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WO2018025899A1 - Solution pour remplir un dispositif microfluidique en plastique - Google Patents

Solution pour remplir un dispositif microfluidique en plastique 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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Prior art keywords
group
general formula
microfluidic device
filling
solution
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English (en)
Japanese (ja)
Inventor
和宏 寺嶌
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Fujifilm Wako Pure Chemical Corp
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Wako Pure Chemical Industries Ltd
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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

Definitions

  • 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

Le problème abordé par la présente invention est de fournir un procédé pour remplir uniformément un dispositif microfluidique en plastique avec une solution aqueuse sans endommager une surface en plastique du dispositif, et une solution utilisée dans ledit procédé. La présente invention concerne une solution contenant un composé ayant un squelette cyclopentahydrophénanthrène représenté par la formule [1] pour remplir un dispositif microfluidique en plastique, et un procédé de remplissage permettant de remplir le dispositif avec la solution.
PCT/JP2017/027994 2016-08-03 2017-08-02 Solution pour remplir un dispositif microfluidique en plastique Ceased WO2018025899A1 (fr)

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JP2008536478A (ja) * 2005-02-15 2008-09-11 アプレラ コーポレイション 高分子電解質被覆イオン交換粒子
JP2009225796A (ja) * 2008-03-19 2009-10-08 F Hoffmann La Roche Ag 修飾されたランダマーの存在下での核酸増幅
JP2010512797A (ja) * 2006-12-19 2010-04-30 シグマ−アルドリッチ・カンパニー 耐熱性ｄｎａポリメラーゼと陰イオン界面活性剤または両性イオン界面活性剤との安定化組成物
JP2013545114A (ja) * 2010-12-09 2013-12-19 アボット ポイント オブ ケア インコーポレイテッド 試料の混合及び分析を容易にするために抗吸着剤を使用する装置及び方法
JP2015514994A (ja) * 2012-04-19 2015-05-21 和光純薬工業株式会社 反応生成物をリアルタイムにサンプリングするための方法
JP2016516562A (ja) * 2013-03-01 2016-06-09 ウェーブ ８０ バイオサイエンシズ インコーポレイテッド マイクロ流体処理のための方法およびシステム

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JP2008536478A (ja) * 2005-02-15 2008-09-11 アプレラ コーポレイション 高分子電解質被覆イオン交換粒子
JP2010512797A (ja) * 2006-12-19 2010-04-30 シグマ−アルドリッチ・カンパニー 耐熱性ｄｎａポリメラーゼと陰イオン界面活性剤または両性イオン界面活性剤との安定化組成物
JP2009225796A (ja) * 2008-03-19 2009-10-08 F Hoffmann La Roche Ag 修飾されたランダマーの存在下での核酸増幅
JP2013545114A (ja) * 2010-12-09 2013-12-19 アボット ポイント オブ ケア インコーポレイテッド 試料の混合及び分析を容易にするために抗吸着剤を使用する装置及び方法
JP2015514994A (ja) * 2012-04-19 2015-05-21 和光純薬工業株式会社 反応生成物をリアルタイムにサンプリングするための方法
JP2016516562A (ja) * 2013-03-01 2016-06-09 ウェーブ ８０ バイオサイエンシズ インコーポレイテッド マイクロ流体処理のための方法およびシステム

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