microfluidic device) of the plastic microfluidic device, and the microfluidic device is It turns out that there is something that breaks.
  • a compound additive
  • an object of the present invention is to provide a method for uniformly filling an aqueous solution into a plastic microfluidic device without breaking the plastic surface of the plastic microfluidic device.
  • the present inventors have found that when a compound having a cyclopentahydrophenanthrene skeleton is contained in an aqueous solution for filling the chamber, The present invention has been completed by finding that not only can an air solution containing the compound be left without leaving air (bubbles) in the device, but also that the plastic surface of the device does not break due to chemical attack. It was.
  • the present invention relates to the following filling solutions and methods.
  • a solution for filling a plastic microfluidic device containing a compound having a cyclopentahydrophenanthrene skeleton represented by the following formula [1] (hereinafter sometimes abbreviated as the filling solution of the present invention).
  • a compound having a cyclopentahydrophenanthrene skeleton is represented by 3-[(3-cholamidopropyl) dimethylammonio] propanesulfonate (CHAPS), 3-[(3-cholamidopropyl) dimethylammonio] -2- Hydroxypropane sulfonate (CHAPSO), N, N-bis (3-D-gluconamidopropyl) coleamide (BIGCHAP), or N, N-bis (3-D-gluconamidopropyl) deoxycholamide (deoxy-BIGCHAP)
  • CHAPSO 3-[(3-cholamidopropyl) dimethylammonio] propanesulfonate
  • CHIGCHAP 3-[(3-cholamidopropyl) dimethylammonio] -2- Hydroxypropane sulfonate
  • BIGCHAP N-bis (3-D-gluconamidopropyl) coleamide
  • deoxy-BIGCHAP N-bis
  • a filling method for filling a plastic microfluidic device with a filling solution for a plastic microfluidic device containing a compound having a cyclopentahydrophenanthrene skeleton represented by the formula [1] (hereinafter referred to as the filling method of the present invention) May be abbreviated).
  • R 3 is a hydrogen atom, a group represented by the general formula [4], or a group represented by the general formula [5], and R 4 is a group represented by the general formula [4], or The filling method according to (12), which is a group represented by the general formula [5].
  • a compound having a cyclopentahydrophenanthrene skeleton is represented by 3-[(3-cholamidopropyl) dimethylammonio] propanesulfonate (CHAPS), 3-[(3-cholamidopropyl) dimethylammonio] -2- Hydroxypropane sulfonate (CHAPSO), N, N-bis (3-D-gluconamidopropyl) coleamide (BIGCHAP), or N, N-bis (3-D-gluconamidopropyl) deoxycholamide (deoxy-BIGCHAP)
  • a PCR method comprising filling a plastic microfluidic device with a PCR reaction solution containing a compound having a cyclopentahydrophenanthrene skeleton represented by the formula [1], and subjecting the device to a PCR reaction.
  • the plastic microfluidic device can be uniformly filled with an aqueous solution without leaving air (bubbles). Therefore, reaction and analysis are not hindered by air (bubbles). Further, the plastic surface of the plastic microfluidic device is not broken by a chemical attack. Furthermore, even if heating and cooling are repeated, there is an effect that the plastic microfluidic device is not broken. Further, the filling solution of the present invention, the filling method of the present invention and the PCR method of the present invention do not require a special apparatus and are not affected by the passage of time.
  • FIG. 1 shows an example of a plastic microfluidic device, which is a schematic view (A) viewed from above and a cross-sectional view (B) in a direction perpendicular to the chamber with respect to the flow direction of the filling solution of the present invention.
  • C1 represents a cross-sectional area in the vertical direction of the chamber with respect to the flow direction of the filling solution of the present invention
  • C2 represents a cross-sectional area in the vertical direction of the channel with respect to the flow direction of the filling solution of the present invention.
  • FIG. 2 is a schematic view of the plastic microfluidic device created in Experimental Example 1 as viewed from above.
  • the filling solution of the present invention is an aqueous solution containing a compound having a cyclopentahydrophenanthrene skeleton represented by the following formula [1].
  • the compound having a cyclopentahydrophenanthrene skeleton according to the present invention is any one having the cyclopentahydrophenanthrene skeleton represented by the formula [1].
  • compounds that do not adversely affect various reactions for example, chemical reactions, biochemical reactions, nucleic acid amplification reactions, etc.
  • various analyzes for example, electrophoresis, chromatography, electroanalysis, etc.
  • a hydrogen atom in the cyclopentahydrophenanthrene skeleton may be substituted with a substituent.
  • Examples of such a compound according to the present invention include an alkyl group having 1 to 3 carbon atoms and a hydroxy group. , A carboxy group, a halogeno group, and / or a cyclopentahydrophenatrene compound having a group represented by the following general formula [10] as a substituent.
  • Z 1 represents an alkylene group having 3 to 5 carbon atoms
  • Z 2 represents a hydroxy group, an —ONa group, or a group represented by the formula [3] described later
  • the alkylene group having 3 to 5 carbon atoms represented by Z 1 in the general formula [10] may be linear, branched or cyclic, but is preferably branched.
  • alkylene group having 3 to 5 carbon atoms represented by Z 1 in the general formula [10] examples include, for example, trimethylene group, propylene group (methylethylene group), ethylmethylene group, dimethylmethylene group, tetramethylene group, 1-methyl Trimethylene group, 2-methyltrimethylene group, 1,2-dimethylethylene group (1-methylpropylene group), 1,1-dimethylethylene group, ethylethylene group, ethylmethylmethylene group, n-propylmethylene group, isopropyl Methylene group, pentamethylene group, 1-methyltetramethylene group, 2-methyltetramethylene group, 1-ethyltrimethylene group, 2-ethyltrimethylene group, n-propylethylene group, isopropylethylene group,
  • Z 2 in the general formula [10] is preferably a group represented by the formula [3] described later.
  • the group represented by the general formula [10] is preferably a group represented by the following general formula [11]. (In the formula, Z 1 is the same as above.
  • Z 2 ′ represents a group represented by the formula [3] described later).
  • a cyclopentahydrophenatrene compound having a methyl group, a hydroxy group, and / or a group represented by the general formula [11] as a substituent is preferable, and a methyl group, a hydroxy group, and a general group are preferable.
  • a cyclopentahydrophenatrene compound having a group represented by the formula [11] as a substituent is more preferred.
  • a compound represented by the following general formula [2] can be mentioned. (Wherein R 1 , R 2 and m are the same as above)
  • R 1 in the general formula [2] is preferably a hydroxy group.
  • M in the general formula [2] is preferably an integer of 1 to 2, and more preferably 2.
  • R 2 in the general formula [2] a group represented by the following general formula [3] is particularly preferable. (Wherein R 3 and R 4 are the same as above)
  • R 3 is a hydrogen atom, a group represented by the following general formula [4], or a group represented by the following general formula [5], and R 4 represents is a group group represented by the general formula [4], or preferably represents a group represented by the following general formula [5], R 4 R 3 is a hydrogen atom is represented by the following general formula [4] In this case, or R 3 and R 4 are more preferably a group represented by the following general formula [5]. (Wherein R 5 , R 6 , Y and n are the same as above) (Wherein R 7 and p are the same as above)
  • the alkyl group having 1 to 3 carbon atoms represented by R 5 and R 6 in the general formula [4] may be either linear or branched, but is preferably linear, for example, a methyl group , An ethyl group, an n-propyl group, and an isopropyl group, a methyl group, an ethyl group, and an n-propyl group are preferable, and a methyl group is particularly preferable.
  • alkylene group having 1 to 6 carbon atoms which may have a hydroxy group as the substituent represented by Y in the general formula [4] those having 1 to 3 carbon atoms are preferable.
  • the alkylene group having 1 to 6 carbon atoms which may have a hydroxy group as the substituent represented by Y in the general formula [4] may be linear, branched or cyclic. A linear one is preferred.
  • methylene group ethylene group, methylmethylene group, trimethylene group, propylene group (methylethylene group), ethylmethylene group, dimethylmethylene group, tetramethylene group, 1-methyltrimethylene group, 2-methyltrimethylene group 1,2-dimethylethylene group (1-methylpropylene group), 1,1-dimethylethylene group, ethylethylene group, ethylmethylmethylene group, n-propylmethylene group, isopropylmethylene group, pentamethylene group, 1- Methyltetramethylene, 2-methyltetramethylene, 1-ethyltrimethylene, 2-ethyltrimethylene, n-propylethylene, isopropylethylene, n-butylmethylene, isobutylmethylene, tert-butylmethylene Group, sec-butylmethylene group, 2,2-di- Tiltrimethylene group, 1,2-dimethyltrimethylene group, 1,3-dimethyltrimethylene group
  • a group is preferable, a methylene group, an ethylene group, a trimethylene group, a hydroxymethylene group, a hydroxyethylene group, and a hydroxytrimethylene group are more preferable, and a trimethylene group and a 2-hydroxytrimethylene group are particularly preferable.
  • n is preferably an integer of 1 to 3, and more preferably 3.
  • n ′ represents an integer of 1 to 3
  • Y ′ represents an alkylene group having 1 to 3 carbon atoms which may have a hydroxy group as a substituent.
  • n ′ is preferably 3.
  • the alkylene group having 1 to 3 carbon atoms which may have a hydroxy group as a substituent represented by Y ′ in the general formula [4 ′] may be linear, branched or cyclic. However, a linear thing is preferable.
  • the alkylene group having 1 to 3 carbon atoms which may have a hydroxy group as a substituent represented by Y ′ is particularly preferably an alkylene group having 3 carbon atoms.
  • a methylene group, an ethylene group, a trimethylene group, a hydroxymethylene group, a hydroxyethylene group, and a hydroxytrimethylene group are preferable, and a trimethylene group and a 2-hydroxytrimethylene group are more preferable.
  • the alkylene group having 1 to 10 carbon atoms and having a hydroxy group as a substituent represented by R 7 in the general formula [5] preferably has 1 to 6 carbon atoms.
  • the alkylene group having 1 to 10 carbon atoms and having a hydroxy group as a substituent represented by R 7 in the general formula [5] may be linear, branched or cyclic, but linear Are preferred.
  • the alkylene group having 1 to 10 carbon atoms and having a hydroxy group as a substituent represented by R 7 in the general formula [5] represents a group in which at least one hydroxy group is bonded to a carbon atom in the alkylene group.
  • X ′ in the general formula [6] is preferably a hydrogen atom.
  • R in the general formula [6] is preferably an integer of 1 to 6.
  • P in the general formula [5] is preferably an integer of 1 to 3.
  • the general formula [2] include sodium cholate, potassium cholate, cholic acid, sodium deoxycholate, potassium deoxycholate, deoxycholic acid, the following general formula [7], and general formula [8]. And the compounds shown. (In the formula, m, n, R 1 and Y ′ are the same as described above.) Wherein m is the same as defined above, R 1 is a hydrogen atom or a hydroxy group, p ′ and p ′′ are each independently an integer of 1 to 3, and r ′ and r ′′ are each Independently an integer from 1 to 6).
  • the general formula [2] include compounds represented by the general formula [7] or the general formula [8], and compounds represented by the following general formula [7 ′] or the general formula [8] Among them, among others, 3-[(3-cholamidopropyl) dimethylammonio] propanesulfonate (CHAPS), 3-[(3-cholamidopropyl) dimethylammonio] -2-hydroxypropanesulfonate (CHAPSO), N, N-bis (3-D-gluconamidopropyl) coleamide (BIGCHAP) and N, N-bis (3-D-gluconamidopropyl) deoxycholamide (deoxy-BIGCHAP) are more preferred.
  • m, n and Y ′ are the same as described above.
  • the compound according to the present invention may be synthesized according to a method known per se, or a commercially available product may be used.
  • the filling solution of the present invention contains the compound according to the present invention in an aqueous solution, and is a solution for filling a plastic microfluidic device.
  • the concentration of the compound according to the present invention is usually 0.0001 w / w% to 20 w / w%, preferably 0, as the concentration in the solution when filling the plastic microfluidic device (in the filling solution of the present invention). 0.005 w / w% to 15 w / w%, more preferably 0.01 w / w% to 10 w / w%, still more preferably 0.1 w / w% to 5 w / w%.
  • the aqueous solution containing the compound according to the present invention may be any aqueous solution usually used in this field, and examples thereof include water and a buffer solution.
  • a buffer solution include a phosphate buffer solution, a Tris buffer solution, a Good buffer solution, a glycine having a buffering action in the vicinity of neutrality of pH 5.0 to 10.0, preferably pH 7.0 to 8.0.
  • examples include a buffer solution and a borate buffer solution.
  • the concentration of the buffering agent in the buffer may be appropriately selected from the range of usually 10 to 500 mM, preferably 10 to 100 mM.
  • reagents used in this field stabilizers such as sugars, nucleic acids, proteins, salts, An antiseptic or the like (hereinafter sometimes abbreviated as “reagents”) may coexist. Specific examples of reagents and the like that coexist when the filling solution of the present invention is used in various reactions are shown below.
  • Tama It may be.
  • the solution used in the various reactions include those containing reagents that coexist when used in the various reactions.
  • those used for biochemical reactions and those used for nucleic acid amplification reactions are preferable, and those used for PCR reactions are particularly preferable.
  • the filling solution of the present invention can be obtained by dissolving (suspending) the compound according to the present invention in an aqueous solution.
  • the solution herein includes a solution in which a solute is dissolved in a solvent and a suspension in which the solute is dispersed in a solvent.
  • the filling solution of the present invention may be prepared, for example, by the following method.
  • the final concentration is usually from 0.0001 w / w% to 20 w / w%, preferably from 0.005 w / w% to 15 w / w%, more preferably from 0.01 w / w% to 10 w / w%, still more preferably 0.
  • the compound according to the present invention in an amount of 1 w / w% to 5 w / w%, for example, has a buffer concentration of usually 10 to 500 mM, preferably 10 to 100 mM, and pH 5.0 to 10.0, preferably pH 7 Dissolve (suspended) in phosphate buffer, Tris buffer, Good's buffer, glycine buffer, or borate buffer, for example, 0.1 to 100 mL, which has a buffering action near neutrality of 0.0 to 8.0 ).
  • plastic microfluidic device may be used as long as it is normally used in this field.
  • a plastic microfluidic device including at least a channel and / or a chamber is preferable. More preferred.
  • the plastic microfluidic device is preferably used for various reactions (for example, chemical reaction, biochemical reaction, nucleic acid amplification reaction, etc.) and various analyzes (for example, electrophoresis, chromatography, electroanalysis, etc.).
  • a channel is a capillary (flow channel) for moving the filling solution of the present invention or used for separation such as electrophoresis, and any channel can be used as long as it can be filled with the filling solution of the present invention.
  • the cross-sectional shape of the channel in the direction perpendicular to the direction in which the filling solution of the present invention flows (fills) may be any, and examples thereof include a trapezoid, a rectangle, a square, an intrinsic circle, and an ellipse.
  • the cross-sectional area of the channel perpendicular to the direction in which the filling solution of the present invention flows (fills) is usually 0.01 mm 2 to 1 mm 2 , preferably 0.05 mm 2 to 0.75 mm 2 , more preferably 0. a .1mm 2 ⁇ 0.5mm 2, flow filling solution of the present invention (filling) the length direction of the channel is normally 0.1 mm ⁇ 100 cm, preferably from 1 mm ⁇ 20 cm, more preferably 5 mm ⁇ 10 cm It is.
  • the end of the channel is connected to a well (a liquid reservoir).
  • the chamber represents a space having a height (depth) and / or a vertical width and / or a cross-sectional area larger than those of the channel.
  • the schematic diagram (FIG. 1) shows the relationship between channels and chambers. Note that the shapes of the chamber and the channel are not limited thereto.
  • the cross-sectional area in the vertical direction of the chamber with respect to the flow direction of the filling solution of the present invention (for example, the total area of C1 and C2 in FIG. 1B, height (depth) ⁇ vertical width) is the filling area of the present invention.
  • the cross-sectional area in the vertical direction of the channel with respect to the direction of flowing (filling) the solution (for example, C2 in FIG.
  • the height (depth) of the chamber is usually 0.001 mm to 10 mm, preferably 0.005 mm to 1 mm, more preferably 0.01 mm to 0.5 mm, and the vertical width is usually 1 mm to 100 mm, preferably 1 mm to 50 mm. More preferably, the width (width in the flowing direction of the filling solution of the present invention) is usually 0.001 mm to 100 mm, preferably 0.005 mm to 50 mm, more preferably 0.01 mm to 20 mm.
  • the chamber may have a pillar (column) that supports the bottom surface and a surface facing the bottom surface and reinforces the strength of the chamber.
  • the pillar may have a cylindrical shape or a polygonal column shape, and the chamber may have one or more pillars.
  • the chamber may have a step, a groove, and a wall inside.
  • the chamber is preferably in communication with a channel, and the channel may be one or more. Therefore, the plastic microfluidic device according to the present invention preferably has at least one chamber, at least one channel communicating with the chamber, and a well connected to an end not communicating with the chamber of the channel. Is particularly useful for such microfluidic devices.
  • the material of the plastic microfluidic device may be any so-called plastic, and examples thereof include polycarbonate, polyethylene, polypropylene, and polyacrylic acid.
  • examples of the polycarbonate include Iupilon (registered trademark, Mitsubishi Gas Chemical Co., Ltd.), Novalex (registered trademark, Mitsubishi Chemical Corporation), Zanta (registered trademark, Mitsubishi Chemical Europe GmbH), and polyethylene includes, for example, spy Ducks (registered trademark, Mitsubishi Chemical Corporation), Nak Safe (registered trademark, NUC Corporation), Nipolon (registered trademark, Tosoh Corporation), etc.
  • examples of polypropylene include Wintec (registered trademark, Japan) Polypro Corporation), Newcon (Registered Trademark, Nippon Polypro Corporation), Funxter (Registered Trademark, Nippon Polypro Corporation), and the like.
  • polyacrylic acid examples include Minlon (Registered Trademark, EI DuPont). ⁇ Dou Nemours & Company), Li San (registered trademark, Arkema France), Leona (registered trademark, Asahi Kasei Co., Ltd.), and the like.
  • polycarbonate is particularly preferable.
  • the microfluidic device is preferably one that can withstand 50 ° C. to 120 ° C., more preferably one that can withstand 80 ° C. to 110 ° C., more preferably one that can withstand 90 ° C. to 105 ° C., and 95 ° C. to 100 ° C. Those that can withstand are particularly preferred.
  • a plastic microfluidic device may be prepared in accordance with a method generally used when producing an analysis chip used in the field of microreactor or microanalysis system, for example, a plastic substrate shaped like a chamber It can be obtained by bonding a plastic film to the film.
  • the method for molding the plastic substrate include a method of manufacturing by injection molding.
  • a method for joining the plastic substrate having the shape of the chamber and the plastic film there may be mentioned a method of joining by thermocompression bonding, ultrasonic waves or laser.
  • Various bonding conditions may be selected depending on the material of the plastic substrate or the plastic film. The specific method is based on, for example, the method described in Patent Document 2-4 (Patent Document 2: WO2012 / 06018, Patent Document 3: JP2013-44528, Patent Document 4: JP2009-226503). It only has to be done.
  • the plastic microfluidic device filled with the filling solution of the present invention includes chemical reactions such as condensation reaction, substitution reaction, hydrolysis reaction, oxidoreductase reaction, transferase reaction, hydrolase reaction, immune reaction (antigen-antibody reaction), etc. Biochemical reaction, polymerase chain reaction (PCR) method (Japanese Patent Laid-Open No.
  • the filling method of the present invention is a method of filling a plastic microfluidic device with an aqueous solution. Specifically, the channel and / or chamber of a plastics microfluidic device is filled with an aqueous solution.
  • the channel and / or chamber of the plastic microfluidic device may be filled in accordance with a method known per se.
  • the filling solution of the present invention is applied to the end of a plastic microfluidic device (the end of a channel or the end of a chamber or a well), utilizing gravity, or utilizing capillary action.
  • a plastic microfluidic device the end of a channel or the end of a chamber or a well
  • capillary action there is a method of filling a plastic microfluidic device, and filling by utilizing capillary action is preferable.
  • the pressure to be applied in the method of filling by applying pressure may be a size that allows the filling solution of the present invention to be filled into the plastic microfluidic device through the channel.
  • the filling solution of the present invention is prepared.
  • the final concentration is usually from 0.0001 w / w% to 20 w / w%, preferably from 0.005 w / w% to 15 w / w%, more preferably from 0.01 w / w% to 10 w / w%, still more preferably 0.
  • a solution is prepared by dissolving the compound of the present invention in an amount of 1 w / w% to 5 w / w% in water or a buffer solution usually used in this field, and is used as the filling solution of the present invention.
  • the filling solution of the present invention is put into the channel and / or chamber of the plastic microfluidic device from the end of the plastic microfluidic device (the end of the channel or the end of the chamber or well) in the microfluidic device.
  • An amount larger than that in which the (channel and / or chamber) is filled is injected, and the filling solution of the present invention is filled into the device (in the channel and / or chamber) by capillary action or gravity.
  • the filling method of this invention may be performed as follows, for example.
  • the filling solution of the present invention is prepared.
  • the final concentration is usually from 0.0001 w / w% to 20 w / w%, preferably from 0.005 w / w% to 15 w / w%, more preferably from 0.01 w / w% to 10 w / w%, still more preferably 0.
  • a solution is prepared by dissolving the compound of the present invention in an amount of 1 w / w% to 5 w / w% in water or a buffer solution usually used in this field, and is used as the filling solution of the present invention.
  • a filling solution of the present invention is injected into a well (injection well) coupled to a plastic microfluidic device in an amount sufficient to fill the microfluidic device (channel and / or chamber).
  • a well coupled to an end of the at least one channel coupled to the chamber not coupled to the chamber from a channel coupled to the injection well through a chamber coupled to the channel;
  • the chamber is filled with the solution by moving the solution to the channel to which the other well (which is referred to as a discharge well) is coupled using capillary action.
  • the inside of the plastic microfluidic device can be uniformly filled with an aqueous solution without leaving air (bubbles), and the plastic surface of the plastic microfluidic device is broken by a chemical attack. Does not occur.
  • the plastic microfluidic device filled with the filling solution of the present invention by the filling method of the present invention does not break even when heating and cooling are repeated.
  • the filling method of the present invention does not require a special device and is not affected by the passage of time.
  • the plastic microfluidic device When the plastic microfluidic device has a chamber and a channel, bubbles may be easily generated due to the shape and / or size thereof.
  • the chamber and the channel The inside of the plastic microfluidic device can be uniformly filled with an aqueous solution without leaving air (bubbles) regardless of the shape of the channel, and no chemical attack occurs. That is, the filling method of the present invention is particularly useful when the plastic microfluidic device has a chamber and a channel.
  • bubbles may be easily generated.
  • the plastic microfluidic device does not leave air (bubbles). Can be uniformly filled with an aqueous solution and no chemical attack occurs, the filling method of the present invention is useful even when the plastic microfluidic device has pillars.
  • the filling method of the present invention is particularly useful when a plastic microfluidic device is heated, in other words, a plastic microfluidic device used under a high temperature condition.
  • a plastic microfluidic device used under a high temperature condition.
  • the PCR method of the present invention is a method of filling a plastic microfluidic device with a PCR reaction solution containing a compound having a cyclopentahydrophenanthrene skeleton represented by the formula [1] and subjecting the device to a PCR reaction.
  • a PCR reaction solution in the PCR method of the present invention (hereinafter sometimes abbreviated as a PCR reaction solution according to the present invention) is represented by the above formula [1] in a PCR reaction solution used for a PCR reaction known per se.
  • Any compound may be used as long as it contains a compound having a cyclopentahydrophenanthrene skeleton, and it conforms to the definition of the filling solution of the present invention except that it contains a reagent contained in a PCR reaction solution known per se.
  • reagents contained in a PCR reaction solution known per se include DNA polymerase, dNTP mix, primer, template DNA, template RNA, metal ion, buffering agent and the like.
  • the PCR method of the present invention is a PCR method according to a method known per se except that a plastic microfluidic device filled with a PCR reaction solution containing a compound having a cyclopentahydrophenanthrene skeleton represented by the formula [1] is used.
  • the PCR method known per se is not limited to the most common polymerase chain reaction (PCR) method (Japanese Patent Laid-Open No. 60-281), but TaqMan (registered trademark) real-time PCR method (for example, US Pat. No. 5,538,848). Description), MGB Eclipse Probe System method (see, for example, the description in US Pat. No.
  • the PCR method of the present invention may be performed as follows, for example.
  • a PCR reaction solution according to the present invention is prepared.
  • the final concentration is usually from 0.0001 w / w% to 20 w / w%, preferably from 0.005 w / w% to 15 w / w%, more preferably from 0.01 w / w% to 10 w / w%, still more preferably 0.
  • the solution thus prepared is prepared as a PCR reaction solution according to the present invention.
  • a PCR reaction solution according to the present invention is injected into a well (referred to as an injection well) coupled to an end of a plastic microfluidic device in an amount sufficient to fill the microfluidic device.
  • a well coupled to an end of the at least one channel coupled to the chamber not coupled to the chamber from a channel coupled to the injection well through a chamber coupled to the channel;
  • the reaction solution is filled into the chamber by moving the reaction solution to a channel to which a well other than the well (to be a discharge well) is coupled using capillary action.
  • a plastic microfluidic device filled with a PCR reaction solution according to the present invention is prepared by, for example, (i) 90 ° C.
  • the PCR method of the present invention by using the inside of the plastic microfluidic device uniformly filled with the PCR reaction solution according to the present invention without causing chemical attack and without leaving air (bubbles).
  • the temperature in the device becomes uniform, and an accurate enzyme reaction can be performed.
  • the plastic microfluidic device can be uniformly filled with the filling solution of the present invention, and the plastic surface of the chamber is not broken by a chemical attack. This is useful when conducting reactions involving temperature changes such as enzymatic reactions and PCR reactions.
  • Example 1 Production of Plastic Microfluidic Device> As shown in FIG. 2, the vertical width is 6 mm, the horizontal width is 15 mm, the depth (height) is 0.35 mm, the channel width is 1 mm, the depth is 0.35 mm, the well diameter is 3 mm, and the pillar diameter is 0.6 mm.
  • a polycarbonate resin was formed by injection molding a polycarbonate resin so that the pillar height was 0.35 mm.
  • a polycarbonate resin film having a thickness of 0.125 mm was placed on a plastic substrate and thermocompression bonded under the conditions of 150 ° C., pressure 250 kg, and pressure bonding time 10 minutes to produce a plastic microfluidic device.
  • the surfactant-containing aqueous solution was introduced into the chamber by moving the surfactant-containing aqueous solution from the well through the chamber to the other well (discharge well) by capillary action.
  • the flow of the surfactant-containing aqueous solution in the chamber at the time of filling and the filling speed were confirmed by visual observation, and evaluated in four stages: ⁇ > ⁇ > ⁇ > ⁇ .
  • Each symbol indicates the following result.
  • ⁇ Aqueous solution is filled very smoothly
  • ⁇ Aqueous solution is filled smoothly
  • ⁇ Aqueous solution is filled (while staying)
  • ⁇ Foaming occurs when filling with aqueous solution
  • Table 1 Show. Also, the filling results were similarly evaluated in the following comparative examples.
  • the aqueous solution can be uniformly filled in the chamber without leaving air (bubbles) in the polycarbonate microfluidic device.
  • the polycarbonate microfluidic device is supplied with air (even if the concentration of the CHAPS-containing aqueous solution is 0.001%, 0.01%, 0.1%, 1%, or 10%). It was found that the aqueous solution could be uniformly filled into the chamber without leaving bubbles.
  • an aqueous solution containing various surfactants having a cyclopentahydrophenatrene skeleton As an aqueous solution containing various surfactants having a cyclopentahydrophenatrene skeleton, an aqueous solution containing CHAPS, CHAPSO, BIGCHAP, or deoxy-BIGCHAP was found to be particularly preferable. It was also found that aqueous solutions containing various surfactants having a cyclopentahydrophenatrene skeleton did not cause cracks due to chemical attack even when heating and cooling of a polycarbonate microfluidic device were repeated.
  • any aqueous solution containing NP-40, polyoxyethylene lauryl ether, Tween 80, MEGA 8, SDS, or CTAB can uniformly fill the polycarbonate chamber.
  • the aqueous solution containing CHAPS (Example 1) is very smoothly filled with the aqueous solution, and the aqueous solution containing NP-40, polyoxyethylene lauryl ether, or MEGA8 is smoothly filled with Tween 80, SDS, or CTAB.
  • the aqueous solution containing was able to be filled, the flow was uneven in the chamber.
  • an aqueous solution containing various surfactants having a cyclopentahydrophenatrene skeleton is an aqueous solution in the plastic microfluidic device without leaving air (bubbles).
  • plastic surface of the plastic microfluidic device could not be broken by chemical attack.
  • a plastic microfluidic device filled with an aqueous solution containing various surfactants having a cyclopentahydrophenatrene skeleton does not break even when heating and cooling are repeated.

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Abstract

The problem addressed by the present invention is to provide a method for uniformly filling a plastic microfluidic device with an aqueous solution without damaging a plastic surface of the device, and a solution used in said method. The present invention relates to a solution containing a compound having a cyclopentahydrophenanthrene skeleton given by the formula [1] for filling a plastic microfluidic device, and a filling method for filling the device with the solution.

Description

プラスチック製マイクロ流体デバイス充填用溶液Solutions for filling plastic microfluidic devices
　本発明は、プラスチック製マイクロ流体デバイス充填用溶液、該溶液を用いたプラスチック製マイクロ流体デバイスへの充填方法、及び該デバイスを用いたＰＣＲ方法に関する。 The present invention relates to a plastic microfluidic device filling solution, a method for filling a plastic microfluidic device using the solution, and a PCR method using the device.
　マイクロ流体デバイスは、該デバイス内に水溶液を充填し、そこで各種反応や分析を行うことが知られている。多くのマイクロ流体デバイスは、部品の互換性、廃棄性の観点からプラスチック材料により作られている。しかし、プラスチック製マイクロ流体デバイスは、表面性状が不均一である為、水溶液を充填する際に気泡の形成が促進され、該デバイスを用いた反応や分析が妨げられる場合がある。その為、プラスチック製マイクロ流体デバイスに気泡等を残存させることなく水溶液で一様に充填させる方法の開発が求められている。 It is known that a microfluidic device is filled with an aqueous solution and performs various reactions and analyzes therein. Many microfluidic devices are made of plastic materials from the viewpoint of component compatibility and disposal. However, since the plastic microfluidic device has non-uniform surface properties, formation of bubbles is promoted when filling with an aqueous solution, and reaction and analysis using the device may be hindered. Therefore, there is a demand for development of a method for uniformly filling a plastic microfluidic device with an aqueous solution without leaving bubbles or the like.
　具体的には、水溶液を含むプラスチック製マイクロ流体デバイス内で酵素反応等の温度変化を伴う化学反応を行う際、ペルチェ素子などの熱源から放出される規定量の熱を、プラスチック製マイクロ流体デバイス内の水溶液へ正確に伝える必要がある。ところが、該デバイス内に空気層や気泡等の水溶液で満たされていない部分があると、水溶液と空気等との比熱の違いによって該デバイス内の温度にむらが生じることにより正確な温度制御ができず、結果として正確な酵素反応等を行うことができない場合がある。 Specifically, when a chemical reaction involving a temperature change such as an enzyme reaction is performed in a plastic microfluidic device containing an aqueous solution, a specified amount of heat released from a heat source such as a Peltier element is transferred to the plastic microfluidic device. It is necessary to convey accurately to the aqueous solution. However, if there is a part in the device that is not filled with an aqueous solution such as an air layer or bubbles, the temperature in the device is uneven due to the difference in specific heat between the aqueous solution and air, so that accurate temperature control can be achieved. As a result, an accurate enzyme reaction or the like may not be performed.
　プラスチック製マイクロ流体デバイスへの水溶液の充填方法としては、例えば、プラスチック表面をプラズマ処理することにより、プラスチック製マイクロ流体デバイスのプラスチック表面（チャンバー内壁面）を均一な親水性にし、該チャンバー内に水溶液を導入する方法が知られている（特許文献１、非特許文献１）。しかし、プラズマ処理によるプラスチック表面の親水化は時間経過とともにその効果が減少する。そのため、製造後長期間保管したプラスチック製マイクロ流体デバイスでは、水溶液を一様に充填させることができなくなるという問題があった。 As a method for filling an aqueous solution into a plastic microfluidic device, for example, the plastic surface (chamber inner wall surface) of the plastic microfluidic device is made uniform hydrophilic by plasma treatment of the plastic surface, and the aqueous solution is put into the chamber. There is known a method of introducing (Patent Document 1, Non-Patent Document 1). However, the effect of hydrophilizing the plastic surface by plasma treatment decreases with time. Therefore, a plastic microfluidic device stored for a long time after manufacture has a problem that it cannot be uniformly filled with an aqueous solution.
特開２０１２－１０２２０５JP2012-102205
　前記の問題を解消するための充填方法としては、有機シリコーン界面活性剤を含有する水溶液を用いる方法が知られている（特表２０１１－５１５６９９）。しかし、該方法を用いても、プラスチック製マイクロ流体デバイス内に気泡が発生する場合があり、より気泡の発生し難い充填方法の開発が必要であった。 As a filling method for solving the above-mentioned problem, a method using an aqueous solution containing an organosilicone surfactant is known (Special Table 2011-515699). However, even if this method is used, bubbles may be generated in the plastic microfluidic device, and it is necessary to develop a filling method in which bubbles are less likely to be generated.
　そこで、本発明者らは、特定の界面活性剤等の化合物（添加物）を含有させた水溶液を用いて、プラスチック製マイクロ流体デバイスを充填する方法を想到した。該充填方法によれば、空気（気泡）を残存させることなくプラスチック製マイクロ流体デバイス内に、水溶液を一様に充填できることを見出した。しかし、特定の界面活性剤の中には、充填された水溶液中の界面活性剤がプラスチック製マイクロ流体デバイスのプラスチック表面（マイクロ流体デバイス内壁面）に対してケミカルアタックを起こし、該マイクロ流体デバイスを破断するものがあることが判明した。 Therefore, the present inventors have conceived a method of filling a plastic microfluidic device with an aqueous solution containing a compound (additive) such as a specific surfactant. It has been found that according to the filling method, the aqueous solution can be uniformly filled in the plastic microfluidic device without leaving air (bubbles). However, among the specific surfactants, the surfactant in the filled aqueous solution causes a chemical attack on the plastic surface (inner wall surface of the microfluidic device) of the plastic microfluidic device, and the microfluidic device is It turns out that there is something that breaks.
　そのため、本発明はプラスチック製マイクロ流体デバイスのプラスチック表面を破断させずに、プラスチック製マイクロ流体デバイス内に水溶液を一様に充填させる方法の提供を課題とする。 Therefore, an object of the present invention is to provide a method for uniformly filling an aqueous solution into a plastic microfluidic device without breaking the plastic surface of the plastic microfluidic device.
　本発明者らは、プラスチック製マイクロ流体デバイスへの充填用溶液に含有させる化合物として種々の化合物を検討した結果、シクロペンタヒドロフェナントレン骨格を有する化合物を該チャンバーに充填させる水溶液に含有させると、該デバイス内に空気（気泡）を残存させることなく該化合物を含有する水溶液を充填できるだけでなく、該デバイスのプラスチック表面に対してケミカルアタックによる破断を生じさせないことを見出し、本発明を完成させるに至った。 As a result of examining various compounds as compounds to be contained in a filling solution for plastic microfluidic devices, the present inventors have found that when a compound having a cyclopentahydrophenanthrene skeleton is contained in an aqueous solution for filling the chamber, The present invention has been completed by finding that not only can an air solution containing the compound be left without leaving air (bubbles) in the device, but also that the plastic surface of the device does not break due to chemical attack. It was.
　本発明は、以下の充填用溶液および方法に関する。
　（１）下記式［１］で示されるシクロペンタヒドロフェナントレン骨格を有する化合物を含む、プラスチック製マイクロ流体デバイスへの充填用溶液（以下、本発明の充填用溶液と略記する場合がある）。
Figure JPOXMLDOC01-appb-I000016
　（２）シクロペンタヒドロフェナントレン骨格を有する化合物が、下記一般式［２］で示されるものである、（１）に記載の溶液
Figure JPOXMLDOC01-appb-I000017

（式中、Ｒ１は水素原子又はヒドロキシ基を表し、Ｒ２はヒドロキシ基、－ＯＮａ基又は下記一般式［３］で示される基を表し、ｍは１～３の整数を表す）
Figure JPOXMLDOC01-appb-I000018
（式中、Ｒ３及びＲ４はそれぞれ独立して水素原子、下記一般式［４］で示される基、又は下記一般式［５］で示される基を表す）
Figure JPOXMLDOC01-appb-I000019
（式中、Ｒ５及びＲ６はそれぞれ独立して炭素数１～３のアルキル基、Ｙは置換基としてヒドロキシ基を有していてもよい炭素数１～６のアルキレン基、ｎは１～６の整数をそれぞれ表す）
Figure JPOXMLDOC01-appb-I000020
（式中、Ｒ７は置換基としてヒドロキシ基を有する炭素数１～１０のアルキレン基、ｐは１～６の整数をそれぞれ表す）。
　（３）Ｒ２が前記一般式［３］で示される基である、（２）に記載の溶液。
　（４）Ｒ３が水素原子、前記一般式［４］で示される基、又は前記一般式［５］で示される基であって、Ｒ４が前記一般式［４］で示される基、又は前記一般式［５］で示される基である、（３）に記載の溶液。
　（５）シクロペンタヒドロフェナントレン骨格を有する化合物が、３－［（３－コールアミドプロピル）ジメチルアンモニオ］プロパンスルホネート（ＣＨＡＰＳ）、３－［（３－コールアミドプロピル）ジメチルアンモニオ］－２－ヒドロキシプロパンスルホネート（ＣＨＡＰＳＯ）、Ｎ，Ｎ－ビス（３－Ｄ－グルコンアミドプロピル）コールアミド（ＢＩＧＣＨＡＰ）、又は、Ｎ，Ｎ－ビス（３－Ｄ－グルコンアミドプロピル）デオキシコールアミド（ｄｅｏｘｙ－ＢＩＧＣＨＡＰ）である、（１）に記載の溶液。
　（６）プラスチック製マイクロ流体デバイスがポリカーボネート製マイクロ流体デバイスである、（１）－（５）に記載の溶液。
　（７）プラスチック製マイクロ流体デバイスがチャンバー又は／及びチャネルを有するものである、（１）－（６）に記載の溶液。
　（８）チャンバー又は／及びチャネルが加温されるものである、（７）に記載の溶液。
　（９）プラスチック製マイクロ流体デバイスへの充填用溶液におけるシクロペンタヒドロフェナントレン骨格を有する化合物の濃度が０．０００１ｗ／ｗ％～２０ｗ／ｗ％である、（１）－（８）に記載の溶液。
　（１０）前記式［１］で示されるシクロペンタヒドロフェナントレン骨格を有する化合物を含むプラスチック製マイクロ流体デバイスへの充填用溶液をプラスチック製マイクロ流体デバイスへ充填する充填方法（以下、本発明の充填方法と略記する場合がある）。
　（１１）シクロペンタヒドロフェナントレン骨格を有する化合物が、前記一般式［２］で示されるものである、（１０）に記載の方法。
　（１２）Ｒ２が前記一般式［３］で示される基である、（１１）に記載の充填方法。
　（１３）Ｒ３が水素原子、前記一般式［４］で示される基、又は前記一般式［５］で示される基であって、Ｒ４が前記一般式［４］で示される基、又は前記一般式［５］で示される基である、（１２）に記載の充填方法。
　（１４）シクロペンタヒドロフェナントレン骨格を有する化合物が、３－［（３－コールアミドプロピル）ジメチルアンモニオ］プロパンスルホネート（ＣＨＡＰＳ）、３－［（３－コールアミドプロピル）ジメチルアンモニオ］－２－ヒドロキシプロパンスルホネート（ＣＨＡＰＳＯ）、Ｎ，Ｎ－ビス（３－Ｄ－グルコンアミドプロピル）コールアミド（ＢＩＧＣＨＡＰ）、又は、Ｎ，Ｎ－ビス（３－Ｄ－グルコンアミドプロピル）デオキシコールアミド（ｄｅｏｘｙ－ＢＩＧＣＨＡＰ）である、（１０）に記載の充填方法。
　（１５）プラスチック製マイクロ流体デバイスがポリカーボネート製マイクロ流体デバイスである、（１０）－（１４）に記載の充填方法。
　（１６）プラスチック製マイクロ流体デバイスがチャンバー又は／及びチャネルを有するものである、（１０）－（１５）に記載の充填方法。
　（１７）チャンバー又は／及びチャネルが加温されるものである、（１６）に記載の充填方法。
　（１８）プラスチック製マイクロ流体デバイスへの充填用溶液におけるシクロペンタヒドロフェナントレン骨格を有する化合物の濃度が０．０００１ｗ／ｗ％～２０ｗ／ｗ％である、（１０）－（１７）に記載の充填方法。
　（１９）前記式［１］で示されるシクロペンタヒドロフェナントレン骨格を有する化合物を含むＰＣＲ反応溶液をプラスチック製マイクロ流体デバイスへ充填し、該デバイスをＰＣＲ反応に付す、ＰＣＲ方法。
　（２０）シクロペンタヒドロフェナントレン骨格を有する化合物が、前記一般式［２］で示されるものである、（１９）に記載のＰＣＲ方法。 The present invention relates to the following filling solutions and methods.
(1) A solution for filling a plastic microfluidic device containing a compound having a cyclopentahydrophenanthrene skeleton represented by the following formula [1] (hereinafter sometimes abbreviated as the filling solution of the present invention).
Figure JPOXMLDOC01-appb-I000016
(2) The solution according to (1), wherein the compound having a cyclopentahydrophenanthrene skeleton is represented by the following general formula [2]
Figure JPOXMLDOC01-appb-I000017

(Wherein R 1 represents a hydrogen atom or a hydroxy group, R 2 represents a hydroxy group, —ONa group or a group represented by the following general formula [3], and m represents an integer of 1 to 3)
Figure JPOXMLDOC01-appb-I000018
(Wherein R 3 and R 4 each independently represent a hydrogen atom, a group represented by the following general formula [4], or a group represented by the following general formula [5])
Figure JPOXMLDOC01-appb-I000019
Wherein R 5 and R 6 are each independently an alkyl group having 1 to 3 carbon atoms, Y is an alkylene group having 1 to 6 carbon atoms which may have a hydroxy group as a substituent, and n is 1 to Each represents an integer of 6)
Figure JPOXMLDOC01-appb-I000020
(Wherein R 7 represents a C 1-10 alkylene group having a hydroxy group as a substituent, and p represents an integer of 1-6).
(3) The solution according to (2), wherein R 2 is a group represented by the general formula [3].
(4) R 3 is a hydrogen atom, a group represented by the general formula [4], or a group represented by the general formula [5], and R 4 is a group represented by the general formula [4], or The solution according to (3), which is a group represented by the general formula [5].
(5) A compound having a cyclopentahydrophenanthrene skeleton is represented by 3-[(3-cholamidopropyl) dimethylammonio] propanesulfonate (CHAPS), 3-[(3-cholamidopropyl) dimethylammonio] -2- Hydroxypropane sulfonate (CHAPSO), N, N-bis (3-D-gluconamidopropyl) coleamide (BIGCHAP), or N, N-bis (3-D-gluconamidopropyl) deoxycholamide (deoxy-BIGCHAP) The solution according to (1), wherein
(6) The solution according to any one of (1) to (5), wherein the plastic microfluidic device is a polycarbonate microfluidic device.
(7) The solution according to any one of (1) to (6), wherein the plastic microfluidic device has a chamber or / and a channel.
(8) The solution according to (7), wherein the chamber or / and the channel are heated.
(9) The solution according to (1) to (8), wherein the concentration of the compound having a cyclopentahydrophenanthrene skeleton in the solution for filling the plastic microfluidic device is 0.0001 w / w% to 20 w / w% .
(10) A filling method for filling a plastic microfluidic device with a filling solution for a plastic microfluidic device containing a compound having a cyclopentahydrophenanthrene skeleton represented by the formula [1] (hereinafter referred to as the filling method of the present invention) May be abbreviated).
(11) The method according to (10), wherein the compound having a cyclopentahydrophenanthrene skeleton is represented by the general formula [2].
(12) The filling method according to (11), wherein R 2 is a group represented by the general formula [3].
(13) R 3 is a hydrogen atom, a group represented by the general formula [4], or a group represented by the general formula [5], and R 4 is a group represented by the general formula [4], or The filling method according to (12), which is a group represented by the general formula [5].
(14) A compound having a cyclopentahydrophenanthrene skeleton is represented by 3-[(3-cholamidopropyl) dimethylammonio] propanesulfonate (CHAPS), 3-[(3-cholamidopropyl) dimethylammonio] -2- Hydroxypropane sulfonate (CHAPSO), N, N-bis (3-D-gluconamidopropyl) coleamide (BIGCHAP), or N, N-bis (3-D-gluconamidopropyl) deoxycholamide (deoxy-BIGCHAP) The filling method according to (10), wherein
(15) The filling method according to (10)-(14), wherein the plastic microfluidic device is a polycarbonate microfluidic device.
(16) The filling method according to any one of (10) to (15), wherein the plastic microfluidic device has a chamber or / and a channel.
(17) The filling method according to (16), wherein the chamber or / and the channel are heated.
(18) The filling according to (10)-(17), wherein the concentration of the compound having a cyclopentahydrophenanthrene skeleton in the filling solution for the plastic microfluidic device is 0.0001 w / w% to 20 w / w% Method.
(19) A PCR method comprising filling a plastic microfluidic device with a PCR reaction solution containing a compound having a cyclopentahydrophenanthrene skeleton represented by the formula [1], and subjecting the device to a PCR reaction.
(20) The PCR method according to (19), wherein the compound having a cyclopentahydrophenanthrene skeleton is represented by the general formula [2].
　本発明の充填用溶液を用いる、本発明の充填方法及び本発明のＰＣＲ方法によれば、空気（気泡）を残存させることなくプラスチック製マイクロ流体デバイス内を水溶液で一様に充填することができる為、反応や分析が空気（気泡）により阻害されることがない。また、プラスチック製マイクロ流体デバイスのプラスチック表面にケミカルアタックによる破断を生じさせることもない。更に、加熱や冷却を繰り返しても、プラスチック製マイクロ流体デバイスに破断を生じさせないという効果を奏する。また、本発明の充填用溶液、本発明の充填方法及び本発明のＰＣＲ方法は、特別な装置を必要とせず、時間経過による影響を受けない。 According to the filling method of the present invention and the PCR method of the present invention using the filling solution of the present invention, the plastic microfluidic device can be uniformly filled with an aqueous solution without leaving air (bubbles). Therefore, reaction and analysis are not hindered by air (bubbles). Further, the plastic surface of the plastic microfluidic device is not broken by a chemical attack. Furthermore, even if heating and cooling are repeated, there is an effect that the plastic microfluidic device is not broken. Further, the filling solution of the present invention, the filling method of the present invention and the PCR method of the present invention do not require a special apparatus and are not affected by the passage of time.
図１はプラスチック製マイクロ流体デバイスの一例であり、上から見た模式図（Ａ）及び本発明の充填用溶液の流れる方向に対するチャンバーの垂直方向の断面図（Ｂ）である。図中、Ｃ１は本発明の充填用溶液の流れる方向に対するチャンバーの垂直方向の断面積を、Ｃ２は本発明の充填用溶液の流れる方向に対するチャネルの垂直方向の断面積をそれぞれ示す。FIG. 1 shows an example of a plastic microfluidic device, which is a schematic view (A) viewed from above and a cross-sectional view (B) in a direction perpendicular to the chamber with respect to the flow direction of the filling solution of the present invention. In the figure, C1 represents a cross-sectional area in the vertical direction of the chamber with respect to the flow direction of the filling solution of the present invention, and C2 represents a cross-sectional area in the vertical direction of the channel with respect to the flow direction of the filling solution of the present invention. 図２は実験例１で作成したプラスチック製マイクロ流体デバイスを上から見た模式図である。FIG. 2 is a schematic view of the plastic microfluidic device created in Experimental Example 1 as viewed from above.
　本発明の充填用溶液は、下記式［１］で示されるシクロペンタヒドロフェナントレン骨格を有する化合物を含む水溶液である。

Figure JPOXMLDOC01-appb-I000021
The filling solution of the present invention is an aqueous solution containing a compound having a cyclopentahydrophenanthrene skeleton represented by the following formula [1].

Figure JPOXMLDOC01-appb-I000021
　本発明に係るシクロペンタヒドロフェナントレン骨格を有する化合物（以下、「本発明に係る化合物」と略記する場合がある）は、前記式［１］で示されるシクロペンタヒドロフェナントレン骨格を有するものであればよいが、各種反応（例えば、化学反応、生化学反応、核酸増幅反応等）や各種分析（例えば、電気泳動、クロマトグラフィー、電気分析等）に悪影響を与えないものが好ましい。本発明に係る化合物は、シクロペンタヒドロフェナントレン骨格中の水素原子が置換基により置換されていてもよく、このような本発明に係る化合物としては、例えば炭素数１～３のアルキル基、ヒドロキシ基、カルボキシ基、ハロゲノ基、又は／及び下記一般式［１０］で示される基等を置換基として有するシクロペンタヒドロフェナトレン化合物等が挙げられる。

Figure JPOXMLDOC01-appb-I000022

（式中、Ｚ１は炭素数３～５のアルキレン基、Ｚ２はヒドロキシ基、－ＯＮａ基又は後述する式［３］で示される基をそれぞれ表す）
　一般式［１０］においてＺ１で示される炭素数３～５のアルキレン基は、直鎖状、分岐状、環状のいずれであってもよいが、分岐状のものが好ましい。以下に一般式［１０］においてＺ１で示される炭素数３～５のアルキレン基の具体例を挙げるが、これらはシクロペンタヒドロフェナントレン骨格の側から命名を行ったものである。一般式［１０］においてＺ１で示される炭素数３～５のアルキレン基としては、例えば、トリメチレン基、プロピレン基（メチルエチレン基）、エチルメチレン基、ジメチルメチレン基、テトラメチレン基、１－メチルトリメチレン基、２－メチルトリメチレン基、１，２－ジメチルエチレン基（１－メチルプロピレン基）、１，１－ジメチルエチレン基、エチルエチレン基、エチルメチルメチレン基、ｎ－プロピルメチレン基、イソプロピルメチレン基、ペンタメチレン基、１－メチルテトラメチレン基、２－メチルテトラメチレン基、１－エチルトリメチレン基、２－エチルトリメチレン基、ｎ－プロピルエチレン基、イソプロピルエチレン基、ｎ－ブチルメチレン基、イソブチルメチレン基、ｔｅｒｔ－ブチルメチレン基、ｓｅｃ－ブチルメチレン基、２，２－ジメチルトリメチレン基、１，２－ジメチルトリメチレン基、１，３－ジメチルトリメチレン基、シクロプロピレン基、シクロブチレン基、シクロペンチレン基が挙げられ、１－メチル－エチレン基（プロピレン基）、１－メチル－トリメチレン基、１－メチル－テトラメチレン基が好ましく、１－メチル－トリメチレン基がより好ましい。
　一般式［１０］におけるＺ２としては、後述する式［３］で示される基が好ましい。
　一般式［１０］で示される基は、下記一般式［１１］で示される基が好ましい。

Figure JPOXMLDOC01-appb-I000023

（式中、Ｚ１は上記と同じ。Ｚ２’は後述する式［３］で示される基を表す）
　本発明に係る化合物としては、中でもメチル基、ヒドロキシ基、又は／及び一般式［１１］で示される基を、置換基として有するシクロペンタヒドロフェナトレン化合物が好ましく、メチル基、ヒドロキシ基、及び一般式［１１］で示される基を、置換基として有するシクロペンタヒドロフェナトレン化合物がより好ましい。より具体的な化合物としては、例えば下記一般式［２］で示される化合物が挙げられる。
Figure JPOXMLDOC01-appb-I000024

（式中、Ｒ１、Ｒ２及びｍは前記と同じ） The compound having a cyclopentahydrophenanthrene skeleton according to the present invention (hereinafter sometimes abbreviated as “compound according to the present invention”) is any one having the cyclopentahydrophenanthrene skeleton represented by the formula [1]. However, those that do not adversely affect various reactions (for example, chemical reactions, biochemical reactions, nucleic acid amplification reactions, etc.) and various analyzes (for example, electrophoresis, chromatography, electroanalysis, etc.) are preferable. In the compound according to the present invention, a hydrogen atom in the cyclopentahydrophenanthrene skeleton may be substituted with a substituent. Examples of such a compound according to the present invention include an alkyl group having 1 to 3 carbon atoms and a hydroxy group. , A carboxy group, a halogeno group, and / or a cyclopentahydrophenatrene compound having a group represented by the following general formula [10] as a substituent.

Figure JPOXMLDOC01-appb-I000022

(Wherein Z 1 represents an alkylene group having 3 to 5 carbon atoms, Z 2 represents a hydroxy group, an —ONa group, or a group represented by the formula [3] described later)
The alkylene group having 3 to 5 carbon atoms represented by Z 1 in the general formula [10] may be linear, branched or cyclic, but is preferably branched. Specific examples of the alkylene group having 3 to 5 carbon atoms represented by Z 1 in the general formula [10] are listed below, and these are named from the cyclopentahydrophenanthrene skeleton side. Examples of the alkylene group having 3 to 5 carbon atoms represented by Z 1 in the general formula [10] include, for example, trimethylene group, propylene group (methylethylene group), ethylmethylene group, dimethylmethylene group, tetramethylene group, 1-methyl Trimethylene group, 2-methyltrimethylene group, 1,2-dimethylethylene group (1-methylpropylene group), 1,1-dimethylethylene group, ethylethylene group, ethylmethylmethylene group, n-propylmethylene group, isopropyl Methylene group, pentamethylene group, 1-methyltetramethylene group, 2-methyltetramethylene group, 1-ethyltrimethylene group, 2-ethyltrimethylene group, n-propylethylene group, isopropylethylene group, n-butylmethylene group , Isobutylmethylene group, tert-butylmethylene group, sec-butyl Examples include tilmethylene group, 2,2-dimethyltrimethylene group, 1,2-dimethyltrimethylene group, 1,3-dimethyltrimethylene group, cyclopropylene group, cyclobutylene group, and cyclopentylene group. An ethylene group (propylene group), a 1-methyl-trimethylene group and a 1-methyl-tetramethylene group are preferable, and a 1-methyl-trimethylene group is more preferable.
Z 2 in the general formula [10] is preferably a group represented by the formula [3] described later.
The group represented by the general formula [10] is preferably a group represented by the following general formula [11].

Figure JPOXMLDOC01-appb-I000023

(In the formula, Z 1 is the same as above. Z 2 ′ represents a group represented by the formula [3] described later).
As the compound according to the present invention, a cyclopentahydrophenatrene compound having a methyl group, a hydroxy group, and / or a group represented by the general formula [11] as a substituent is preferable, and a methyl group, a hydroxy group, and a general group are preferable. A cyclopentahydrophenatrene compound having a group represented by the formula [11] as a substituent is more preferred. As a more specific compound, for example, a compound represented by the following general formula [2] can be mentioned.
Figure JPOXMLDOC01-appb-I000024

(Wherein R 1 , R 2 and m are the same as above)
　一般式［２］におけるＲ１としては、ヒドロキシ基が好ましい。 R 1 in the general formula [2] is preferably a hydroxy group.
　一般式［２］におけるｍとしては、１～２の整数が好ましく、２がより好ましい。 M in the general formula [2] is preferably an integer of 1 to 2, and more preferably 2.
　一般式［２］におけるＲ２としては、下記一般式［３］で示される基が特に好ましい。
Figure JPOXMLDOC01-appb-I000025
（式中、Ｒ３及びＲ４は前記と同じ） As R 2 in the general formula [2], a group represented by the following general formula [3] is particularly preferable.
Figure JPOXMLDOC01-appb-I000025
(Wherein R 3 and R 4 are the same as above)
　一般式［３］におけるＲ３及びＲ４としては、Ｒ３が水素原子、下記一般式［４］で示される基、又は下記一般式［５］で示される基であって、Ｒ４が下記一般式［４］で示される基、又は下記一般式［５］で示される基である場合が好ましく、Ｒ３が水素原子であってＲ４が下記一般式［４］で示される基である場合、或いはＲ３及びＲ４が下記一般式［５］で示される基である場合がより好ましい。

Figure JPOXMLDOC01-appb-I000026
（式中、Ｒ５、Ｒ６、Ｙ及びｎは前記と同じ）
Figure JPOXMLDOC01-appb-I000027
（式中、Ｒ７及びｐは前記と同じ） As R 3 and R 4 in the general formula [3], R 3 is a hydrogen atom, a group represented by the following general formula [4], or a group represented by the following general formula [5], and R 4 represents is a group group represented by the general formula [4], or preferably represents a group represented by the following general formula [5], R 4 R 3 is a hydrogen atom is represented by the following general formula [4] In this case, or R 3 and R 4 are more preferably a group represented by the following general formula [5].

Figure JPOXMLDOC01-appb-I000026
(Wherein R 5 , R 6 , Y and n are the same as above)
Figure JPOXMLDOC01-appb-I000027
(Wherein R 7 and p are the same as above)
　一般式［４］におけるＲ５及びＲ６で示される炭素数１～３のアルキル基としては、直鎖状、分岐状のいずれであってもよいが直鎖状のものが好ましく、例えばメチル基、エチル基、ｎ－プロピル基、イソプロピル基が挙げられ、メチル基、エチル基、ｎ－プロピル基が好ましく、メチル基が特に好ましい。 The alkyl group having 1 to 3 carbon atoms represented by R 5 and R 6 in the general formula [4] may be either linear or branched, but is preferably linear, for example, a methyl group , An ethyl group, an n-propyl group, and an isopropyl group, a methyl group, an ethyl group, and an n-propyl group are preferable, and a methyl group is particularly preferable.
　一般式［４］におけるＹで示される置換基としてヒドロキシ基を有していてもよい炭素数１～６のアルキレン基としては、炭素数１～３のものが好ましい。また、当該一般式［４］におけるＹで示される置換基としてヒドロキシ基を有していてもよい炭素数１～６のアルキレン基は、直鎖状、分枝状又は環状のいずれであってもよいが、直鎖状のものが好ましい。具体的には、メチレン基、エチレン基、メチルメチレン基、トリメチレン基、プロピレン基（メチルエチレン基）、エチルメチレン基、ジメチルメチレン基、テトラメチレン基、１－メチルトリメチレン基、２－メチルトリメチレン基、１，２－ジメチルエチレン基（１－メチルプロピレン基）、１，１－ジメチルエチレン基、エチルエチレン基、エチルメチルメチレン基、ｎ－プロピルメチレン基、イソプロピルメチレン基、ペンタメチレン基、１－メチルテトラメチレン基、２－メチルテトラメチレン基、１－エチルトリメチレン基、２－エチルトリメチレン基、ｎ－プロピルエチレン基、イソプロピルエチレン基、ｎ－ブチルメチレン基、イソブチルメチレン基、ｔｅｒｔ－ブチルメチレン基、ｓｅｃ－ブチルメチレン基、２，２－ジメチルトリメチレン基、１，２－ジメチルトリメチレン基、１，３－ジメチルトリメチレン基、ヘキサメチレン基、１－メチルペンタメチレン基、２－メチルペンタメチレン基、３－メチルペンタメチレン基、１－エチルテトラメチレン基、２－エチルテトラメチレン基、１－ｎ－プロピルトリメチレン基、１－イソプロピルトリメチレン基、２－ｎ－プロピルトリメチレン基、２－イソプロピルトリメチレン基、ｎ－ブチルエチレン基、イソブチルエチレン基、ｔｅｒｔ－ブチルエチレン基、ｓｅｃ－ブチルエチレン基、ｎ－ペンチルメチレン基、イソペンチルメチレン基、ネオペンチルメチレン基、ｔｅｒｔ－ペンチルメチレン基、ｓｅｃ－ペンチルメチレン基、シクロプロピレン基、シクロブチレン基、シクロペンチレン基、シクロヘキシレン基、ヒドロキシメチレン基、ヒドロキシエチレン基、ヒドロキシトリメチレン基、ヒドロキシプロピレン基、ヒドロキシエチルメチレン基、ヒドロキシテトラメチレン基、ヒドロキシ１－メチルトリメチレン基、ヒドロキシ２－メチルトリメチレン基、ヒドロキシ１，２－ジメチルエチレン基、ヒドロキシ１，１－ジメチルエチレン基、ヒドロキシエチルエチレン基、ヒドロキシｎ－プロピルメチレン基、ヒドロキシイソプロピルメチレン基、ヒドロキシペンタメチレン基、ヒドロキシ１－メチルテトラメチレン基、ヒドロキシ２－メチルテトラメチレン基、ヒドロキシ１－エチルトリメチレン基、ヒドロキシ２－エチルトリメチレン基、ヒドロキシｎ－プロピルエチレン基、ヒドロキシイソプロピルエチレン基、ヒドロキシｎ－ブチルメチレン基、ヒドロキシイソブチルメチレン基、ヒドロキシｔｅｒｔ－ブチルメチレン基、ヒドロキシｓｅｃ－ブチルメチレン基、ヒドロキシ２，２－ジメチルトリメチレン基、ヒドロキシ１，２－ジメチルトリメチレン基、ヒドロキシ１，３－ジメチルトリメチレン基、ヒドロキシヘキサメチレン基、ヒドロキシ１－メチルペンタメチレン基、ヒドロキシ２－メチルペンタメチレン基、ヒドロキシ３－メチルペンタメチレン基、ヒドロキシ１－エチルテトラメチレン基、ヒドロキシ２－エチルテトラメチレン基、ヒドロキシ１－ｎ－プロピルトリメチレン基、ヒドロキシ１－イソプロピルトリメチレン基、ヒドロキシ２－ｎ－プロピルトリメチレン基、ヒドロキシ２－イソプロピルトリメチレン基、ヒドロキシｎ－ブチルエチレン基、ヒドロキシイソブチルエチレン基、ヒドロキシｔｅｒｔ－ブチルエチレン基、ヒドロキシｓｅｃ－ブチルエチレン基、ヒドロキシｎ－ペンチルメチレン基、ヒドロキシイソペンチルメチレン基、ヒドロキシネオペンチルメチレン基、ヒドロキシｔｅｒｔ－ペンチルメチレン基、ヒドロキシｓｅｃ－ペンチルメチレン基等が挙げられる。中でも、メチレン基、エチレン基、メチルメチレン基、トリメチレン基、プロピレン基、エチルメチレン基、ジメチルメチレン基、シクロプロピレン基、ヒドロキシメチレン基、ヒドロキシエチレン基、ヒドロキシトリメチレン基、ヒドロキシプロピレン基、ヒドロキシエチルメチレン基が好ましく、メチレン基、エチレン基、トリメチレン基、ヒドロキシメチレン基、ヒドロキシエチレン基、ヒドロキシトリメチレン基がより好ましく、トリメチレン基、２－ヒドロキシトリメチレン基が特に好ましい。 As the alkylene group having 1 to 6 carbon atoms which may have a hydroxy group as the substituent represented by Y in the general formula [4], those having 1 to 3 carbon atoms are preferable. In addition, the alkylene group having 1 to 6 carbon atoms which may have a hydroxy group as the substituent represented by Y in the general formula [4] may be linear, branched or cyclic. A linear one is preferred. Specifically, methylene group, ethylene group, methylmethylene group, trimethylene group, propylene group (methylethylene group), ethylmethylene group, dimethylmethylene group, tetramethylene group, 1-methyltrimethylene group, 2-methyltrimethylene group 1,2-dimethylethylene group (1-methylpropylene group), 1,1-dimethylethylene group, ethylethylene group, ethylmethylmethylene group, n-propylmethylene group, isopropylmethylene group, pentamethylene group, 1- Methyltetramethylene, 2-methyltetramethylene, 1-ethyltrimethylene, 2-ethyltrimethylene, n-propylethylene, isopropylethylene, n-butylmethylene, isobutylmethylene, tert-butylmethylene Group, sec-butylmethylene group, 2,2-di- Tiltrimethylene group, 1,2-dimethyltrimethylene group, 1,3-dimethyltrimethylene group, hexamethylene group, 1-methylpentamethylene group, 2-methylpentamethylene group, 3-methylpentamethylene group, 1- Ethyltetramethylene group, 2-ethyltetramethylene group, 1-n-propyltrimethylene group, 1-isopropyltrimethylene group, 2-n-propyltrimethylene group, 2-isopropyltrimethylene group, n-butylethylene group, Isobutylethylene group, tert-butylethylene group, sec-butylethylene group, n-pentylmethylene group, isopentylmethylene group, neopentylmethylene group, tert-pentylmethylene group, sec-pentylmethylene group, cyclopropylene group, cyclobutylene Group, cyclopentylene group, cyclohexane Silene group, hydroxymethylene group, hydroxyethylene group, hydroxytrimethylene group, hydroxypropylene group, hydroxyethylmethylene group, hydroxytetramethylene group, hydroxy1-methyltrimethylene group, hydroxy2-methyltrimethylene group, hydroxy1,2 -Dimethylethylene group, hydroxy 1,1-dimethylethylene group, hydroxyethylethylene group, hydroxy n-propylmethylene group, hydroxyisopropylmethylene group, hydroxypentamethylene group, hydroxy1-methyltetramethylene group, hydroxy-2-methyltetramethylene group Group, hydroxy 1-ethyltrimethylene group, hydroxy 2-ethyltrimethylene group, hydroxy n-propylethylene group, hydroxyisopropylethylene group, hydroxy n-butyl Lumethylene group, hydroxyisobutylmethylene group, hydroxy tert-butylmethylene group, hydroxysec-butylmethylene group, hydroxy2,2-dimethyltrimethylene group, hydroxy1,2-dimethyltrimethylene group, hydroxy1,3-dimethyltrimethylene group Group, hydroxy hexamethylene group, hydroxy 1-methyl pentamethylene group, hydroxy 2-methyl pentamethylene group, hydroxy 3-methyl pentamethylene group, hydroxy 1-ethyl tetramethylene group, hydroxy 2-ethyl tetramethylene group, hydroxy 1- n-propyl trimethylene group, hydroxy 1-isopropyl trimethylene group, hydroxy 2-n-propyl trimethylene group, hydroxy 2-isopropyl trimethylene group, hydroxy n-butylethylene group, Roxyisobutylethylene group, hydroxy tert-butylethylene group, hydroxy sec-butylethylene group, hydroxy n-pentylmethylene group, hydroxyisopentylmethylene group, hydroxyneopentylmethylene group, hydroxytert-pentylmethylene group, hydroxysec-pentylmethylene group Groups and the like. Among them, methylene group, ethylene group, methylmethylene group, trimethylene group, propylene group, ethylmethylene group, dimethylmethylene group, cyclopropylene group, hydroxymethylene group, hydroxyethylene group, hydroxytrimethylene group, hydroxypropylene group, hydroxyethylmethylene A group is preferable, a methylene group, an ethylene group, a trimethylene group, a hydroxymethylene group, a hydroxyethylene group, and a hydroxytrimethylene group are more preferable, and a trimethylene group and a 2-hydroxytrimethylene group are particularly preferable.
　一般式［４］におけるｎとしては１～３の整数が好ましく、３がより好ましい。 In the general formula [4], n is preferably an integer of 1 to 3, and more preferably 3.
　一般式［４］の好ましい具体例としては、例えば下記一般式［４’］で示される基が挙げられる。
Figure JPOXMLDOC01-appb-I000028

（式中、ｎ’は１～３の整数を表し、Ｙ’は置換基としてヒドロキシ基を有していてもよい炭素数１～３のアルキレン基を表す。） Preferable specific examples of the general formula [4] include, for example, a group represented by the following general formula [4 ′].
Figure JPOXMLDOC01-appb-I000028

(In the formula, n ′ represents an integer of 1 to 3, and Y ′ represents an alkylene group having 1 to 3 carbon atoms which may have a hydroxy group as a substituent.)
　一般式［４’］におけるｎ’は、３が好ましい。 In the general formula [4 ′], n ′ is preferably 3.
　一般式［４’］においてＹ’で示される置換基としてヒドロキシ基を有していてもよい炭素数１～３のアルキレン基は、直鎖状、分枝状又は環状のいずれであってもよいが、直鎖状のものが好ましい。一般式［４’］においてＹ’で示される置換基としてヒドロキシ基を有していてもよい炭素数１～３のアルキレン基は、炭素数３のアルキレン基が特に好ましい。具体的には、メチレン基、エチレン基、メチルメチレン基、トリメチレン基、プロピレン基（メチルエチレン基）、エチルメチレン基、ジメチルメチレン基、シクロプロピレン基、ヒドロキシメチレン基、ヒドロキシエチレン基、ヒドロキシトリメチレン基、ヒドロキシプロピレン基、ヒドロキシエチルメチレン基等が挙げられる。中でも、メチレン基、エチレン基、トリメチレン基、ヒドロキシメチレン基、ヒドロキシエチレン基、ヒドロキシトリメチレン基が好ましく、トリメチレン基、２－ヒドロキシトリメチレン基がより好ましい。 The alkylene group having 1 to 3 carbon atoms which may have a hydroxy group as a substituent represented by Y ′ in the general formula [4 ′] may be linear, branched or cyclic. However, a linear thing is preferable. In the general formula [4 ′], the alkylene group having 1 to 3 carbon atoms which may have a hydroxy group as a substituent represented by Y ′ is particularly preferably an alkylene group having 3 carbon atoms. Specifically, methylene group, ethylene group, methylmethylene group, trimethylene group, propylene group (methylethylene group), ethylmethylene group, dimethylmethylene group, cyclopropylene group, hydroxymethylene group, hydroxyethylene group, hydroxytrimethylene group , Hydroxypropylene group, hydroxyethylmethylene group and the like. Among these, a methylene group, an ethylene group, a trimethylene group, a hydroxymethylene group, a hydroxyethylene group, and a hydroxytrimethylene group are preferable, and a trimethylene group and a 2-hydroxytrimethylene group are more preferable.
　一般式［５］におけるＲ７で示される置換基としてヒドロキシ基を有する炭素数１～１０のアルキレン基は、炭素数１～６のものが好ましい。また、当該一般式［５］におけるＲ７で示される置換基としてヒドロキシ基を有する炭素数１～１０のアルキレン基は、直鎖状、分岐状、環状のいずれであってもよいが直鎖状のものが好ましい。一般式［５］におけるＲ７で示される置換基としてヒドロキシ基を有する炭素数１～１０のアルキレン基は、アルキレン基中の炭素原子に少なくとも一つ以上のヒドロキシ基が結合したものを表す。具体的には、ヒドロキシメチレン基、ヒドロキシエチレン基、ヒドロキシトリメチレン基、ヒドロキシプロピレン基、ヒドロキシエチルメチレン基、ヒドロキシテトラメチレン基、ヒドロキシ１－メチルトリメチレン基、ヒドロキシ２－メチルトリメチレン基、ヒドロキシ１，２－ジメチルエチレン基、ヒドロキシ１，１－ジメチルエチレン基、ヒドロキシエチルエチレン基、ヒドロキシｎ－プロピルメチレン基、ヒドロキシイソプロピルメチレン基、ヒドロキシペンタメチレン基、ヒドロキシ１－メチルテトラメチレン基、ヒドロキシ２－メチルテトラメチレン基、ヒドロキシ１－エチルトリメチレン基、ヒドロキシ２－エチルトリメチレン基、ヒドロキシｎ－プロピルエチレン基、ヒドロキシイソプロピルエチレン基、ヒドロキシｎ－ブチルメチレン基、ヒドロキシイソブチルメチレン基、ヒドロキシｔｅｒｔ－ブチルメチレン基、ヒドロキシｓｅｃ－ブチルメチレン基、ヒドロキシ２，２－ジメチルトリメチレン基、ヒドロキシ１，２－ジメチルトリメチレン基、ヒドロキシ１，３－ジメチルトリメチレン基、ヒドロキシヘキサメチレン基、ヒドロキシ１－メチルペンタメチレン基、ヒドロキシ２－メチルペンタメチレン基、ヒドロキシ３－メチルペンタメチレン基、ヒドロキシ１－エチルテトラメチレン基、ヒドロキシ２－エチルテトラメチレン基、ヒドロキシ１－ｎ－プロピルトリメチレン基、ヒドロキシ１－イソプロピルトリメチレン基、ヒドロキシ２－ｎ－プロピルトリメチレン基、ヒドロキシ２－イソプロピルトリメチレン基、ヒドロキシｎ－ブチルエチレン基、ヒドロキシイソブチルエチレン基、ヒドロキシｔｅｒｔ－ブチルエチレン基、ヒドロキシｓｅｃ－ブチルエチレン基、ヒドロキシｎ－ペンチルメチレン基、ヒドロキシイソペンチルメチレン基、ヒドロキシネオペンチルメチレン基、ヒドロキシｔｅｒｔ－ペンチルメチレン基、ヒドロキシｓｅｃ－ペンチルメチレン基、ヒドロキシヘプタメチレン基、ヒドロキシオクタメチレン基、ヒドロキシノナメチレン基、ヒドロキシデカメチレン基等が挙げられる。好ましい具体例としては、例えば下記一般式［６］で示されるものが挙げられる。
Figure JPOXMLDOC01-appb-I000029
（式中、ｒは１～１０の整数を表し、Ｘ’は水素原子又はヒドロキシ基を表す。） The alkylene group having 1 to 10 carbon atoms and having a hydroxy group as a substituent represented by R 7 in the general formula [5] preferably has 1 to 6 carbon atoms. In addition, the alkylene group having 1 to 10 carbon atoms and having a hydroxy group as a substituent represented by R 7 in the general formula [5] may be linear, branched or cyclic, but linear Are preferred. The alkylene group having 1 to 10 carbon atoms and having a hydroxy group as a substituent represented by R 7 in the general formula [5] represents a group in which at least one hydroxy group is bonded to a carbon atom in the alkylene group. Specifically, hydroxymethylene group, hydroxyethylene group, hydroxytrimethylene group, hydroxypropylene group, hydroxyethylmethylene group, hydroxytetramethylene group, hydroxy1-methyltrimethylene group, hydroxy2-methyltrimethylene group, hydroxy-1 , 2-dimethylethylene group, hydroxy 1,1-dimethylethylene group, hydroxyethylethylene group, hydroxy n-propylmethylene group, hydroxyisopropylmethylene group, hydroxypentamethylene group, hydroxy1-methyltetramethylene group, hydroxy-2-methyl Tetramethylene group, hydroxy 1-ethyltrimethylene group, hydroxy 2-ethyltrimethylene group, hydroxy n-propylethylene group, hydroxyisopropylethylene group, hydroxy n-butyl Lumethylene group, hydroxyisobutylmethylene group, hydroxy tert-butylmethylene group, hydroxysec-butylmethylene group, hydroxy2,2-dimethyltrimethylene group, hydroxy1,2-dimethyltrimethylene group, hydroxy1,3-dimethyltrimethylene group Group, hydroxy hexamethylene group, hydroxy 1-methyl pentamethylene group, hydroxy 2-methyl pentamethylene group, hydroxy 3-methyl pentamethylene group, hydroxy 1-ethyl tetramethylene group, hydroxy 2-ethyl tetramethylene group, hydroxy 1- n-propyl trimethylene group, hydroxy 1-isopropyl trimethylene group, hydroxy 2-n-propyl trimethylene group, hydroxy 2-isopropyl trimethylene group, hydroxy n-butylethylene group, Droxyisobutylethylene group, hydroxy tert-butylethylene group, hydroxy sec-butylethylene group, hydroxy n-pentylmethylene group, hydroxyisopentylmethylene group, hydroxyneopentylmethylene group, hydroxytert-pentylmethylene group, hydroxysec-pentyl Examples include a methylene group, a hydroxyheptamethylene group, a hydroxyoctamethylene group, a hydroxynonamethylene group, and a hydroxydecamethylene group. Preferable specific examples include those represented by the following general formula [6].
Figure JPOXMLDOC01-appb-I000029
(In the formula, r represents an integer of 1 to 10, and X ′ represents a hydrogen atom or a hydroxy group.)
　一般式［６］におけるＸ’は水素原子が好ましい。 X ′ in the general formula [6] is preferably a hydrogen atom.
　一般式［６］におけるｒは１～６の整数が好ましい。 R in the general formula [6] is preferably an integer of 1 to 6.
　一般式［５］におけるｐは１～３の整数が好ましい。 P in the general formula [5] is preferably an integer of 1 to 3.
　一般式［５］の好ましい具体例としては、下記一般式５’で示される基が挙げられる。
Figure JPOXMLDOC01-appb-I000030
（式中、ｐ及びｒは前記と同じである。） Preferable specific examples of the general formula [5] include a group represented by the following general formula 5 ′.
Figure JPOXMLDOC01-appb-I000030
(Wherein p and r are the same as above)
　一般式［２］の具体例としては、コール酸ナトリウム、コール酸カリウム、コール酸、デオキシコール酸ナトリウム、デオキシコール酸カリウム、デオキシコール酸、下記一般式［７］、及び一般式［８］で示される化合物が挙げられる。
Figure JPOXMLDOC01-appb-I000031
（式中、ｍ、ｎ、Ｒ１、及びＹ’は前記と同じである。）


Figure JPOXMLDOC01-appb-I000032

（式中、ｍは前記と同じであり、Ｒ１は水素原子又はヒドロキシ基であり、ｐ’及びｐ’’はそれぞれ独立して１～３の整数であり、ｒ’及びｒ’’はそれぞれ独立して１～６の整数である）。 Specific examples of the general formula [2] include sodium cholate, potassium cholate, cholic acid, sodium deoxycholate, potassium deoxycholate, deoxycholic acid, the following general formula [7], and general formula [8]. And the compounds shown.
Figure JPOXMLDOC01-appb-I000031
(In the formula, m, n, R 1 and Y ′ are the same as described above.)


Figure JPOXMLDOC01-appb-I000032

Wherein m is the same as defined above, R 1 is a hydrogen atom or a hydroxy group, p ′ and p ″ are each independently an integer of 1 to 3, and r ′ and r ″ are each Independently an integer from 1 to 6).
　一般式［２］の好ましい具体例としては、前記一般式［７］又は一般式［８］で示される化合物が挙げられ、下記一般式［７’］又は一般式［８］で示される化合物が好ましく、中でも、３－［（３－コールアミドプロピル）ジメチルアンモニオ］プロパンスルホネート（ＣＨＡＰＳ）、３－［（３－コールアミドプロピル）ジメチルアンモニオ］－２－ヒドロキシプロパンスルホネート（ＣＨＡＰＳＯ）、Ｎ，Ｎ－ビス（３－Ｄ－グルコンアミドプロピル）コールアミド（ＢＩＧＣＨＡＰ）及びＮ，Ｎ－ビス（３－Ｄ－グルコンアミドプロピル）デオキシコールアミド（ｄｅｏｘｙ－ＢＩＧＣＨＡＰ）がより好ましい。

Figure JPOXMLDOC01-appb-I000033


（式中、ｍ、ｎ及びＹ’は前記と同じである。） Preferable specific examples of the general formula [2] include compounds represented by the general formula [7] or the general formula [8], and compounds represented by the following general formula [7 ′] or the general formula [8] Among them, among others, 3-[(3-cholamidopropyl) dimethylammonio] propanesulfonate (CHAPS), 3-[(3-cholamidopropyl) dimethylammonio] -2-hydroxypropanesulfonate (CHAPSO), N, N-bis (3-D-gluconamidopropyl) coleamide (BIGCHAP) and N, N-bis (3-D-gluconamidopropyl) deoxycholamide (deoxy-BIGCHAP) are more preferred.

Figure JPOXMLDOC01-appb-I000033


(In the formula, m, n and Y ′ are the same as described above.)
　本発明に係る化合物は、自体公知の方法に準じて合成しても、市販のものを用いてもよい。 The compound according to the present invention may be synthesized according to a method known per se, or a commercially available product may be used.
　本発明の充填用溶液は、本発明に係る化合物を水溶液に含有させたものであり、プラスチック製マイクロ流体デバイスに充填させるための溶液である。 The filling solution of the present invention contains the compound according to the present invention in an aqueous solution, and is a solution for filling a plastic microfluidic device.
　本発明に係る化合物の濃度は、プラスチック製マイクロ流体デバイスに充填させる際の溶液中（本発明の充填用溶液中）の濃度として、通常０．０００１ｗ／ｗ％～２０ｗ／ｗ％、好ましくは０．００５ｗ／ｗ％～１５ｗ／ｗ％、より好ましくは０．０１ｗ／ｗ％～１０ｗ／ｗ％、更に好ましくは０．１ｗ／ｗ％～５ｗ／ｗ％である。 The concentration of the compound according to the present invention is usually 0.0001 w / w% to 20 w / w%, preferably 0, as the concentration in the solution when filling the plastic microfluidic device (in the filling solution of the present invention). 0.005 w / w% to 15 w / w%, more preferably 0.01 w / w% to 10 w / w%, still more preferably 0.1 w / w% to 5 w / w%.
　本発明に係る化合物を含有させる水溶液としては、通常この分野において用いられるものであればよく、例えば水、緩衝液等が挙げられる。このような緩衝液としては、例えばｐＨ５．０～１０．０、好ましくはｐＨ７．０～８．０の中性付近に緩衝作用を有する、リン酸緩衝液、トリス緩衝液、グッド緩衝液、グリシン緩衝液、ホウ酸緩衝液等が挙げられる。また、緩衝液中の緩衝剤濃度は、通常１０～５００ｍＭ、好ましくは１０～１００ｍＭの範囲から適宜選択すればよい。また、本発明の充填用溶液中には、本発明に係る化合物の他に、自体公知の方法に準じて、この分野で用いられる試薬類、糖類、核酸、タンパク質、塩類等の安定化剤、防腐剤等（以下、試薬類と略記する場合がある）を共存させてもよい。本発明の充填用溶液を各種反応に用いる場合に共存させる試薬類等の具体例を以下に示す。
［化学反応に用いる場合］
縮合反応に用いる場合：縮合剤、カルボン酸、アミノ化合物
置換反応に用いる場合：求核性物質、求電子性物質
加水分解反応に用いる場合：エステル化合物、塩基性物質
［生化学反応に用いる場合］
オキシドレダクターゼ反応に用いる場合：酸化酵素、還元酵素、酵素基質、緩衝剤
トランスフェラーゼ反応に用いる場合：トランスアミナーゼ、キナーゼ、酵素基質、緩衝剤
ヒドロラーゼ反応に用いる場合：リパーゼ、プロテアーゼ、ヌクレアーゼ、酵素基質、緩衝剤
免疫反応（抗原抗体反応）に用いる場合：抗原、抗体、緩衝剤
［核酸増幅反応に用いる場合］
ＰＣＲ反応に用いる場合：ＤＮＡポリメラーゼ、ｄＮＴＰ　ｍｉｘ、プライマー、鋳型ＤＮＡ、鋳型ＲＮＡ、金属イオン、緩衝剤
ＬＡＭＰ反応に用いる場合：ＤＮＡポリメラーゼ、ｄＮＴＰ　ｍｉｘ、プライマー、標的ＤＮＡ、標的ＲＮＡ、金属イオン、緩衝剤
ＩＣＡＮ反応に用いる場合：ＤＮＡポリメラーゼ、ＲＮａｓｅＨ、ｄＮＴＰ　ｍｉｘ、プライマー、金属イオン、緩衝剤
ＬＣＲ反応に用いる場合：標的ＤＮＡ、オリゴヌクレオチドプローブ、耐熱性ＤＮＡリガーゼ、金属イオン、緩衝剤
ＳＤＡ反応に用いる場合：ＤＮＡポリメラーゼ、制限酵素、プライマー、ｄＮＴＰ　ｍｉｘ、標的ＤＮＡ、標的ＲＮＡ、金属イオン、緩衝剤
　また、本発明の充填用溶液は、上記各種反応に用いられる溶液に本発明に係る化合物を上記濃度含有させたものであってもよい。該各種反応に用いられる溶液としては、例えば、上記各種反応に用いる場合に共存させる試薬類等を含むもの等が挙げられる。
　本発明の充填用溶液は、該各種反応に用いられるものの中でも、生化学反応に用いるもの、核酸増幅反応に用いられるものが好ましく、ＰＣＲ反応に用いられるものが特に好ましい。 The aqueous solution containing the compound according to the present invention may be any aqueous solution usually used in this field, and examples thereof include water and a buffer solution. Examples of such a buffer solution include a phosphate buffer solution, a Tris buffer solution, a Good buffer solution, a glycine having a buffering action in the vicinity of neutrality of pH 5.0 to 10.0, preferably pH 7.0 to 8.0. Examples include a buffer solution and a borate buffer solution. Further, the concentration of the buffering agent in the buffer may be appropriately selected from the range of usually 10 to 500 mM, preferably 10 to 100 mM. Further, in the filling solution of the present invention, in addition to the compound according to the present invention, in accordance with a method known per se, reagents used in this field, stabilizers such as sugars, nucleic acids, proteins, salts, An antiseptic or the like (hereinafter sometimes abbreviated as “reagents”) may coexist. Specific examples of reagents and the like that coexist when the filling solution of the present invention is used in various reactions are shown below.
[When used for chemical reaction]
When used for condensation reaction: When used for substitution reaction of condensing agent, carboxylic acid, amino compound: When used for hydrolysis reaction of nucleophilic substance and electrophilic substance: Ester compound, basic substance [When used for biochemical reaction]
When used for oxidoreductase reaction: For oxidase, reductase, enzyme substrate, buffer transferase reaction: For transaminase, kinase, enzyme substrate, buffer hydrolase reaction: Lipase, protease, nuclease, enzyme substrate, buffer When used for immune reaction (antigen-antibody reaction): antigen, antibody, buffer [when used for nucleic acid amplification reaction]
When used for PCR reaction: DNA polymerase, dNTP mix, primer, template DNA, template RNA, metal ion, buffer When used for LAMP reaction: DNA polymerase, dNTP mix, primer, target DNA, target RNA, metal ion, buffer When used for ICAN reaction: When used for DNA polymerase, RNaseH, dNTP mix, primer, metal ion, buffer LCR reaction: When used for target DNA, oligonucleotide probe, thermostable DNA ligase, metal ion, buffer SDA reaction: DNA polymerase, restriction enzyme, primer, dNTP mix, target DNA, target RNA, metal ion, buffer In addition, the filling solution of the present invention contains the above-mentioned concentration of the compound of the present invention in the solution used for the above various reactions. Tama It may be. Examples of the solution used in the various reactions include those containing reagents that coexist when used in the various reactions.
Among the solutions used for the various reactions, those used for biochemical reactions and those used for nucleic acid amplification reactions are preferable, and those used for PCR reactions are particularly preferable.
　本発明の充填用溶液は、前記の本発明に係る化合物を水溶液に溶解（懸濁）させることにより得られる。本明細書における溶液は、溶質が溶媒に溶解した溶液、および溶質が溶媒に分散した懸濁液を含む。本発明の充填用溶液は、具体的には例えば以下の方法で調製すればよい。
　終濃度が通常０．０００１ｗ／ｗ％～２０ｗ／ｗ％、好ましくは０．００５ｗ／ｗ％～１５ｗ／ｗ％、より好ましくは０．０１ｗ／ｗ％～１０ｗ／ｗ％、更に好ましくは０．１ｗ／ｗ％～５ｗ／ｗ％となる量の本発明に係る化合物を、例えば緩衝剤濃度が通常１０～５００ｍＭ、好ましくは１０～１００ｍＭであって、ｐＨ５．０～１０．０、好ましくはｐＨ７．０～８．０の中性付近に緩衝作用を有する、リン酸緩衝液、トリス緩衝液、グッド緩衝液、グリシン緩衝液、又はホウ酸緩衝液、例えば０．１ｍＬ～１００ｍＬに溶解（懸濁）させることにより得られる。 The filling solution of the present invention can be obtained by dissolving (suspending) the compound according to the present invention in an aqueous solution. The solution herein includes a solution in which a solute is dissolved in a solvent and a suspension in which the solute is dispersed in a solvent. Specifically, the filling solution of the present invention may be prepared, for example, by the following method.
The final concentration is usually from 0.0001 w / w% to 20 w / w%, preferably from 0.005 w / w% to 15 w / w%, more preferably from 0.01 w / w% to 10 w / w%, still more preferably 0. The compound according to the present invention in an amount of 1 w / w% to 5 w / w%, for example, has a buffer concentration of usually 10 to 500 mM, preferably 10 to 100 mM, and pH 5.0 to 10.0, preferably pH 7 Dissolve (suspended) in phosphate buffer, Tris buffer, Good's buffer, glycine buffer, or borate buffer, for example, 0.1 to 100 mL, which has a buffering action near neutrality of 0.0 to 8.0 ).
　プラスチック製マイクロ流体デバイスは、通常この分野で用いられるものであればよく、例えば少なくともチャネル及び／又はチャンバーを含むプラスチック製マイクロ流体デバイスが挙げられ、少なくともチャンバーを含むものが好ましく、チャネル及びチャンバーを含むものがより好ましい。プラスチック製マイクロ流体デバイスは、各種反応（例えば、化学反応、生化学反応、核酸増幅反応等）や各種分析（例えば、電気泳動、クロマトグラフィー、電気分析等）に用いられるものが好ましい。 Any plastic microfluidic device may be used as long as it is normally used in this field. For example, a plastic microfluidic device including at least a channel and / or a chamber is preferable. More preferred. The plastic microfluidic device is preferably used for various reactions (for example, chemical reaction, biochemical reaction, nucleic acid amplification reaction, etc.) and various analyzes (for example, electrophoresis, chromatography, electroanalysis, etc.).
　チャネルとは、本発明の充填用溶液等を移動する為又は電気泳動等の分離に用いる為等の毛細管（流路）であり、本発明の充填用溶液を充填し得るものであれば何れでもよい。本発明の充填用溶液を流す（充填する）方向に垂直な方向のチャネルの断面の形状はいずれでもよく、例えば、台形、長方形、正方形、真性円、楕円等が挙げられる。本発明の充填用溶液を流す（充填する）方向に垂直な方向のチャネルの断面積としては、通常０．０１ｍｍ２～１ｍｍ２、好ましくは０．０５ｍｍ２～０．７５ｍｍ２、より好ましくは０．１ｍｍ２～０．５ｍｍ２であり、本発明の充填用溶液を流す（充填する）方向のチャネルの長さは、通常０．１ｍｍ～１００ｃｍ、好ましくは１ｍｍ～２０ｃｍ、より好ましくは５ｍｍ～１０ｃｍである。一般的には、チャネルの端部は、ウェル（液だめ）と接続している。 A channel is a capillary (flow channel) for moving the filling solution of the present invention or used for separation such as electrophoresis, and any channel can be used as long as it can be filled with the filling solution of the present invention. Good. The cross-sectional shape of the channel in the direction perpendicular to the direction in which the filling solution of the present invention flows (fills) may be any, and examples thereof include a trapezoid, a rectangle, a square, an intrinsic circle, and an ellipse. The cross-sectional area of the channel perpendicular to the direction in which the filling solution of the present invention flows (fills) is usually 0.01 mm 2 to 1 mm 2 , preferably 0.05 mm 2 to 0.75 mm 2 , more preferably 0. a .1mm 2 ~ 0.5mm 2, flow filling solution of the present invention (filling) the length direction of the channel is normally 0.1 mm ~ 100 cm, preferably from 1 mm ~ 20 cm, more preferably 5 mm ~ 10 cm It is. In general, the end of the channel is connected to a well (a liquid reservoir).
　チャンバーとは、チャネルよりも高さ（深さ）及び／又は縦幅及び／又は断面積が大きい空間を表す。チャネルとチャンバーとの関係を模式図（図１）に示す。なお、チャンバー及びチャネルの形状はこれに限定されない。本発明の充填用溶液の流れる方向に対するチャンバーの垂直方向の断面積（例えば図１（Ｂ）におけるＣ１とＣ２との総面積、高さ（深さ）×縦幅）は、本発明の充填用溶液を流す（充填する）方向に対するチャネルの垂直方向の断面積（例えば図１（Ｂ）におけるＣ２）に対して１．５倍以上であればよく、２倍以上が好ましく、２．５倍以上がより好ましい。
　チャンバーは、高さ（深さ）が通常０．００１ｍｍ～１０ｍｍ、好ましくは０．００５ｍｍ～１ｍｍ、より好ましくは、０．０１ｍｍ～０．５ｍｍ、縦幅が通常１ｍｍ～１００ｍｍ、好ましくは１ｍｍ～５０ｍｍ、より好ましくは１ｍｍ～１０ｍｍ、横幅（本発明の充填用溶液の流れる方向の幅）が通常０．００１ｍｍ～１００ｍｍ、好ましくは０．００５ｍｍ～５０ｍｍ、より好ましくは、０．０１ｍｍ～２０ｍｍ、容量が通常１μＬ～１０００μＬ、好ましくは１μＬ～１００μＬ、より好ましくは１０μＬ～１００μＬであり、ＰＣＲ等の各種反応や分析用途で用いられるものが好ましく、ＰＣＲ用途で用いられるものが特に好ましい。
　チャンバーは、底面と底面と向かい合う面を支持し、チャンバーの強度を補強するピラー（柱）を有していてもよい。該ピラーの形状は円柱状、多角柱状などいずれであってもよく、またチャンバーが有するピラーは一つであっても複数であってもよい。また、チャンバーは、内部に段差や溝、壁があってもよい。チャンバーはチャネルと連通しているのが好ましく、該チャネルは１つでも２つ以上でもよい。
　従って、本発明に係るプラスチック製マイクロ流体デバイスは少なくとも１つのチャンバー、該チャンバーに連通する少なくとも１つのチャネル及び該チャネルのチャンバーと連通していない端部に接続するウェルを有するものが好ましく、本発明はこのようなマイクロ流体デバイスに特に有用である。 The chamber represents a space having a height (depth) and / or a vertical width and / or a cross-sectional area larger than those of the channel. The schematic diagram (FIG. 1) shows the relationship between channels and chambers. Note that the shapes of the chamber and the channel are not limited thereto. The cross-sectional area in the vertical direction of the chamber with respect to the flow direction of the filling solution of the present invention (for example, the total area of C1 and C2 in FIG. 1B, height (depth) × vertical width) is the filling area of the present invention. The cross-sectional area in the vertical direction of the channel with respect to the direction of flowing (filling) the solution (for example, C2 in FIG. 1B) may be 1.5 times or more, preferably 2 times or more, and 2.5 times or more Is more preferable.
The height (depth) of the chamber is usually 0.001 mm to 10 mm, preferably 0.005 mm to 1 mm, more preferably 0.01 mm to 0.5 mm, and the vertical width is usually 1 mm to 100 mm, preferably 1 mm to 50 mm. More preferably, the width (width in the flowing direction of the filling solution of the present invention) is usually 0.001 mm to 100 mm, preferably 0.005 mm to 50 mm, more preferably 0.01 mm to 20 mm. Usually, 1 μL to 1000 μL, preferably 1 μL to 100 μL, more preferably 10 μL to 100 μL, and those used for various reactions such as PCR and analytical applications are preferable, and those used for PCR are particularly preferable.
The chamber may have a pillar (column) that supports the bottom surface and a surface facing the bottom surface and reinforces the strength of the chamber. The pillar may have a cylindrical shape or a polygonal column shape, and the chamber may have one or more pillars. The chamber may have a step, a groove, and a wall inside. The chamber is preferably in communication with a channel, and the channel may be one or more.
Therefore, the plastic microfluidic device according to the present invention preferably has at least one chamber, at least one channel communicating with the chamber, and a well connected to an end not communicating with the chamber of the channel. Is particularly useful for such microfluidic devices.
　プラスチック製マイクロ流体デバイスの素材は、所謂プラスチックであればいずれでもよく、例えば、ポリカーボネート、ポリエチレン、ポリプロピレン、ポリアクリル酸等が挙げられる。ポリカーボネートとしては、例えばユーピロン（登録商標、三菱瓦斯化学株式会社）、ノバレックス（登録商標、三菱化学株式会社）、ザンター（登録商標、ミツビシ　ケミカル　ヨーロッパ　ゲーエムベーハー）等が挙げられ、ポリエチレンとしては、例えばスパイダックス（登録商標、三菱化学株式会社）、ナックセーフ（登録商標、株式会社ＮＵＣ）、ニポロン（登録商標、東ソ－株式会社）等が挙げられ、ポリプロピレンとしては、例えばウィンテック（登録商標、日本ポリプロ株式会社）、ニューコン（登録商標、日本ポリプロ株式会社）、ファンクスター（登録商標、日本ポリプロ株式会社）等が挙げられ、ポリアクリル酸としては、例えばミンロン（登録商標、イー・アイ・デュポン・ドウ・ヌムール・アンド・カンパニー）、リルサン（登録商標、アルケマ　フランス）、レオナ（登録商標、旭化成株式会社）等が挙げられる。
　これらの中でも、特にＰＣＲ反応、ＬＡＭＰ反応、ＩＣＡＮ反応、ＬＣＲ反応、ＳＤＡ反応等の核酸増幅反応に用いる場合には、該マイクロ流体デバイス内に温度を加える必要がある為、その素材は、熱耐性を有するものが好ましく、この点からもポリカーボネートが特に好ましい。
　該マイクロ流体デバイスは、５０℃～１２０℃に耐えられるものが好ましく、８０℃～１１０℃に耐えられるものがより好ましく、９０℃～１０５℃に耐えられるものが更に好ましく、９５℃～１００℃に耐えられるものが特に好ましい。 The material of the plastic microfluidic device may be any so-called plastic, and examples thereof include polycarbonate, polyethylene, polypropylene, and polyacrylic acid. Examples of the polycarbonate include Iupilon (registered trademark, Mitsubishi Gas Chemical Co., Ltd.), Novalex (registered trademark, Mitsubishi Chemical Corporation), Zanta (registered trademark, Mitsubishi Chemical Europe GmbH), and polyethylene includes, for example, spy Ducks (registered trademark, Mitsubishi Chemical Corporation), Nak Safe (registered trademark, NUC Corporation), Nipolon (registered trademark, Tosoh Corporation), etc., and examples of polypropylene include Wintec (registered trademark, Japan) Polypro Corporation), Newcon (Registered Trademark, Nippon Polypro Corporation), Funxter (Registered Trademark, Nippon Polypro Corporation), and the like. Examples of polyacrylic acid include Minlon (Registered Trademark, EI DuPont).・ Dou Nemours & Company), Li San (registered trademark, Arkema France), Leona (registered trademark, Asahi Kasei Co., Ltd.), and the like.
Among these, in particular, when used for nucleic acid amplification reactions such as PCR reaction, LAMP reaction, ICAN reaction, LCR reaction, SDA reaction, etc., it is necessary to add temperature to the microfluidic device. In view of this, polycarbonate is particularly preferable.
The microfluidic device is preferably one that can withstand 50 ° C. to 120 ° C., more preferably one that can withstand 80 ° C. to 110 ° C., more preferably one that can withstand 90 ° C. to 105 ° C., and 95 ° C. to 100 ° C. Those that can withstand are particularly preferred.
　プラスチック製マイクロ流体デバイスは、マイクロリアクターやマイクロアナリシスシステムの分野等において使用する分析チップを作製する際に一般に行われている方法に準じて作成すればよく、例えばチャンバーの形を型取ったプラスチック基板にプラスチック製のフイルムを接合させることにより得られる。プラスチック基板の成形方法としては、射出形成により製造する方法が挙げられる。チャンバーの形を型取ったプラスチック基板とプラスチック製フイルムとの接合方法としては、熱圧着や超音波、レーザーにより接合する方法が挙げられる。各種接合条件は、プラスチック基板やプラスチック製フイルムの素材によって、適切なものを選択すればよい。前記の具体的な方法は、例えば特許文献２－４（特許文献２：ＷＯ２０１２／０６０１８、特許文献３：特開２０１３－４４５２８、特許文献４：特開２００９－２２６５０３）に記載の方法に準じてなされればよい。 A plastic microfluidic device may be prepared in accordance with a method generally used when producing an analysis chip used in the field of microreactor or microanalysis system, for example, a plastic substrate shaped like a chamber It can be obtained by bonding a plastic film to the film. Examples of the method for molding the plastic substrate include a method of manufacturing by injection molding. As a method for joining the plastic substrate having the shape of the chamber and the plastic film, there may be mentioned a method of joining by thermocompression bonding, ultrasonic waves or laser. Various bonding conditions may be selected depending on the material of the plastic substrate or the plastic film. The specific method is based on, for example, the method described in Patent Document 2-4 (Patent Document 2: WO2012 / 06018, Patent Document 3: JP2013-44528, Patent Document 4: JP2009-226503). It only has to be done.
　本発明の充填用溶液により充填されたプラスチック製マイクロ流体デバイスは、縮合反応、置換反応、加水分解反応等の化学反応やオキシドレダクターゼ反応、トランスフェラーゼ反応、ヒドロラーゼ反応、免疫反応（抗原抗体反応）等の生化学反応、ポリメラーゼ連鎖反応（ＰＣＲ）法（特開昭６０－２８１号公報）、ＬＡＭＰ（Ｌｏｏｐ－ｍｅｄｉａｔｅｄ　Ｉｓｏｔｈｅｒｍａｌ　Ａｍｐｌｉｆｉｃａｔｉｏｎ）法（Ｔｓｕｇｕｎｏｒｉ　Ｎｏｔｏｍｉ　ｅｔ　ａｌ．，　Ｎｕｃｌｅｉｃ　Ａｃｉｄ　Ｒｅｓ．，　２８，　ｅ６３，　２０００）、ＩＣＡＮ（登録商標）（Ｉｓｏｔｈｅｒｍａｌ　ａｎｄ　Ｃｈｉｍｅｒｉｃ　ｐｒｉｍｅｒ－ｉｎｉｔｉａｔｅｄ　Ａｍｐｌｉｆｉｃａｔｉｏｎ　ｏｆ　Ｎｕｃｌｅｉｃ　ａｃｉｄｓ）法（臨床病理，　５１（１１），　１０６１－１０６７，　２００３，　Ｎｏｖ）、ＬＣＲ（ｌｉｇａｓｅ　ｃｈａｉｎ　ｒｅａｃｔｉｏｎ）法（特開平４－２１１３９９号）、ＳＤＡ（ｓｔｒａｎｄ　ｄｉｓｐｌａｃｅｍｅｎｔ　ａｍｐｌｉｆｉｃａｔｉｏｎ）法（特開平８－１９３９４号）等の核酸増幅反応等に用いることができ、好ましくは生化学反応や核酸増幅反応、より好ましくはＰＣＲ反応に用いられる。 The plastic microfluidic device filled with the filling solution of the present invention includes chemical reactions such as condensation reaction, substitution reaction, hydrolysis reaction, oxidoreductase reaction, transferase reaction, hydrolase reaction, immune reaction (antigen-antibody reaction), etc. Biochemical reaction, polymerase chain reaction (PCR) method (Japanese Patent Laid-Open No. Sho 60-281), LAMP (Loop-mediated Isolation Amplification) method (Tsungunori Notomi et al., Nucleic Acid Res., 28, e63, 2000) ICAN (registered trademark) (Isothermal and Chimeric primer-initiated Amplification of Nucleic Acids) (Clinical Pathology, 51 (11), 1061-1067, 2003, Nov), LCR (ligase chain reaction) method (Japanese Patent Laid-Open No. 4-21399), SDA (strand displacement amplification) method (Japanese Patent Laid-Open No. 8-19394), etc. It is preferably used for a biochemical reaction or a nucleic acid amplification reaction, more preferably a PCR reaction.
　本発明の充填方法は、プラスチック製マイクロ流体デバイスを水溶液で充填させる方法である。具体的には、プラスチックス製マイクロ流体デバイスのチャネル及び／又はチャンバーを水溶液で充填させる方法である。本発明の方法は、本発明の充填用溶液を用いる以外は、自体公知の方法に準じてプラスチック製マイクロ流体デバイスのチャネル及び／又はチャンバーを充填させればよい。 The filling method of the present invention is a method of filling a plastic microfluidic device with an aqueous solution. Specifically, the channel and / or chamber of a plastics microfluidic device is filled with an aqueous solution. In the method of the present invention, except for using the filling solution of the present invention, the channel and / or chamber of the plastic microfluidic device may be filled in accordance with a method known per se.
　例えば、本発明の充填用溶液をプラスチック製マイクロ流体デバイスの端部（チャネルの端部又はチャンバーの端部或いはウェル）に圧力を印加して、又は重力を利用して、或いは毛管現象を利用することによりプラスチック製マイクロ流体デバイスに充填させる方法が挙げられ、毛管現象を利用することによる充填が好ましい。 For example, the filling solution of the present invention is applied to the end of a plastic microfluidic device (the end of a channel or the end of a chamber or a well), utilizing gravity, or utilizing capillary action. Thus, there is a method of filling a plastic microfluidic device, and filling by utilizing capillary action is preferable.
　圧力を印加することにより充填する方法における印加する圧力は、本発明の充填用溶液を、チャネルを通じてプラスチック製マイクロ流体デバイスに充填させることが可能な大きさであればよい。 The pressure to be applied in the method of filling by applying pressure may be a size that allows the filling solution of the present invention to be filled into the plastic microfluidic device through the channel.
　本発明の充填方法は、例えば以下のように行えばよい。
　（１）本発明の充填用溶液を調製する。
　終濃度が通常０．０００１ｗ／ｗ％～２０ｗ／ｗ％、好ましくは０．００５ｗ／ｗ％～１５ｗ／ｗ％、より好ましくは０．０１ｗ／ｗ％～１０ｗ／ｗ％、更に好ましくは０．１ｗ／ｗ％～５ｗ／ｗ％となる量の本発明に係る化合物を、水やこの分野において通常用いられる緩衝液に溶解させた溶液を調製し、本発明の充填用溶液とする。
　（２）プラスチック製マイクロ流体デバイスの端部（チャネルの端部又はチャンバーの端部或いはウェル）からプラスチック製マイクロ流体デバイスのチャネル及び／又はチャンバーに、本発明の充填用溶液を該マイクロ流体デバイス内（チャネル及び／又はチャンバー内）が満たされる量以上注入し、毛管現象又は重力により本発明の充填用溶液を該デバイス内（チャネル及び／又はチャンバー内）へ充填する。 What is necessary is just to perform the filling method of this invention as follows, for example.
(1) The filling solution of the present invention is prepared.
The final concentration is usually from 0.0001 w / w% to 20 w / w%, preferably from 0.005 w / w% to 15 w / w%, more preferably from 0.01 w / w% to 10 w / w%, still more preferably 0. A solution is prepared by dissolving the compound of the present invention in an amount of 1 w / w% to 5 w / w% in water or a buffer solution usually used in this field, and is used as the filling solution of the present invention.
(2) The filling solution of the present invention is put into the channel and / or chamber of the plastic microfluidic device from the end of the plastic microfluidic device (the end of the channel or the end of the chamber or well) in the microfluidic device. An amount larger than that in which the (channel and / or chamber) is filled is injected, and the filling solution of the present invention is filled into the device (in the channel and / or chamber) by capillary action or gravity.
　また、図２のようにチャンバー、チャネル及びウェルを有するプラスチック製マイクロ流体デバイスを用いる場合、本発明の充填方法は、例えば以下のように行えばよい。
　（１）本発明の充填用溶液を調製する。
　終濃度が通常０．０００１ｗ／ｗ％～２０ｗ／ｗ％、好ましくは０．００５ｗ／ｗ％～１５ｗ／ｗ％、より好ましくは０．０１ｗ／ｗ％～１０ｗ／ｗ％、更に好ましくは０．１ｗ／ｗ％～５ｗ／ｗ％となる量の本発明に係る化合物を、水やこの分野において通常用いられる緩衝液に溶解させた溶液を調製し、本発明の充填用溶液とする。
　（２）プラスチック製マイクロ流体デバイスに結合したウェル（注入用ウェルとする）に、本発明の充填用溶液を該マイクロ流体デバイス内（チャネル及び／又はチャンバー内）が満たされる量以上注入する。
　（３）注入用ウェルが結合するチャネルから、該チャネルと結合したチャンバーを通して、該チャンバーに結合した少なくとも一つのチャネルの該チャンバーと結合していない端部に結合するウェルであって前記注入用ウェル以外のウェル（排出用ウェルとする）が結合するチャネルまで、毛管現象を利用して該溶液を移動させることにより、該溶液を該チャンバーへ充填する。 Moreover, when using the plastic microfluidic device which has a chamber, a channel, and a well like FIG. 2, the filling method of this invention may be performed as follows, for example.
(1) The filling solution of the present invention is prepared.
The final concentration is usually from 0.0001 w / w% to 20 w / w%, preferably from 0.005 w / w% to 15 w / w%, more preferably from 0.01 w / w% to 10 w / w%, still more preferably 0. A solution is prepared by dissolving the compound of the present invention in an amount of 1 w / w% to 5 w / w% in water or a buffer solution usually used in this field, and is used as the filling solution of the present invention.
(2) A filling solution of the present invention is injected into a well (injection well) coupled to a plastic microfluidic device in an amount sufficient to fill the microfluidic device (channel and / or chamber).
(3) a well coupled to an end of the at least one channel coupled to the chamber not coupled to the chamber from a channel coupled to the injection well through a chamber coupled to the channel; The chamber is filled with the solution by moving the solution to the channel to which the other well (which is referred to as a discharge well) is coupled using capillary action.
　本発明の充填方法によれば、空気（気泡）を残存させることなくプラスチック製マイクロ流体デバイス内を水溶液で一様に充填することができ、プラスチック製マイクロ流体デバイスのプラスチック表面にケミカルアタックによる破断が生じない。特に、本発明の充填方法により本発明の充填用溶液を充填したプラスチック製マイクロ流体デバイスは、加熱や冷却を繰り返しても、破断が生じない。また、本発明の充填方法は、特別な装置を必要とせず、時間経過による影響を受けない。 According to the filling method of the present invention, the inside of the plastic microfluidic device can be uniformly filled with an aqueous solution without leaving air (bubbles), and the plastic surface of the plastic microfluidic device is broken by a chemical attack. Does not occur. In particular, the plastic microfluidic device filled with the filling solution of the present invention by the filling method of the present invention does not break even when heating and cooling are repeated. Moreover, the filling method of the present invention does not require a special device and is not affected by the passage of time.
　プラスチック製マイクロ流体デバイスがチャンバー及びチャネルを有している場合、これらの形状及び／又は大きさに起因して気泡が発生しやすくなる場合があるが、本発明の充填方法によれば当該チャンバー及びチャネルの形状によらず空気（気泡）を残存させることなくプラスチック製マイクロ流体デバイス内を水溶液で一様に充填することができ、またケミカルアタックも生じない。即ち、プラスチック製マイクロ流体デバイスがチャンバー及びチャネルを有している場合に本発明の充填方法は特に有用である。
　また、プラスチック製マイクロ流体デバイスがピラーを有している場合も気泡が発生しやすくなる場合があるが、本発明の充填方法によれば空気（気泡）を残存させることなくプラスチック製マイクロ流体デバイス内を水溶液で一様に充填することができ、またケミカルアタックも生じないことから、プラスチック製マイクロ流体デバイスがピラーを有している場合にも本発明の充填方法は有用である。 When the plastic microfluidic device has a chamber and a channel, bubbles may be easily generated due to the shape and / or size thereof. According to the filling method of the present invention, the chamber and the channel The inside of the plastic microfluidic device can be uniformly filled with an aqueous solution without leaving air (bubbles) regardless of the shape of the channel, and no chemical attack occurs. That is, the filling method of the present invention is particularly useful when the plastic microfluidic device has a chamber and a channel.
In addition, when the plastic microfluidic device has pillars, bubbles may be easily generated. However, according to the filling method of the present invention, the plastic microfluidic device does not leave air (bubbles). Can be uniformly filled with an aqueous solution and no chemical attack occurs, the filling method of the present invention is useful even when the plastic microfluidic device has pillars.
　本発明の充填方法は、プラスチック製マイクロ流体デバイスを加温する場合、言い換えれば高い温度条件下で使用されるプラスチック製マイクロ流体デバイスに特に有用であり、プラスチック製マイクロ流体デバイスを加温する温度としては、５０℃～１２０℃が好ましく、８０℃～１１０℃がより好ましく、９０℃～１０５℃が更に好ましく、９５℃～１００℃が特に好ましい。 The filling method of the present invention is particularly useful when a plastic microfluidic device is heated, in other words, a plastic microfluidic device used under a high temperature condition. Is preferably 50 ° C to 120 ° C, more preferably 80 ° C to 110 ° C, still more preferably 90 ° C to 105 ° C, and particularly preferably 95 ° C to 100 ° C.
　本発明のＰＣＲ方法は、前記式［１］で示されるシクロペンタヒドロフェナントレン骨格を有する化合物を含むＰＣＲ反応溶液をプラスチック製マイクロ流体デバイスへ充填し、該デバイスをＰＣＲ反応に付す方法である。本発明のＰＣＲ方法におけるＰＣＲ反応溶液（以下、本発明に係るＰＣＲ反応溶液と略記する場合がある）は、自体公知のＰＣＲ反応に用いられるＰＣＲ反応溶液中に、前記式［１］で示されるシクロペンタヒドロフェナントレン骨格を有する化合物を含有させたものであればいずれでもよく、自体公知のＰＣＲ反応溶液に含まれる試薬等を含むこと以外は、本発明の充填用溶液の定義に準ずる。自体公知のＰＣＲ反応溶液に含まれる試薬としては、例えば、ＤＮＡポリメラーゼ、ｄＮＴＰ　ｍｉｘ、プライマー、鋳型ＤＮＡ、鋳型ＲＮＡ、金属イオン、緩衝剤等が挙げられる。 The PCR method of the present invention is a method of filling a plastic microfluidic device with a PCR reaction solution containing a compound having a cyclopentahydrophenanthrene skeleton represented by the formula [1] and subjecting the device to a PCR reaction. A PCR reaction solution in the PCR method of the present invention (hereinafter sometimes abbreviated as a PCR reaction solution according to the present invention) is represented by the above formula [1] in a PCR reaction solution used for a PCR reaction known per se. Any compound may be used as long as it contains a compound having a cyclopentahydrophenanthrene skeleton, and it conforms to the definition of the filling solution of the present invention except that it contains a reagent contained in a PCR reaction solution known per se. Examples of reagents contained in a PCR reaction solution known per se include DNA polymerase, dNTP mix, primer, template DNA, template RNA, metal ion, buffering agent and the like.
　本発明のＰＣＲ方法において、前記式［１］で示されるシクロペンタヒドロフェナントレン骨格を有する化合物を含むＰＣＲ反応溶液をプラスチック製マイクロ流体デバイスへ充填する際の全ての条件は、前記の本発明の充填方法に準ずる。 In the PCR method of the present invention, all conditions for filling a PCR microfluidic device with a PCR reaction solution containing a compound having a cyclopentahydrophenanthrene skeleton represented by the above formula [1] are the same as those of the above-described filling of the present invention. Follow the method.
　本発明のＰＣＲ方法は、前記式［１］で示されるシクロペンタヒドロフェナントレン骨格を有する化合物を含むＰＣＲ反応溶液を充填したプラスチック製マイクロ流体デバイスを用いる以外は、自体公知の方法に準じてＰＣＲ法を行えばよい。自体公知のＰＣＲ法としては、最も一般的なポリメラーゼ連鎖反応（ＰＣＲ）法（特開昭６０－２８１号公報）に限定されず、ＴａｑＭａｎ（登録商標）リアルタイムＰＣＲ法（例えば米国特許第５５３８８４８号の記載参照）、ＭＧＢ　Ｅｃｌｉｐｓｅ　Ｐｒｏｂｅ　Ｓｙｓｔｅｍ法（例えば米国特許第５，８０１，１５５号の記載参照）、Ｍｏｌｅｃｕｌａｒ　Ｂｅａｃｏｎｓ　Ｐｒｏｂｅ　Ｔｅｃｈｎｏｌｏｇｙ法（例えば米国特許第５９２５５１７号の記載参照）、ＬＵＸ　Ｆｌｕｏｒｏｇｅｎｉｃ　Ｐｒｉｍｅｒ法（Ｉｎｖｉｔｒｏｇｅｎ　Ｃｏｒｐｏｒａｔｉｏｎ）、Ｑｕｅｎｃｈｉｎｇ　ｐｒｏｂｅ－ＰＣＲ（ＱＰ）法（例えば米国特許第６，４９２，１２１号の記載参照）等のリアルタイムＰＣＲ法等の改良された公知のＰＣＲ法が挙げられる。 The PCR method of the present invention is a PCR method according to a method known per se except that a plastic microfluidic device filled with a PCR reaction solution containing a compound having a cyclopentahydrophenanthrene skeleton represented by the formula [1] is used. Just do. The PCR method known per se is not limited to the most common polymerase chain reaction (PCR) method (Japanese Patent Laid-Open No. 60-281), but TaqMan (registered trademark) real-time PCR method (for example, US Pat. No. 5,538,848). Description), MGB Eclipse Probe System method (see, for example, the description in US Pat. No. 5,801,155), Molecular Beacons Probe Technology method (see, for example, the description in US Pat. No. 5,925,517), LUX Fluorogenic Prime method (see, for example, US Pat. No. 5,925,517). Improved known P such as real-time PCR method such as Quenching probe-PCR (QP) method (for example, see description in US Pat. No. 6,492,121) R method, and the like.
　本発明のＰＣＲ方法は、例えば以下のように行えばよい。
（１）本発明に係るＰＣＲ反応溶液を調製する。
　終濃度が通常０．０００１ｗ／ｗ％～２０ｗ／ｗ％、好ましくは０．００５ｗ／ｗ％～１５ｗ／ｗ％、より好ましくは０．０１ｗ／ｗ％～１０ｗ／ｗ％、更に好ましくは０．１ｗ／ｗ％～５ｗ／ｗ％となる量の本発明に係る化合物を、ＰＣＲ反応溶液（ＤＮＡポリメラーゼ、ｄＮＴＰ　ｍｉｘ、プライマー、鋳型ＤＮＡ、鋳型ＲＮＡ、金属イオン、緩衝剤等を含む溶液）に溶解させた溶液を調製し、本発明に係るＰＣＲ反応溶液とする。
（２）プラスチック製マイクロ流体デバイスの端部に結合したウェル（注入用ウェルとする）に、本発明に係るＰＣＲ反応溶液を該マイクロ流体デバイス内が満たされる量以上注入する。
（３）注入用ウェルが結合するチャネルから、該チャネルと結合したチャンバーを通して、該チャンバーに結合した少なくとも一つのチャネルの該チャンバーと結合していない端部に結合するウェルであって前記注入用ウェル以外のウェル（排出用ウェルとする）が結合するチャネルまで、毛管現象を利用して該反応溶液を移動させることにより、該反応溶液を該チャンバーへ充填する。
（４）本発明に係るＰＣＲ反応溶液を充填したプラスチック製マイクロ流体デバイスを、例えば（ｉ）９０℃～９８℃で１０秒～５分、（ｉｉ）「９０℃～９８℃で１０秒～１分、４５℃～６５℃で１０秒～１分、６８℃～７２℃で１０秒～２分」を１サイクルとして１５サイクル～４０サイクル、（ｉｉｉ）６８℃～７２℃で３０秒～５分、反応させてＰＣＲ反応を行う。 The PCR method of the present invention may be performed as follows, for example.
(1) A PCR reaction solution according to the present invention is prepared.
The final concentration is usually from 0.0001 w / w% to 20 w / w%, preferably from 0.005 w / w% to 15 w / w%, more preferably from 0.01 w / w% to 10 w / w%, still more preferably 0. Dissolve the compound of the present invention in an amount of 1 w / w% to 5 w / w% in a PCR reaction solution (a solution containing DNA polymerase, dNTP mix, primer, template DNA, template RNA, metal ion, buffer, etc.). The solution thus prepared is prepared as a PCR reaction solution according to the present invention.
(2) A PCR reaction solution according to the present invention is injected into a well (referred to as an injection well) coupled to an end of a plastic microfluidic device in an amount sufficient to fill the microfluidic device.
(3) a well coupled to an end of the at least one channel coupled to the chamber not coupled to the chamber from a channel coupled to the injection well through a chamber coupled to the channel; The reaction solution is filled into the chamber by moving the reaction solution to a channel to which a well other than the well (to be a discharge well) is coupled using capillary action.
(4) A plastic microfluidic device filled with a PCR reaction solution according to the present invention is prepared by, for example, (i) 90 ° C. to 98 ° C. for 10 seconds to 5 minutes, (ii) “90 ° C. to 98 ° C. for 10 seconds to 1 Minutes, 45 ° C to 65 ° C for 10 seconds to 1 minute, 68 ° C to 72 ° C for 10 seconds to 2 minutes ", 15 cycles to 40 cycles, (iii) 68 ° C to 72 ° C for 30 seconds to 5 minutes Then, the PCR reaction is performed.
　本発明のＰＣＲ方法によれば、ケミカルアタックを生じずに、また空気（気泡）を残存させることなく本発明に係るＰＣＲ反応溶液で一様に充填されたプラスチック製マイクロ流体デバイス内を用いることで、該デバイス内の温度が均一となり、正確な酵素反応を行うことができる。 According to the PCR method of the present invention, by using the inside of the plastic microfluidic device uniformly filled with the PCR reaction solution according to the present invention without causing chemical attack and without leaving air (bubbles). The temperature in the device becomes uniform, and an accurate enzyme reaction can be performed.
　本発明の充填方法は、プラスチック製マイクロ流体デバイスを本発明の充填用溶液で一様に充填させることができ、該チャンバーのプラスチック表面に対してケミカルアタックによる破断を生じさせないことから、該チャンバー内で酵素反応、ＰＣＲ反応等の温度変化を伴う反応を行う場合において有用である。 In the filling method of the present invention, the plastic microfluidic device can be uniformly filled with the filling solution of the present invention, and the plastic surface of the chamber is not broken by a chemical attack. This is useful when conducting reactions involving temperature changes such as enzymatic reactions and PCR reactions.
　以下、実施例および比較例に基づいて本発明を具体的に説明するが、本発明はこれらの例によって何ら限定されない。 Hereinafter, the present invention will be specifically described based on examples and comparative examples, but the present invention is not limited to these examples.
　＜実験例１　プラスチック製マイクロ流体デバイスの作成＞
　図２に示すように、縦幅６ｍｍ、横幅１５ｍｍ、深さ（高さ）０．３５ｍｍ、チャネルの幅が１ｍｍ、深さが０．３５ｍｍ、ウェルの直径が３ｍｍ、ピラーの直径が０．６ｍｍ、ピラーの高さが０．３５ｍｍとなるようにポリカーボネート製樹脂を射出成形により成形し、プラスチック基板を作成した。厚さ０．１２５ｍｍのポリカーボネート製樹脂フィルムをプラスチック基板に重ねて、１５０℃、圧力２５０ｋｇ、圧着時間１０分の条件で熱圧着させてプラスチック製マイクロ流体デバイスを作成した。 <Experimental Example 1 Production of Plastic Microfluidic Device>
As shown in FIG. 2, the vertical width is 6 mm, the horizontal width is 15 mm, the depth (height) is 0.35 mm, the channel width is 1 mm, the depth is 0.35 mm, the well diameter is 3 mm, and the pillar diameter is 0.6 mm. A polycarbonate resin was formed by injection molding a polycarbonate resin so that the pillar height was 0.35 mm. A polycarbonate resin film having a thickness of 0.125 mm was placed on a plastic substrate and thermocompression bonded under the conditions of 150 ° C., pressure 250 kg, and pressure bonding time 10 minutes to produce a plastic microfluidic device.
　＜実施例１－１１　界面活性剤含有水溶液のチャンバーへの充填＞
　（１）界面活性剤含有水溶液の調製
　下記のシクロペンタヒドロフェナトレン骨格を有する各種界面活性剤と水とを混合して下記表１に示す所定濃度（ｗ／ｗ％）の界面活性剤含有水溶液を調製した。
　［シクロペンタヒドロフェナトレン骨格を有する各種界面活性剤］
・３－［（３－コールアミドプロピル）ジメチルアンモニオ］プロパンスルホネート（ＣＨＡＰＳ）（（株）同仁化学研究所製）
・３－［（３－コールアミドプロピル）ジメチルアンモニオ］－２－ヒドロキシプロパンスルホネート（ＣＨＡＰＳＯ）（（株）同仁化学研究所製）
・Ｎ，Ｎ－ビス（３－Ｄ－グルコンアミドプロピル）コールアミド（ＢＩＧＣＨＡＰ）（（株）同仁化学研究所製）
・Ｎ，Ｎ－ビス（３－Ｄ－グルコンアミドプロピル）デオキシコールアミド（ｄｅｏｘｙ－ＢＩＧＣＨＡＰ）（（株）同仁化学研究所製）
・コール酸ナトリウム（和光純薬工業（株）製）
・デオキシコール酸ナトリウム（和光純薬工業（株）製）
　（２）界面活性剤含有水溶液のチャンバーへの充填
　得られた界面活性剤含有水溶液２５μＬをピペットマンにて、実験例１で作成したポリカーボネート製マイクロ流体デバイスの一方のウェル（注入用ウェル）から注入し、毛管現象にて界面活性剤含有水溶液を該ウェルからチャンバーを通してもう一方のウェル（排出用ウェル）に移動させることにより、チャンバー内に界面活性剤含有水溶液を導入した。
　目視にて充填時のチャンバー内の界面活性剤含有水溶液の流れ、充填速度について確認をし、◎＞○＞△＞×の４段階で評価した。各記号は、それぞれ以下の結果であったことを示す。
　◎：水溶液が非常に滑らかに充填される
　○：水溶液が滑らかに充填される
　△：水溶液が（滞留しながら）充填される
　×：水溶液充填時に泡がみが発生する
下記表１にその結果を示す。また、以下の比較例でも充填結果を同様に評価した。
　（３）ケミカルアタックの影響の確認
　界面活性剤含有水溶液が充填されたポリカーボネート製マイクロ流体デバイスを加温し、該水溶液のポリカーボネート製マイクロ流体デバイスに対するケミカルアタックの影響を確認した。即ち、前記（２）で得られた１％ＣＨＡＰＳ含有水溶液を充填させたポリカーボネート製マイクロ流体デバイスを、「９７℃　６秒、６１℃　６秒」を１サイクルとして４０サイクル加温と冷却を繰り返した。反応後のポリカーボネート製マイクロ流体デバイスのプラスチック表面の状態を、顕微鏡（ＹＨ－Ｚ１００Ｒ）（キーエンス製）を用いて観察し、クラックの有無を確認した。その結果を下記表１に（２）で得られた結果と併せて示す。
　（４）結果 <Example 1-11 Filling of chamber with surfactant-containing aqueous solution>
(1) Preparation of surfactant-containing aqueous solution Surfactant-containing aqueous solution having a predetermined concentration (w / w%) shown in Table 1 below by mixing water with various surfactants having the following cyclopentahydrophenatrene skeleton. Was prepared.
[Various surfactants having cyclopentahydrophenatrene skeleton]
・ 3-[(3-Choleamidopropyl) dimethylammonio] propanesulfonate (CHAPS) (manufactured by Dojindo Laboratories)
・ 3-[(3-Choleamidopropyl) dimethylammonio] -2-hydroxypropanesulfonate (CHAPSO) (manufactured by Dojindo Laboratories)
・ N, N-bis (3-D-gluconamidopropyl) coleamide (BIGCHAP) (manufactured by Dojindo Laboratories)
N, N-bis (3-D-gluconamidopropyl) deoxycholamide (deoxy-BIGCHAP) (manufactured by Dojindo Laboratories)
・ Sodium cholate (Wako Pure Chemical Industries, Ltd.)
・ Sodium deoxycholate (manufactured by Wako Pure Chemical Industries, Ltd.)
(2) Filling the chamber with the surfactant-containing aqueous solution 25 μL of the obtained surfactant-containing aqueous solution was injected from one well (injection well) of the polycarbonate microfluidic device created in Experimental Example 1 with Pipetteman. Then, the surfactant-containing aqueous solution was introduced into the chamber by moving the surfactant-containing aqueous solution from the well through the chamber to the other well (discharge well) by capillary action.
The flow of the surfactant-containing aqueous solution in the chamber at the time of filling and the filling speed were confirmed by visual observation, and evaluated in four stages: ◎>◯>Δ> ×. Each symbol indicates the following result.
◎: Aqueous solution is filled very smoothly ○: Aqueous solution is filled smoothly △: Aqueous solution is filled (while staying) ×: Foaming occurs when filling with aqueous solution The results are shown in Table 1 below. Show. Also, the filling results were similarly evaluated in the following comparative examples.
(3) Confirmation of influence of chemical attack The polycarbonate microfluidic device filled with the surfactant-containing aqueous solution was heated, and the influence of the chemical attack on the polycarbonate microfluidic device of the aqueous solution was confirmed. That is, the polycarbonate microfluidic device filled with the 1% CHAPS-containing aqueous solution obtained in the above (2) was repeatedly heated and cooled for 40 cycles with “97 ° C. 6 seconds, 61 ° C. 6 seconds” as one cycle. . The state of the plastic surface of the polycarbonate microfluidic device after the reaction was observed using a microscope (YH-Z100R) (manufactured by Keyence) to confirm the presence or absence of cracks. The results are shown in Table 1 below together with the results obtained in (2).
(4) Results
Figure JPOXMLDOC01-appb-T000034
 　シクロペンタヒドロフェナトレン骨格を有する各種界面活性剤を含有する水溶液を用いると、いずれもポリカーボネート製マイクロ流体デバイスに、空気（気泡）を残存させることなく一様にチャンバー内に該水溶液を充填できることが判った。
　また、ＣＨＡＰＳ含有水溶液を用いると、ＣＨＡＰＳ含有水溶液の濃度が０．００１％、０．０１％、０．１％、１％及び１０％のいずれの場合でも、ポリカーボネート製マイクロ流体デバイスに、空気（気泡）を残存させることなく一様にチャンバー内に該水溶液を充填できることが判った。
　シクロペンタヒドロフェナトレン骨格を有する各種界面活性剤を含有する水溶液としては、ＣＨＡＰＳ、ＣＨＡＰＳＯ、ＢＩＧＣＨＡＰ、又はｄｅｏｘｙ－ＢＩＧＣＨＡＰを含有する水溶液が特に好ましいことが判った。
　また、シクロペンタヒドロフェナトレン骨格を有する各種界面活性剤を含有する水溶液は、ポリカーボネート製マイクロ流体デバイスの加熱及び冷却を繰り返しても、いずれもケミカルアタックによるクラックを生じさせないことが判った。
Figure JPOXMLDOC01-appb-T000034
 When an aqueous solution containing various surfactants having a cyclopentahydrophenatrene skeleton is used, the aqueous solution can be uniformly filled in the chamber without leaving air (bubbles) in the polycarbonate microfluidic device. understood.
In addition, when a CHAPS-containing aqueous solution is used, the polycarbonate microfluidic device is supplied with air (even if the concentration of the CHAPS-containing aqueous solution is 0.001%, 0.01%, 0.1%, 1%, or 10%). It was found that the aqueous solution could be uniformly filled into the chamber without leaving bubbles.
As an aqueous solution containing various surfactants having a cyclopentahydrophenatrene skeleton, an aqueous solution containing CHAPS, CHAPSO, BIGCHAP, or deoxy-BIGCHAP was found to be particularly preferable.
It was also found that aqueous solutions containing various surfactants having a cyclopentahydrophenatrene skeleton did not cause cracks due to chemical attack even when heating and cooling of a polycarbonate microfluidic device were repeated.
　＜比較例１－１４　シクロペンタヒドロフェナトレン骨格を有さない化合物含有水溶液のチャンバーへの充填＞
　実施例１－１１におけるシクロペンタヒドロフェナトレン骨格を有する各種界面活性剤の代わりに下記表２に記載の各化合物と水とを混合して下記表２に示す所定濃度（ｗ／ｗ％）の化合物含有水溶液を調製し、界面活性剤含有水溶液の代わりに用いた以外は、実施例１－１１と同様の方法により実験を行った。
　なお、比較例１－１４で用いた化合物はいずれもシクロペンタヒドロフェナトレン骨格を有していないものである。
　チャンバーへの充填結果及びチャンバー表面への影響を下記表２に纏めて示す。

　［シクロペンタヒドロフェナトレン骨格を有さない化合物］
・４－ノニルフェニルポリ（エチレングリコール）（ＮＰ－４０）（シグマアルドリッチ合同会社製）
・ポリオキシエチレンラウリルエーテル（和光純薬工業（株）製）
・モノオレイン酸ポリオキシエチレンソルビタン（Ｔｗｅｅｎ８０）（和光純薬工業（株）製）
・ｎ－オクタノイル－Ｎ－メチル－Ｄ－グルカミン（ＭＥＧＡ８）（（株）同仁化学研究所製）
・ドデシル硫酸ナトリウム（ＳＤＳ）（和光純薬工業（株）製）
・臭化セチルトリメチルアンモニウム（ＣＴＡＢ）（和光純薬工業（株）製）
・モノラウリン酸ポリオキシエチレンソルビタン（Ｔｗｅｅｎ２０）（和光純薬工業（株）製）
・Ｎ，Ｏ－ビス（トリメチルシリル）アセトアミド（ＢＳＡ）（シグマアルドリッチ合同会社製）
・グリセリン（和光純薬工業（株）製）
・ラウリルジメチルアミノ酢酸ベタイン（アンヒトール２４Ｂ）（花王（株）製）
・Ｃｈｅｍｇａｒｄ　Ｓ－１００（ＣＨＥＭＧＵＡＲＤ，ＩＮＣ．製）
・ＫＦ－６４２（信越化学工業（株）製）
<Comparative Example 1-14 Filling of chamber with compound-containing aqueous solution having no cyclopentahydrophenatrene skeleton>
Instead of the various surfactants having a cyclopentahydrophenatrene skeleton in Example 1-11, each compound shown in Table 2 below and water were mixed to obtain a predetermined concentration (w / w%) shown in Table 2 below. An experiment was conducted in the same manner as in Example 1-11 except that a compound-containing aqueous solution was prepared and used instead of the surfactant-containing aqueous solution.
Note that none of the compounds used in Comparative Examples 1-14 have a cyclopentahydrophenatrene skeleton.
The results of filling the chamber and the effect on the chamber surface are summarized in Table 2 below.

[Compounds without cyclopentahydrophenatrene skeleton]
・ 4-Nonylphenyl poly (ethylene glycol) (NP-40) (manufactured by Sigma-Aldrich LLC)
・ Polyoxyethylene lauryl ether (Wako Pure Chemical Industries, Ltd.)
・ Polyoxyethylene sorbitan monooleate (Tween 80) (manufactured by Wako Pure Chemical Industries, Ltd.)
N-octanoyl-N-methyl-D-glucamine (MEGA8) (manufactured by Dojindo Laboratories)
・ Sodium dodecyl sulfate (SDS) (Wako Pure Chemical Industries, Ltd.)
・ Cetyltrimethylammonium bromide (CTAB) (manufactured by Wako Pure Chemical Industries, Ltd.)
・ Polyoxyethylene sorbitan monolaurate (Tween 20) (manufactured by Wako Pure Chemical Industries, Ltd.)
・ N, O-bis (trimethylsilyl) acetamide (BSA) (manufactured by Sigma-Aldrich LLC)
・ Glycerin (Wako Pure Chemical Industries, Ltd.)
・ Lauryldimethylaminoacetic acid betaine (Amphithol 24B) (manufactured by Kao Corporation)
・ Chemguard S-100 (manufactured by CHEMGUARD, INC.)
・ KF-642 (manufactured by Shin-Etsu Chemical Co., Ltd.)
Figure JPOXMLDOC01-appb-T000035
　ＮＰ－４０、ポリオキシエチレンラウリルエーテル、Ｔｗｅｅｎ８０、ＭＥＧＡ８、ＳＤＳ、又はＣＴＡＢを含有する水溶液はいずれもポリカーボネート製チャンバーに対し一様に該水溶液を充填できることが判った。しかし、ＣＨＡＰＳを含有する水溶液（実施例１）は水溶液が非常に滑らかに充填され、ＮＰ－４０、ポリオキシエチレンラウリルエーテル、又はＭＥＧＡ８を含有する水溶液は滑らかに充填され、Ｔｗｅｅｎ８０、ＳＤＳ、又はＣＴＡＢを含有する水溶液は充填できるもののチャンバー内で不均一な流れとなっていた。
　ＮＰ－４０、ポリオキシエチレンラウリルエーテル、又はＭＥＧＡ８を含有する水溶液を用いると、ポリカーボネート製マイクロ流体デバイスの加熱及び冷却を繰り返すと、いずれもケミカルアタックによるクラックを生じさせることが判った。
　また、Ｔｗｅｅｎ２０、ＢＳＡ、グリセリン、アンヒトール２４Ｂ、Ｃｈｅｍｇａｒｄ　Ｓ－１００、又はＫＦ－６４２を含有する水溶液は、いずれもポリカーボネート製チャンバーに対し一様に該水溶液を充填することができないことが判った。
　実施例１－１１及び比較例１－１４の結果から、シクロペンタヒドロフェナトレン骨格を有する各種界面活性剤を含有する水溶液は、空気（気泡）を残存させることなくプラスチック製マイクロ流体デバイス内を水溶液で一様に充填することができ、プラスチック製マイクロ流体デバイスのプラスチック表面にケミカルアタックによる破断を生じさせないことが判った。
　特に、シクロペンタヒドロフェナトレン骨格を有する各種界面活性剤を含有する水溶液を充填したプラスチック製マイクロ流体デバイスは、加熱や冷却を繰り返しても、破断が生じないことが判った。
Figure JPOXMLDOC01-appb-T000035
It has been found that any aqueous solution containing NP-40, polyoxyethylene lauryl ether, Tween 80, MEGA 8, SDS, or CTAB can uniformly fill the polycarbonate chamber. However, the aqueous solution containing CHAPS (Example 1) is very smoothly filled with the aqueous solution, and the aqueous solution containing NP-40, polyoxyethylene lauryl ether, or MEGA8 is smoothly filled with Tween 80, SDS, or CTAB. Although the aqueous solution containing was able to be filled, the flow was uneven in the chamber.
It was found that when an aqueous solution containing NP-40, polyoxyethylene lauryl ether, or MEGA8 was used, repeated heating and cooling of the polycarbonate microfluidic device caused cracks due to chemical attack.
It was also found that any aqueous solution containing Tween 20, BSA, glycerin, amphital 24B, Chemgard S-100, or KF-642 cannot uniformly fill the polycarbonate chamber.
Based on the results of Example 1-11 and Comparative Example 1-14, an aqueous solution containing various surfactants having a cyclopentahydrophenatrene skeleton is an aqueous solution in the plastic microfluidic device without leaving air (bubbles). It was found that the plastic surface of the plastic microfluidic device could not be broken by chemical attack.
In particular, it has been found that a plastic microfluidic device filled with an aqueous solution containing various surfactants having a cyclopentahydrophenatrene skeleton does not break even when heating and cooling are repeated.

Claims (20)

  1. 下記式［１］で示されるシクロペンタヒドロフェナントレン骨格を有する化合物を含む、プラスチック製マイクロ流体デバイスへの充填用溶液。
    Figure JPOXMLDOC01-appb-I000001
    A solution for filling a plastic microfluidic device comprising a compound having a cyclopentahydrophenanthrene skeleton represented by the following formula [1].
    Figure JPOXMLDOC01-appb-I000001
  2. シクロペンタヒドロフェナントレン骨格を有する化合物が、下記一般式［２］で示されるものである、請求項１に記載の溶液
    Figure JPOXMLDOC01-appb-I000002


    （式中、Ｒ１は水素原子又はヒドロキシ基を表し、Ｒ２はヒドロキシ基、－ＯＮａ基又は下記一般式［３］で示される基を表し、ｍは１～３の整数を表す）
    Figure JPOXMLDOC01-appb-I000003
    （式中、Ｒ３及びＲ４はそれぞれ独立して水素原子、下記一般式［４］で示される基、又は下記一般式［５］で示される基を表す）
    Figure JPOXMLDOC01-appb-I000004
    （式中、Ｒ５及びＲ６はそれぞれ独立して炭素数１～３のアルキル基、Ｙは置換基としてヒドロキシ基を有していてもよい炭素数１～６のアルキレン基、ｎは１～６の整数をそれぞれ表す）
    Figure JPOXMLDOC01-appb-I000005
    （式中、Ｒ７は置換基としてヒドロキシ基を有する炭素数１～１０のアルキレン基、ｐは１～６の整数をそれぞれ表す）。     The solution according to claim 1, wherein the compound having a cyclopentahydrophenanthrene skeleton is represented by the following general formula [2].
    Figure JPOXMLDOC01-appb-I000002


    (Wherein R 1 represents a hydrogen atom or a hydroxy group, R 2 represents a hydroxy group, —ONa group or a group represented by the following general formula [3], and m represents an integer of 1 to 3)
    Figure JPOXMLDOC01-appb-I000003
    (Wherein R 3 and R 4 each independently represent a hydrogen atom, a group represented by the following general formula [4], or a group represented by the following general formula [5])
    Figure JPOXMLDOC01-appb-I000004
    Wherein R 5 and R 6 are each independently an alkyl group having 1 to 3 carbon atoms, Y is an alkylene group having 1 to 6 carbon atoms which may have a hydroxy group as a substituent, and n is 1 to Each represents an integer of 6)
    Figure JPOXMLDOC01-appb-I000005
    (Wherein R 7 represents a C 1-10 alkylene group having a hydroxy group as a substituent, and p represents an integer of 1-6).
  3. Ｒ２が前記一般式［３］で示される基である、請求項２に記載の溶液。 The solution according to claim 2, wherein R 2 is a group represented by the general formula [3].
  4. Ｒ３が水素原子、前記一般式［４］で示される基、又は前記一般式［５］で示される基であって、Ｒ４が前記一般式［４］で示される基、又は前記一般式［５］で示される基である、請求項３に記載の溶液。 R 3 is a hydrogen atom, a group represented by the general formula [4], or a group represented by the general formula [5], and R 4 is a group represented by the general formula [4], or the general formula The solution according to claim 3, which is a group represented by [5].
  5. シクロペンタヒドロフェナントレン骨格を有する化合物が、３－［（３－コールアミドプロピル）ジメチルアンモニオ］プロパンスルホネート（ＣＨＡＰＳ）、３－［（３－コールアミドプロピル）ジメチルアンモニオ］－２－ヒドロキシプロパンスルホネート（ＣＨＡＰＳＯ）、Ｎ，Ｎ－ビス（３－Ｄ－グルコンアミドプロピル）コールアミド（ＢＩＧＣＨＡＰ）、又は、Ｎ，Ｎ－ビス（３－Ｄ－グルコンアミドプロピル）デオキシコールアミド（ｄｅｏｘｙ－ＢＩＧＣＨＡＰ）である、請求項１に記載の溶液。 Compounds having a cyclopentahydrophenanthrene skeleton include 3-[(3-cholamidopropyl) dimethylammonio] propanesulfonate (CHAPS), 3-[(3-cholamidopropyl) dimethylammonio] -2-hydroxypropanesulfonate (CHAPSO), N, N-bis (3-D-gluconamidopropyl) coleamide (BIGCHAP), or N, N-bis (3-D-gluconamidopropyl) deoxycholamide (deoxy-BIGCHAP). The solution according to claim 1.
  6. プラスチック製マイクロ流体デバイスがポリカーボネート製マイクロ流体デバイスである、請求項１に記載の溶液。 The solution of claim 1, wherein the plastic microfluidic device is a polycarbonate microfluidic device.
  7. プラスチック製マイクロ流体デバイスがチャンバー又は／及びチャネルを有するものである、請求項１に記載の溶液。 The solution according to claim 1, wherein the plastic microfluidic device has a chamber or / and a channel.
  8. チャンバー又は／及びチャネルが加温されるものである、請求項７に記載の溶液。 The solution according to claim 7, wherein the chamber or / and the channel are heated.
  9. プラスチック製マイクロ流体デバイスへの充填用溶液におけるシクロペンタヒドロフェナントレン骨格を有する化合物の濃度が０．０００１ｗ／ｗ％～２０ｗ／ｗ％である、請求項１に記載の溶液。 The solution according to claim 1, wherein the concentration of the compound having a cyclopentahydrophenanthrene skeleton in the solution for filling the plastic microfluidic device is 0.0001 w / w% to 20 w / w%.
  10. 下記式［１］で示されるシクロペンタヒドロフェナントレン骨格を有する化合物を含むプラスチック製マイクロ流体デバイスへの充填用溶液をプラスチック製マイクロ流体デバイスへ充填する充填方法。
    Figure JPOXMLDOC01-appb-I000006
    A filling method for filling a plastic microfluidic device with a filling solution for a plastic microfluidic device containing a compound having a cyclopentahydrophenanthrene skeleton represented by the following formula [1].
    Figure JPOXMLDOC01-appb-I000006
  11. シクロペンタヒドロフェナントレン骨格を有する化合物が、下記一般式［２］で示されるものである、請求項１０に記載の充填方法。
    Figure JPOXMLDOC01-appb-I000007

    （式中、Ｒ１は水素原子又はヒドロキシ基を表し、Ｒ２はヒドロキシ基、－ＯＮａ基又は下記一般式［３］で示される基を表し、ｍは１～３の整数を表す）
    Figure JPOXMLDOC01-appb-I000008
    （式中、Ｒ３及びＲ４はそれぞれ独立して水素原子、下記一般式［４］で示される基、又は下記一般式［５］で示される基を表す）
    Figure JPOXMLDOC01-appb-I000009
    （式中、Ｒ５及びＲ６はそれぞれ独立して炭素数１～３のアルキル基、Ｙは置換基としてヒドロキシ基を有していてもよい炭素数１～６のアルキレン基、ｎは１～６の整数をそれぞれ表す）
    Figure JPOXMLDOC01-appb-I000010
    （式中、Ｒ７は置換基としてヒドロキシ基を有する炭素数１～１０のアルキレン基、ｐは１～６の整数をそれぞれ表す）。     The filling method according to claim 10, wherein the compound having a cyclopentahydrophenanthrene skeleton is represented by the following general formula [2].
    Figure JPOXMLDOC01-appb-I000007

    (Wherein R 1 represents a hydrogen atom or a hydroxy group, R 2 represents a hydroxy group, —ONa group or a group represented by the following general formula [3], and m represents an integer of 1 to 3)
    Figure JPOXMLDOC01-appb-I000008
    (Wherein R 3 and R 4 each independently represent a hydrogen atom, a group represented by the following general formula [4], or a group represented by the following general formula [5])
    Figure JPOXMLDOC01-appb-I000009
    Wherein R 5 and R 6 are each independently an alkyl group having 1 to 3 carbon atoms, Y is an alkylene group having 1 to 6 carbon atoms which may have a hydroxy group as a substituent, and n is 1 to Each represents an integer of 6)
    Figure JPOXMLDOC01-appb-I000010
    (Wherein R 7 represents a C 1-10 alkylene group having a hydroxy group as a substituent, and p represents an integer of 1-6).
  12. Ｒ２が前記一般式［３］で示される基である、請求項１１に記載の充填方法。 The filling method according to claim 11, wherein R 2 is a group represented by the general formula [3].
  13. Ｒ３が水素原子、前記一般式［４］で示される基、又は前記一般式［５］で示される基であって、Ｒ４が前記一般式［４］で示される基、又は前記一般式［５］で示される基である、請求項１２に記載の充填方法。 R 3 is a hydrogen atom, a group represented by the general formula [4], or a group represented by the general formula [5], and R 4 is a group represented by the general formula [4], or the general formula The filling method according to claim 12, which is a group represented by [5].
  14. シクロペンタヒドロフェナントレン骨格を有する化合物が、３－［（３－コールアミドプロピル）ジメチルアンモニオ］プロパンスルホネート（ＣＨＡＰＳ）、３－［（３－コールアミドプロピル）ジメチルアンモニオ］－２－ヒドロキシプロパンスルホネート（ＣＨＡＰＳＯ）、Ｎ，Ｎ－ビス（３－Ｄ－グルコンアミドプロピル）コールアミド（ＢＩＧＣＨＡＰ）、又は、Ｎ，Ｎ－ビス（３－Ｄ－グルコンアミドプロピル）デオキシコールアミド（ｄｅｏｘｙ－ＢＩＧＣＨＡＰ）である、請求項１０に記載の充填方法。 Compounds having a cyclopentahydrophenanthrene skeleton include 3-[(3-cholamidopropyl) dimethylammonio] propanesulfonate (CHAPS), 3-[(3-cholamidopropyl) dimethylammonio] -2-hydroxypropanesulfonate (CHAPSO), N, N-bis (3-D-gluconamidopropyl) coleamide (BIGCHAP), or N, N-bis (3-D-gluconamidopropyl) deoxycholamide (deoxy-BIGCHAP). The filling method according to claim 10.
  15. プラスチック製マイクロ流体デバイスがポリカーボネート製マイクロ流体デバイスである、請求項１０に記載の充填方法。 The filling method according to claim 10, wherein the plastic microfluidic device is a polycarbonate microfluidic device.
  16. プラスチック製マイクロ流体デバイスがチャンバー又は／及びチャネルを有するものである、請求項１０に記載の充填方法。 The filling method according to claim 10, wherein the plastic microfluidic device has a chamber or / and a channel.
  17. チャンバー又は／及びチャネルが加温されるものである、請求項１６に記載の充填方法。 The filling method according to claim 16, wherein the chamber or / and the channel are heated.
  18. プラスチック製マイクロ流体デバイスへの充填用溶液におけるシクロペンタヒドロフェナントレン骨格を有する化合物の濃度が０．０００１ｗ／ｗ％～２０ｗ／ｗ％である、請求項１０に記載の充填方法。 The filling method according to claim 10, wherein the concentration of the compound having a cyclopentahydrophenanthrene skeleton in the filling solution for the plastic microfluidic device is 0.0001 w / w% to 20 w / w%.
  19. 下記式［１］で示されるシクロペンタヒドロフェナントレン骨格を有する化合物を含むＰＣＲ反応溶液をプラスチック製マイクロ流体デバイスへ充填し、該デバイスをＰＣＲ反応に付す、ＰＣＲ方法。
    Figure JPOXMLDOC01-appb-I000011
    A PCR method comprising filling a plastic microfluidic device with a PCR reaction solution containing a compound having a cyclopentahydrophenanthrene skeleton represented by the following formula [1], and subjecting the device to a PCR reaction.
    Figure JPOXMLDOC01-appb-I000011
  20. シクロペンタヒドロフェナントレン骨格を有する化合物が、下記一般式［２］で示されるものである、請求項１９に記載のＰＣＲ方法。
    Figure JPOXMLDOC01-appb-I000012

    （式中、Ｒ１は水素原子又はヒドロキシ基を表し、Ｒ２はヒドロキシ基、－ＯＮａ基又は下記一般式［３］で示される基を表し、ｍは１～３の整数を表す）
    Figure JPOXMLDOC01-appb-I000013
    （式中、Ｒ３及びＲ４はそれぞれ独立して水素原子、下記一般式［４］で示される基、又は下記一般式［５］で示される基を表す）
    Figure JPOXMLDOC01-appb-I000014
    （式中、Ｒ５及びＲ６はそれぞれ独立して炭素数１～３のアルキル基、Ｙは置換基としてヒドロキシ基を有していてもよい炭素数１～６のアルキレン基、ｎは１～６の整数をそれぞれ表す）
    Figure JPOXMLDOC01-appb-I000015
    （式中、Ｒ７は置換基としてヒドロキシ基を有する炭素数１～１０のアルキレン基、ｐは１～６の整数をそれぞれ表す）。     The PCR method according to claim 19, wherein the compound having a cyclopentahydrophenanthrene skeleton is represented by the following general formula [2].
    Figure JPOXMLDOC01-appb-I000012

    (Wherein R 1 represents a hydrogen atom or a hydroxy group, R 2 represents a hydroxy group, —ONa group or a group represented by the following general formula [3], and m represents an integer of 1 to 3)
    Figure JPOXMLDOC01-appb-I000013
    (Wherein R 3 and R 4 each independently represent a hydrogen atom, a group represented by the following general formula [4], or a group represented by the following general formula [5])
    Figure JPOXMLDOC01-appb-I000014
    Wherein R 5 and R 6 are each independently an alkyl group having 1 to 3 carbon atoms, Y is an alkylene group having 1 to 6 carbon atoms which may have a hydroxy group as a substituent, and n is 1 to Each represents an integer of 6)
    Figure JPOXMLDOC01-appb-I000015
    (Wherein R 7 represents a C 1-10 alkylene group having a hydroxy group as a substituent, and p represents an integer of 1-6).
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