JP2005249399A - Micro-fluid element and its manufacturing method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a manufacturing method of a micro-fluid element for forming with the uniform thickness on the passage surface, a porous resin layer capable of immobilizing, for example, a protein such as an enzyme or an antigen, or a catalyst most suitably in great numbers without clogging the passage on the inner surface of the fine passage of the micro-fluid element. <P>SOLUTION: The porous resin layer having many pores on the surface is formed beforehand on a support, and a hollow part having the porous resin layer on the bottom surface is formed on the porous resin layer by using an active energy ray-curing composition. Then, a member working as a lid is fixed on the recessed part to form the passage, to thereby enable to form the porous resin layer easily with the uniform thickness on the passage surface. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a microfluidic device having a porous resin layer having a three-dimensional network structure on the inner surface of a flow path, and a method for manufacturing the same.

  In recent years, in various fields including medical diagnosis and biochemical tests, attempts to use microfluidic devices have been started to analyze components of trace fluids containing DNA, biochemical substances, and the like.

  A microfluidic device is also called a microfluidic device, a microfluidic device, a microfabricated device, a lab on chip, or a micro total analytical system (μ-TAS), By performing the reaction and analysis in a fine capillary channel inside, it is possible to speed up the reaction and analysis, reduce the required sample amount, and reduce waste.

  Using such a microfluidic device, when an enzyme, catalyst, functional group, etc. is immobilized on the inner surface of the flow channel and reacted with a sample in the fluid, a probe such as a DNA fragment of a specific sequence is immobilized. When detecting DNA or the like in a sample, it is important to immobilize more enzymes, catalysts, or probes such as DNA fragments in order to improve the reaction rate and analysis sensitivity.

  In order to increase the amount of the functional group, (bio) chemical substance, biological substance, or the like as described above in the flow path, it is preferable to make the flow path porous. The flow path in which a porous body is formed is a method in which a range from the surface of a silicon or aluminum plate to a certain depth is made porous by etching and heat treatment, and a lid is fixed to the porous surface. A formed flow path is known in which the entire interior is a porous body (see Patent Document 1). However, since the entire interior of the flow channel is a porous body, and the liquid flowing through the flow channel flows through the pores of the porous body, several hundreds of fluids are required to flow through the flow channel at a sufficient flow rate. High pressure over KPa was required. For this reason, the microfluidic device body and the connection port for introducing the liquid need to have a sturdy structure that can withstand high pressure. In addition, these inorganic materials are limited in terms of the type of functional groups introduced on the surface, and the immobilization density of the functional groups is small, so that even if they are made porous, they are insufficient. Furthermore, since silicon and metal are optically opaque and dyes and fluorescent dyes fixed inside the porous body cannot be observed from the outside, they did not contribute to improving the sensitivity of light absorption or fluorescence detection. And since it is also optically opaque, in the fluorescence measurement, the amount of scattering of the excitation light is large, and the excitation light that is not completely cut by the filter enters the light receiving side, so the baseline of the measured fluorescence intensity is As a result, the S / N ratio was lowered and the reliability was lowered. Furthermore, since silicon and metal have high thermal conductivity, it is difficult to provide a temperature gradient in the flow path, and there are restrictions on use as a microfluidic device.

  On the other hand, the resin (organic polymer) has many types of functional groups that can be introduced onto the surface, and the immobilization density of the functional groups can be increased, which is preferable as a material constituting the inner surface of the flow path. (See Patent Document 2). However, in a method for forming a groove-like flow path as known as a method for manufacturing a resin microfluidic device (see, for example, Patent Document 3), fine irregularities are formed on the surface of a substrate by an electronic etching method. The active energy ray-curable compound is applied on the surface, and the active energy ray-curable compound is cured by irradiating the energy ray to the portion other than the portion that forms the flow path, and the uncured active energy ray is cured in the unirradiated portion. Although a method of forming a groove-like flow path having fine irregularities on the bottom surface by removing a functional compound has been described, the flow path bottom surface of the microfluidic device is simply for imparting hydrophilicity. Only the unevenness is provided, and the three-dimensional network porous layer is not formed. Although it is not known whether the microfluidic device on the inner surface having such irregularities has a larger amount to fix the probe than the microfluidic device that does not treat the inner surface, according to the confirmation experiment of the present inventors. The degree of increase was small and not enough.

  On the other hand, as a method for increasing the amount of enzyme or catalyst immobilized on the surface of a plate or the like rather than on the surface of the flow path, a method of forming a thin porous layer on the surface of the plate and immobilizing it is disclosed ( Patent Document 2). However, a method for providing such a porous layer on one surface of the fine channel inner surface of the microfluidic device has not been known so far.

  Further, a hydrophilic solution can be obtained by applying a mixed solution of an energy ray curable resin, a chain polymer and a solvent to a substrate, irradiating energy, and then contacting the chain polymer with a non-solvent to cause phase separation. A method for producing a porous film is disclosed (see Patent Document 4). However, with respect to this method as well, a method for providing such a resin with a uniform thickness on the inner surface of a fine channel of a microfluidic device has not been known.

JP-A-6-169756 JP 2000-2705 A JP 2000-46797 A JP-A-10-007,835

  The problem to be solved by the present invention is that, without blocking the flow path on the inner surface of the fine flow path of the microfluidic device, for example, proteins such as enzymes and antigens, or catalysts, etc. can be optimally immobilized. A microfluidic device in which a porous resin layer having a three-dimensional network structure is formed on the surface of the flow channel with a uniform thickness, and a microfluidic device in which the porous resin layer is formed in an arbitrary position in the flow direction of the flow channel Another object of the present invention is to provide a microfluidic device in which the porous tree layer is formed in a part of a cross section of a flow path, and a method for manufacturing the microfluidic device.

  As a result of intensive studies on a method for solving the above problems, the inventors of the present invention formed a porous resin layer having a three-dimensional network structure on the surface in advance on the support, and cured active energy rays on the porous resin layer. The above-mentioned problem can be solved by forming a recess having a three-dimensional network structure porous resin layer on the bottom surface using an adhesive composition and then forming a flow path by fixing a member serving as a lid to the recess. As a result, the present invention has been completed.

  That is, in a microfluidic device comprising a support, a porous resin layer having a three-dimensional network structure, a flow path and a lid, (I) having the porous resin layer on the support, and (II) the porous The resin layer is filled with a cured resin of the active energy ray curable resin composition (X) impregnated except for the flow path portion, and (III) the cured resin of the active energy ray curable resin composition (X) is formed in the flow path. A porous resin layer having a three-dimensional network structure that is not filled with an active material formed on the porous resin layer having a three-dimensional network structure that is filled with a cured resin of the active energy ray-curable resin composition (X). The present invention provides a microfluidic device characterized in that a cured resin layer of an energy ray curable resin composition (X) and a lid portion are used as wall surfaces to form a hollow shape.

  The present invention also includes (1) a step of forming a porous resin layer having a three-dimensional network structure having a large number of pores on the surface of the support, and (2) active energy ray polymerizable property on the porous resin layer. Applying the active energy ray-curable composition (X) containing the compound (a) to form an uncured coating film of the composition (X), and the uncured coating film other than the portion to be the flow path Is irradiated with an active energy ray to form a cured or semi-cured coating film of the composition (X), and the uncured composition (X) in the non-irradiated portion is removed to form a porous three-dimensional network structure Forming a recess having a resin layer exposed on the bottom surface; and (3) fixing another member serving as a lid to the recess of the member having the recess to form the recess as a hollow flow path. A method of manufacturing a microfluidic device, preferably a three-dimensional network structure on the surface of the support The step of forming the porous resin layer is compatible with the active energy ray-polymerizable compound (b) and the compound (b) on the support, but is not compatible with the polymer formed from the compound (b). After applying an active energy ray-curable film-forming solution (J) containing an insoluble poor solvent (R), the film-forming solution (J) is irradiated with active energy rays to form a tertiary on the surface of the support. The present invention provides a method for manufacturing a microfluidic device, which is a step of forming a porous resin layer having an original network structure.

  Since the microfluidic device of the present invention has a thin porous resin layer fixed to the inner surface of the flow path, the functional group, (bio) chemical substance, or even in a fine region without clogging the flow path, A large amount of biological material can be immobilized. In addition, since the porous resin layer has a three-dimensional network structure, the porous resin layer has a very large surface area and can immobilize a large amount of probes. Further, a rectangular or trapezoidal shape having a porous resin layer having a three-dimensional network structure formed on one or two surfaces of the inner surface of the flow path, in particular, an average distance to the inner wall facing the porous resin layer is Those in the range of 1 to 50 μm can effectively generate an affinity between the analyte to be moved through the porous portion and the immobilized probe. For this reason, when the microfluidic device of the present invention is used, processing such as accurate and rapid synthesis, separation, and analysis becomes possible. Especially, it can utilize suitably also for DNA analysis by making the fixed probe into an oligonucleotide.

  When the microfluidic device of the present invention is used as a member for liquid chromatography, high-speed analysis is possible even when the developing solution is flowed at a low pressure, so that the separation column, the connecting portion with the developing solution introduction pipe, etc. It is not necessary to have a high pressure resistance, and it is easy to incorporate in μ-TAS, and in that case, it is not necessary to make the entire μ-TAS device into a sturdy structure. In addition, the substance to be separated can be preferably separated. In addition, the amount of (bio) chemical substances that have functional groups and molecular recognition functions can be greatly increased compared to conventional methods, so that the allowable amount of sample introduction is increased, quantitativeness and accuracy are improved, and sensitivity is improved. , Can be planned. Furthermore, since it is highly productive and can be manufactured at low cost, it can also be used in disposable applications.

  In addition, since the porous resin layer having a three-dimensional network structure can be formed only on the bottom surface of the flow path, the surface of the porous resin layer can be optically observed through the flow path, and high sensitivity. In addition, highly quantitative measurement is possible.

  Further, when the microfluidic device of the present invention is used as a member for affinity electrophoresis, analysis can be performed without using a sol or gel as an electrophoresis medium, so that a complicated preparation operation is required prior to use. It is unnecessary, has little performance degradation during market distribution, and can be stored in a dry state. Therefore, storage and market distribution are easy. In addition, the amount of (bio) chemical substances that have functional groups and molecular recognition functions can be greatly increased compared to conventional methods, so that the allowable amount of sample introduction is increased, quantitativeness and accuracy are improved, and sensitivity is improved. Is planned. Furthermore, since the porous resin layer having a three-dimensional network structure can be formed only on the bottom surface of the flow path, the surface of the porous resin layer can be optically observed through the flow path, and high sensitivity. In addition, highly quantitative measurement is possible.

  When the microfluidic device of the present invention is used as a member for microarrays for DNA analysis, immunodiagnosis, etc., the amount of probes immobilized can be greatly increased compared to the conventional method, so that detection sensitivity is improved and quantitativeness is improved. The analysis can be speeded up. In addition, since a porous resin layer having a three-dimensional network structure is formed as a spot in the flow path and a different probe can be fixed to each spot, multi-item analysis can be performed with a single flow path. Furthermore, since the porous resin layer having a three-dimensional network structure can be formed only on the bottom surface of the flow path, the surface of the porous resin layer can be optically observed through the flow path, and high sensitivity. In addition, highly quantitative measurement is possible.

  When the microfluidic device of the present invention is used as a reaction tank or reaction tube of a microreactor, the amount of catalyst, enzyme, etc. can be increased, so that the reaction rate and yield can be improved. In addition, a porous resin layer with a three-dimensional network structure can be formed in any area in the flow path, and different catalysts can be fixed in each area, making it possible to perform multistage reactions in a single flow path. is there.

  By using the manufacturing method of the present invention, the difficulty and limitation of forming a porous resin layer later in the narrow channel can be released, so that the channel on the inner surface of the micro channel of the microfluidic device In addition, it is possible to easily manufacture a microfluidic device having a porous resin layer having a three-dimensional network structure with a uniform thickness on the surface of the channel. In addition, the thickness, pore shape, and pore diameter of the porous resin layer can be easily adjusted to optimum values for the purpose of use. Furthermore, it is also easy to provide a porous resin layer forming portion having a three-dimensional network structure having an arbitrary length, for example, a spot-like porous resin layer portion, in a part of the flow direction of the flow path. Further, the fluid in the flow channel does not leak through the porous resin layer portion other than the porous resin layer on the bottom surface of the flow channel.

  According to the production method of the present invention, the pore diameter and thickness of the porous resin layer having a three-dimensional network structure formed on the inner surface of the fine channel can be easily adjusted to an optimum value for the purpose of use. The specific surface area is large, many substances can be immobilized, and analysis and detection of (bio) chemistry with high sensitivity and accuracy can be performed in a short time with a very small amount of test solution. Moreover, since a porous resin layer having a three-dimensional network structure can be formed only on the bottom surface of the flow path, the surface of the porous resin layer can be optically observed through the flow path, and used for analysis. In some cases, highly sensitive measurement is possible. Furthermore, it is easy to provide a porous resin layer forming portion having an arbitrary length, for example, a spot-like porous resin layer portion, in a part of the flow direction of the flow path.

  In addition, after the step of forming the porous resin layer having a three-dimensional network structure, by providing a step of surface-treating or modifying the surface of the porous resin layer, a functional group having reactivity on the surface of the porous resin layer is provided. Groups can be introduced and reacted with these functional groups to fix various (bio) chemical substances and biological substances to the surface of the porous resin layer by covalent bonds. Of course, these substances can also be fixed by adsorption using ionic bonds or hydrophobic bonds. At this time, as described above, a plurality of porous resin layer spots can be formed, and different types and concentrations of functional groups can be introduced into the plurality of porous spots, or different types of substances can be fixed easily. .

  Furthermore, when the viscosity of the composition (X) at 25 ° C. is set to 30 to 3000 mPa · s, when the composition (X) is applied onto the porous resin layer having a three-dimensional network structure, the composition (X ) Rapidly penetrates into the porous resin layer, and a microfluidic device in which the fluid in the flow channel does not leak through the porous resin layer portion other than the porous resin layer on the bottom surface of the flow channel can be easily manufactured. Further, by setting the viscosity within the above range, the composition (X) is completely removed from the porous resin layer when the uncured composition (X) in the non-irradiated part is removed after irradiation with the active energy ray. The

The method for producing a microfluidic device having a porous resin layer having a three-dimensional network structure on the inner surface of the flow path of the present invention has at least the following steps (1) to (3).
(1) forming a three-dimensional network structure porous resin layer having a large number of pores on the surface of the support;
(2) An active energy ray-curable composition (X) containing the active energy ray-polymerizable compound (a) is applied on the porous resin layer, and the composition (X) is uncured. Forming a film, irradiating the uncured coating film other than the portion to be formed with the flow path with active energy rays to form a cured or semi-cured coating film of the composition (X), Removing the composition (X) to form a recess in which a porous resin layer having a three-dimensional network structure is exposed on the bottom surface;
(3) A step of fixing another member serving as a lid to the recess of the member having the recess to form the recess as a hollow flow path.

  The method for forming a porous resin layer having a three-dimensional network structure having a large number of pores in step (1) is arbitrary as long as the porous resin layer can be formed. For example, the following four methods are preferably used. be able to. The three-dimensional network structure here refers to a structure in which pores (voids) and a matrix resin are connected in a three-dimensional direction, and the pores are opened on the surface of the resin porous layer. For example, a structure in which cellular cavities are continuously connected to each other (also referred to as a sponge structure), or a structure in which voids between resin particles fixed to each other become continuous pores (both an aggregated particle structure or a sintered body structure) This is an intermediate structure between these two, and the structure in which the pores and the resin are almost equivalent and the phases are continuous with each other (also referred to as a modulation structure or a giroid structure), and a non-woven structure (Also referred to as a mat-like structure).

  A first method for forming a porous resin layer having a three-dimensional network structure is an active energy ray polymerizable compound (b) (hereinafter referred to as a polymerizable compound (b)) on a support. An active energy ray-curable film-forming solution (J) containing a poor solvent (R) that is compatible with the compound (b) but not compatible with the polymer produced from the compound (b) (hereinafter, The film-forming solution is referred to as a film-forming solution (J).) Then, the film-forming solution (J) is irradiated with active energy rays to polymerize the compound (b) and cause phase separation. Is a method of forming a porous resin layer having a three-dimensional network structure (hereinafter, this method is referred to as a reaction-induced phase separation method). In this method, the produced polymer becomes incompatible with the poor solvent (R) due to the polymerization of the compound (b), and the polymer and the poor solvent (R) undergo phase separation, and the inside of the polymer or between the polymers. In this state, the poor solvent (R) is incorporated. By removing this poor solvent (R), the region occupied by the poor solvent (R) becomes pores, and a porous resin layer having a three-dimensional network structure can be formed.

  The polymerizable compound (b) is polymerized by active energy rays in the presence or absence of a polymerization initiator, and is an addition polymerizable compound or a polymerizable carbon- Those having a carbon double bond are preferred, and among them, a highly reactive (meth) acrylic compound or vinyl ether, or a maleimide compound that cures even in the absence of a photopolymerization initiator is preferred. Furthermore, since it is possible to increase the shape retention in a semi-cured state and to increase the strength after curing, it is preferably a compound that forms a crosslinked polymer by polymerization. Therefore, a compound having two or more polymerizable carbon-carbon double bonds in one molecule (hereinafter referred to as “having two or more addition polymerizable functional groups in one molecule” is “polyfunctional”. More preferably).

  As such a polymerizable compound (b), for example, a (meth) acrylic monomer, a maleimide monomer, or a polymerizable oligomer having a (meth) acryloyl group or a maleimide group in a molecular chain (also referred to as a prepolymer). ) Etc. can be used.

  Examples of the (meth) acrylic monomer include diethylene glycol di (meth) acrylate, neopentyl glycol di (meth) acrylate, 1,6-hexanediol di (meth) acrylate, and 2,2′-bis (4- (meth) ) Acryloyloxypolyethyleneoxyphenyl) propane, 2,2′-bis (4- (meth) acryloyloxypolypropyleneoxyphenyl) propane, hydroxydipivalic acid neopentyl glycol di (meth) acrylate, dicyclopentanyl diacrylate, bis Bifunctional monomers such as (acryloxyethyl) hydroxyethyl isocyanurate and N-methylenebisacrylamide; trimethylolpropane tri (meth) acrylate, trimethylolethane tri (meth) acrylate, tris Acryloxy) isocyanurate, caprolactone-modified tris (acryloxyethyl) isocyanurate and other trifunctional monomers; pentaerythritol tetra (meth) acrylate and other tetrafunctional monomers; dipentaerythritol hexa (meth) acrylate and other functional monomers Can be mentioned.

  Examples of maleimide monomers include 4,4′-methylenebis (N-phenylmaleimide), 2,3-bis (2,4,5-trimethyl-3-thienyl) maleimide, 1,2-bismaleimide ethane, 1 , 6-bismaleimide hexane, triethylene glycol bismaleimide, N, N′-m-phenylene dimaleimide, m-tolylene dimaleimide, N, N′-1,4-phenylene dimaleimide, N, N′-diphenylmethane di Maleimide, N, N′-diphenyl ether dimaleimide, N, N′-diphenylsulfone dimaleimide, 1,4-bis (maleimidoethyl) -1,4-diazoniabicyclo- [2,2,2] octane dichloride, 4 , 4′-isopropylidenediphenyl dicyanate, N, N ′-(methylenedi-p-phenyle ) Bifunctional maleimides such as dimaleimide; N-(9-acridinyl) maleimide having a polymerizable functional group other than the maleimide group and a maleimide group such as maleimide. These maleimide monomers can be copolymerized with a compound having a polymerizable carbon / carbon double bond, such as vinyl monomers, vinyl ethers, and acrylic monomers.

  Examples of the polymerizable oligomer having a (meth) acryloyl group or a maleimide group in the molecular chain include those having a mass average molecular weight of 500 to 50,000. For example, (meth) acrylic acid ester of epoxy resin, ( Examples thereof include (meth) acrylic acid esters, (meth) acrylic acid esters of polybutadiene resins, and polyurethane resins having a (meth) acryloyl group at the molecular terminals.

  These polymerizable compounds (b) can be used alone or in admixture of two or more. Monofunctional (meth) acrylic monomers, monofunctional maleimide monomers, etc. for the purpose of adjusting viscosity, adhesiveness and adhesiveness in semi-cured state, or adding functions such as reactivity and hydrophilicity These may be used in combination with a monofunctional monomer. For example, you may add the below-mentioned amphiphilic compound (c).

  Examples of the monofunctional (meth) acrylic monomer include methyl methacrylate, alkyl (meth) acrylate, isobornyl (meth) acrylate, alkoxy polyethylene glycol (meth) acrylate, phenoxydialkyl (meth) acrylate, and phenoxy polyethylene glycol (meth) acrylate. , Alkylphenoxypolyethylene glycol (meth) acrylate, nonylphenoxypolypropylene glycol (meth) acrylate, hydroxyalkyl (meth) acrylate, glycerol acrylate methacrylate, butanediol mono (meth) acrylate, 2-hydroxy-3-phenoxypropyl acrylate, 2- Acryloyloxyethyl-2-hydroxypropyl acrylate, ethylene oxide Phthalic acid acrylate, w-carboxyaprolactone monoacrylate, 2-acryloyloxypropyl hydrogen phthalate, 2-acryloyloxyethyl succinic acid, acrylic acid dimer, 2-acryloyloxypropylpyrihydrohydrogen phthalate, fluorine-substituted alkyl ( (Meth) acrylate, chlorine-substituted alkyl (meth) acrylate, sulfonic acid sodaethoxy (meth) acrylate, sulfonic acid-2-methylpropane-2-acrylamide, phosphoric ester group-containing (meth) acrylate, glycidyl (meth) acrylate, 2- Isocyanatoethyl (meth) acrylate, (meth) acryloyl chloride, (meth) acrylaldehyde, sulfonate group-containing (meth) acrylate, silano group-containing ( ) Acrylate, ((di) alkyl) amino group-containing (meth) acrylate, quaternary ((di) alkyl) ammonium group-containing (meth) acrylate, (N-alkyl) acrylamide, (N, N-dialkyl) acrylamide, Examples include achloroyl morpholine.

Examples of monofunctional maleimide monomers include N-alkylmaleimides such as N-methylmaleimide, N-ethylmaleimide, N-butylmaleimide and N-dodecylmaleimide; N-alicyclic maleimides such as N-cyclohexylmaleimide; N -Benzylmaleimide; N-phenylmaleimide, N- (alkylphenyl) maleimide, N-dialkoxyphenylmaleimide, N- (2-chlorophenyl) maleimide, 2,3-dichloro-N- (2,6-diethylphenyl) maleimide N- (substituted or unsubstituted phenyl) maleimide such as 2,3-dichloro-N- (2-ethyl-6-methylphenyl) maleimide; N-benzyl-2,3-dichloromaleimide, N- (4′- Halogens such as fluorophenyl) -2,3-dichloromaleimide Maleimide having a hydroxyl group such as hydroxyphenylmaleimide; maleimide having a carboxy group such as N- (4-carboxy-3-hydroxyphenyl) maleimide; maleimide having an alkoxy group such as N-methoxyphenylmaleimide; N- [ Maleimides having amino groups such as 3- (diethylamino) propyl] maleimide; polycyclic aromatic maleimides such as N- (1-pyrenyl) maleimide; N- (dimethylamino-4-methyl-3-coumarinyl) maleimide, N- And maleimide having a heterocyclic ring such as (4-anilino-1-naphthyl) maleimide.
As these monofunctional monomers, functional groups that can serve as anchors for immobilizing (bio) chemical substances and biological substances and ionic functional groups, for example, functional groups that can be preferably introduced into a porous resin layer having a three-dimensional network structure described later. It is also preferable to use a monomer having a functional group exemplified as a group in the molecule.

  The poor solvent (R) used in the reaction-induced phase separation method is compatible with the polymerizable compound (b), but the polymer produced from the polymerizable compound (b) is not compatible (dissolves with each other). Use things. The degree of compatibility between the poor solvent (R) and the polymerizable compound (b) may be such that a uniform film-forming solution (J) can be obtained. The poor solvent (R) may be a single solvent or a mixed solvent. In the case of a mixed solvent, the constituent component alone is not compatible with the polymerizable compound (b), or a polymerizable compound. The polymer of (b) may be dissolved. Examples of such poor solvent (R) include alkyl esters of fatty acids such as methyl decanoate, methyl octoate and diisobutyl adipate; ketones such as diisobutyl ketone; alcohols such as decanol; 2-propanol and ethanol And a mixture of alcohol and water, such as a mixture of water and water.

  In the reaction-induced phase separation method, the pore size and strength of the obtained porous resin layer having a three-dimensional network structure change depending on the content of the compound (b) contained in the film-forming solution (J). As the content of the compound (b) increases, the strength of the porous resin layer improves, but the pore diameter tends to decrease. A preferable content of the compound (b) is in the range of 15 to 50% by mass, more preferably in the range of 25 to 40% by mass. When the content of the compound (b) is 15% by mass or less, the strength of the porous resin layer is lowered, and when the content of the compound (b) is 50% by mass or more, the pore diameter of the porous part is adjusted. It becomes difficult.

  Various additives such as a polymerization initiator, a solvent, a surfactant, a polymerization inhibitor, or a polymerization retarder are added to the film-forming solution (J) in order to adjust the polymerization rate, degree of polymerization, or pore size distribution. May be.

  The photopolymerization initiator is not particularly limited as long as it is active with respect to active energy rays and can polymerize the polymerizable compound (b), and includes a radical polymerization initiator, an anionic polymerization initiator, Cationic polymerization initiators can be used, for example, acetophenones such as p-tert-butyltrichloroacetophenone, 2,2′-diethoxyacetophenone, 2-hydroxy-2-methyl-1-phenylpropan-1-one; benzophenone Ketones such as 4,4'-bisdimethylaminobenzophenone, 2-chlorothioxanthone, 2-methylthioxanthone, 2-ethylthioxanthone, 2-isopropylthioxanthone; benzoin, benzoin methyl ether, benzoin isopropyl ether, benzoin isobutyl ether, etc. Benzoy Ethers; and the azide such as N- azide sulfonyl phenyl maleimide; benzyl dimethyl ketal, benzil ketals, such as hydroxycyclohexyl phenyl ketone. A polymerizable photopolymerization initiator such as a maleimide compound can also be used.

  Polymerization retarders and polymerization inhibitors include α-methylstyrene, 2,4-diphenyl-4-methyl-1-pentene and other active energy ray polymerizable compounds such as vinyl monomers having a low polymerization rate; tert-butylphenol, etc. Hindantophenols and the like.

  The solvent to be added is not particularly limited, and examples thereof include alcohols such as ethanol, ketones such as acetone, amide solvents such as N, N-dimethylformamide, and chlorine solvents such as methylene chloride. .

  In addition, in order to provide functions such as coatability and smoothness, or to adjust pattern resolution and hydrophilicity during pattern formation by photolithography, known and commonly used surfactants, hydrophobic compounds, thickening agents, etc. Agents, modifiers, colorants, fluorescent dyes, ultraviolet absorbers, enzymes, proteins, cells, catalysts and the like can also be added.

  The support that can be used in the reaction-induced layer separation method may be any support that is not substantially affected by the membrane-forming solution (J) or the active energy ray used and does not cause, for example, dissolution or decomposition.

  Examples of such a support include polymers; glass; crystals such as quartz; ceramics; semiconductors such as silicon; metals and the like. Among these, polymers are particularly preferable. The polymer used for the support may be a homopolymer or a copolymer, and may be a thermoplastic polymer or a thermosetting polymer. The support may be composed of a polymer blend or a polymer alloy, or may be a laminate or other complex. Further, the support may contain additives such as a modifier, a colorant, a filler, and a reinforcing material.

  When reaction-induced phase separation is used, the aggregated particulate structure in which particulate polymers having a diameter of about 0.1 μm to 1 μm are aggregated with each other, or a sponge-like structure in which bubbles having a diameter of about 0.1 μm to 5 μm are connected to each other. A porous resin layer having a three-dimensional network structure can be formed. In the reaction-induced phase separation method, a so-called isotropic membrane is usually formed in which the pore diameter is uniform in the thickness direction of the membrane, but a volatile solvent is added to the membrane-forming solution (J). After adding and applying, by volatilizing and removing a part thereof before irradiation with active energy rays, a so-called heterogeneous film (also referred to as an asymmetric film) having a pore size distribution in the film thickness direction can be formed. . At this time, by adding a volatile good solvent, a layer having a small pore diameter (also referred to as a dense layer) can be formed on the contact surface with the support on which the film-forming solution (J) is applied. By adding a poor solvent or a non-solvent, a dense layer can be formed on the surface opposite to the support. By the reaction-induced phase separation method, for example, a porous resin layer having a three-dimensional network structure with a pore size of 0.05 to 5 μm can be formed.

  In this reaction-induced phase separation method, when the porous resin layer having a three-dimensional network structure is formed in a limited region or a plurality of regions, for example, (a) active energy ray-curable resin composition (X) By applying to a part of the support by a silk screen method or the like, and (b) applying the pattern exposure after applying the active energy ray-curable resin composition (X) to the entire support. The porous resin layer can be formed on a part of the support. In the first method, the functional group can be easily introduced into the porous resin layer by using the compound (b) having a functional group.

  The second method for forming a porous resin layer having a three-dimensional network structure is that the support is not dissolved or swollen after contacting the support with a solvent (S) capable of dissolving or swelling the support. The solvent (S) is a method of forming the porous resin layer by washing and removing the solvent (S) using a compatible solvent (T) (hereinafter, this method is referred to as “surface swelling method”). is there. In this method, a polymer that dissolves or swells with a solvent is used as a support, a solvent is brought into contact with the surface of the polymer to dissolve or swell part of the support, and then the polymer and By washing with an incompatible solvent, the polymer is aggregated in a network and a porous resin layer having a three-dimensional network structure is formed.

  Examples of the support in the surface swelling method include styrene polymers such as polystyrene, poly-α-methylstyrene, polystyrene / maleic acid copolymer, polystyrene / acrylonitrile copolymer; and polysulfone systems such as porsulfone and polyethersulfone. Polymers; (meth) acrylic polymers such as polymethyl methacrylate and polyacrylonitrile; polymaleimide polymers; polycarbonate polymers such as bisphenol A polycarbonate, bisphenol F polycarbonate and bisphenol Z polycarbonate; cellulose acetate and methylcellulose Cellulose polymers such as: polyurethane polymers; polyamide polymers; polyimide polymers.

  The solvent (S) in the surface swelling method is not particularly limited as long as it can dissolve or swell the support, and examples thereof include amide solvents such as N, N-dimethylformamide and N, N-dimethylacetamide, dimethyl Examples include chlorinated solvents such as sulfoxide and methylene chloride. These solvents (S) can also be mixed and used as a mixed solvent.

  The solvent (T) is miscible with the solvent (S) and does not dissolve the support. Examples of the solvent (T) include water, alcohols such as propanol, and a mixture of water and alcohol.

  Examples of the method of bringing the support into contact with the solvent (S) include immersion of the support in the solvent (S), spraying of the solvent (S) onto the support surface, casting, and the like.

  Examples of the method of cleaning the solvent (S) with the solvent (T) include a method of cleaning by immersing in the solvent (T), or a method of cleaning by spraying the solvent (T). A method of immersing the whole body in the solvent (T) is preferable.

  The porous resin layer having a three-dimensional network structure manufactured by the surface swelling method is integrated with the support and can form a sponge-like or aggregated particle structure. The thickness of the porous resin layer can be controlled by the contact time between the support and the solvent (S). The shorter the contact time, the thinner the porous resin layer. The contact time between the support and the solvent (S) needs to be appropriately adjusted depending on the material and thickness of the support to be used, the kind of the solvent, and the like. When the contact time is too short, the dissolution of the support does not proceed sufficiently and pores are not sufficiently formed. Moreover, when the contact time is too long, the strength of the support decreases.

  In this surface swelling method, when the porous resin layer having a three-dimensional network structure is formed in a limited region or a plurality of regions, for example, a portion other than the portion where the porous resin layer is formed is covered with a masking tape. In this way, a porous resin layer having a three-dimensional network structure can be formed on a part of the support.

  A third method for forming a porous resin layer having a three-dimensional network structure is to apply a film-forming solution (K) obtained by dissolving a chain polymer (P) in a solvent (U) to a support, The chain polymer (P) is made porous by bringing the polymer into contact with a solvent (V) that does not dissolve or swell the chain polymer (P) and is compatible with the solvent (U). To form a porous resin layer having a three-dimensional network structure on the surface of the support (hereinafter, this method is referred to as a wet method).

  As the chain polymer (P) that can be used in the wet method, a polymer that dissolves in a solvent (U) to form a porous resin layer having a three-dimensional network structure can be used. A styrene polymer, a sulfone polymer, A chain polymer (P) such as a vinyl polymer, an amide polymer, an imide polymer, a cellulose polymer, a polycarbonate, and an acrylic polymer is preferable because it can reduce the cost and is easy to handle.

  The solvent (U) in the wet method can be the same solvent as the solvent (S) that can be used in the surface swelling method, and the solvent (V) can be the same solvent as the solvent (T) in the surface swelling method. .

  Moreover, you may add various additives, such as an additive which can be used in the above-mentioned reaction induction type | formula phase separation method, to a film forming liquid (K) as needed.

  The support that can be used in the wet method is arbitrary, and it is preferably one that is not substantially attacked by the film-forming solution (K) obtained by dissolving the chain polymer (P) in the solvent (U). 3), a porous resin layer having a three-dimensional network structure can be formed by a mechanism in which the swelling method is added to the wet method. Examples of such a support include polymers; glass; crystals such as quartz; ceramics; semiconductors such as silicon; metals and the like. Among these, polymers are particularly preferable.

  The porous structure obtained by the wet method may be a sponge-like structure, an aggregated particle-like structure, a guiloid structure, or a complex-shaped structure having macrovoids.

  When the wet method is used, a heterogeneous film (asymmetric film) having a dense layer is usually formed on the opposite side of the coating support, but salts and other low molecular compounds (pore forming agents), etc. It is also possible to obtain an isotropic film by addition of the above or by adjusting the boiling point of a poor solvent or a good solvent. Further, by adjusting the concentration of the chain polymer (P), the amount of the solvent added, etc., a porous resin layer having a three-dimensional network structure with a pore size of 0.005 to 2 μm can be formed.

  In this wet method, when the porous resin layer having a three-dimensional network structure is formed in a limited region or in a plurality of regions, for example, it is manufactured only on a portion where the porous resin resin layer is formed by screen printing or the like. A method of applying a membrane liquid (K) or a method of covering a portion other than a portion where the porous resin layer is formed with a masking tape, and a porous resin layer having a three-dimensional network structure on a part of the support. Can be formed.

  A fourth method for forming a porous resin layer having a three-dimensional network structure is to uniformly mix an active energy ray-polymerizable compound (d), a chain polymer (Q), and a solvent (W) for dissolving both of them. The film-forming solution (L) is applied to a support, irradiated with active energy rays to polymerize the polymerizable compound (d) in the solution, and then the support and the chain polymer (Q) are combined. A porous polymer layer having a three-dimensional network structure is formed on the surface of the support by aggregating the chain polymer into a porous state by bringing it into contact with a solvent (N) that is not dissolved and is compatible with the solvent (W). (Hereinafter, this method is referred to as an energy beam-wet method).

  In this method, as described in the section of the active energy ray polymerizable compound (b), the active energy ray polymerizable compound (d) is added with a hydroxyl group, an amino group, a carboxyl group, an aldehyde group, an epoxy group or any other functional group. By using the active energy ray-polymerizable compound having the above, these functional groups can be introduced into the surface of the porous resin layer having a three-dimensional network structure more efficiently than the polymerization inducing layer separation method.

  The active energy ray polymerizable compound (d) is preferably a crosslinkable compound, and can be suitably selected from those exemplified as the active energy ray polymerizable compound (b). The cured product of the active energy ray polymerizable compound (d) may be soluble or insoluble in the solvent (W), but the functional group of the compound (d) is efficiently arranged on the pore surface. In order to do so, it is preferably soluble. When the cured product of the compound (d) is a crosslinked polymer, “soluble” is replaced with “gelling” and “insoluble” is replaced with “not gelling” (the same applies hereinafter).

  Further, the compound (d) may be soluble or insoluble in the solvent (N), but in order to efficiently arrange the functional group of the compound (d) on the pore surface. Is preferably gelled when the cured product of the compound (d) is a cross-linked polymer, and in the case of a non-cross-linked polymer, it is preferably insoluble to avoid loss.

  The chain polymer (Q) is also arbitrary, and for example, those exemplified as the chain polymer (P) can be used. When the chain polymer (Q) is used by mixing two or more chain polymers (Q), the pore diameter on the surface of the porous resin layer having a three-dimensional network structure is not excessively reduced, It is preferable because macrovoids are hardly formed inside the porous resin layer having a three-dimensional network structure.

  The solvent (W) may or may not dissolve or swell the polymer produced from the active energy ray polymerizable compound (d). This is preferable because the group can be fixed to the surface of the pores with high density. The solvent (W) can be suitably selected from those exemplified as the solvent (S).

  The solvent (N) is preferably not dissolved when the polymer produced from the active energy ray polymerizable compound (d) is a non-crosslinked polymer, but when the polymer is a crosslinked polymer. The swell may or may not swell. The solvent (N) can be suitably selected from those exemplified as the solvent (T).

  In the fourth method, when the porous resin layer having a three-dimensional network structure is formed in a limited region or a plurality of regions, for example, only on the portion where the porous resin layer is formed by screen printing or the like. Forming the porous resin layer on a part of the support by applying the film-forming solution (L) or by covering the part other than the part where the porous resin layer is formed with a masking tape. I can do it.

  In the fourth method, by using the active energy ray-polymerizable compound (d) having a functional group, functional groups are easily introduced at a high density on the pore surface of the porous resin layer having a three-dimensional network structure. it can.

  The porous resin layer having a three-dimensional network structure that can be formed by the method exemplified above may be formed on the entire surface of the support, or may be formed on a part of the support. In the latter case, a flow path that passes through both the formed portion and the non-formed portion of the porous resin layer may be formed. By doing so, it is possible to form a microfluidic device having a porous resin layer portion having a three-dimensional network structure in a part of the bottom surface of the flow path and not in other portions. At this time, by setting the plane dimension of the porous resin layer having a three-dimensional network structure formed on a part of the support to be larger than the plane dimension of the flow path, the position of the porous resin layer and the flow path is determined. It is not necessary to match the strictness and manufacturing becomes easy. The porous resin layer having a three-dimensional network structure other than the flow path portion is filled with a cured product of the active energy ray-curable resin composition (X). For example, exact alignment is performed by forming the porous resin layer in the form of n (n is a positive integer) parallel lines on the support, and forming the flow path in a direction perpendicular to the porous resin layer. The n-dimensional three-dimensional network structure porous resin layer spots can be formed in the flow path.

  The shape of the support used in the four methods exemplified above is not particularly limited, and any shape can be used according to the purpose of use. Examples thereof include sheet-like (including film-like, ribbon-like, and belt-like), plate-like, roll-like, and spherical shapes, and the composition (X) can be easily applied thereon, and the active energy. From the viewpoint that it is easy to irradiate a line, it is preferable that the coated surface has a planar shape or a quadric surface shape.

  The support may be surface-treated both in the case of a polymer and other materials. Surface treatment is aimed at preventing dissolution by reaction-induced phase separation method or wet method film-forming solution, and improving wettability of film-forming solution and improving adhesion of porous resin layer with 3D network structure. And so on.

  The surface treatment method of the support is arbitrary, for example, a composition containing a compound selected from the group of compounds listed as the polymerizable compound (a) is applied to the surface of the support and cured by irradiation with active energy rays. Treatment, corona treatment, plasma treatment, flame treatment, acid or alkali treatment, sulfonation treatment, fluorination treatment, primer treatment with silane coupling agent, surface graft polymerization, application of surfactant or release agent, rubbing And physical treatment such as sandblasting.

  According to the method exemplified above, a porous resin layer having a sponge-like structure, an aggregated particle-like structure, a guiloid structure, a shape having a macrovoid, or a mixed shape structure on the surface of the support. Can be formed. Further, since the obtained porous resin layer having a three-dimensional network structure has a large surface area as the surface of a large number of pores, a large amount of catalyst, enzyme, DNA, sugar chain, cell, protein, etc. can be immobilized.

  When the surface of a porous resin layer with a three-dimensional network structure is made hydrophobic, proteins such as enzymes and antigens can be hydrophobically interacted with the porous resin with a three-dimensional network structure without introducing functional groups into the porous surface. It can be fixed to the surface of the layer. On the other hand, when protein, DNA, sugar chain or the like is immobilized, a functional group (for example, amino group, carboxyl group, hydroxyl group, epoxy group, etc.) having reactivity on the pore surface of the porous resin layer having a three-dimensional network structure in advance. Aldehyde group, isocyanato group, —COCl group, etc.), and then reacts the amino group, hydroxyl group, phosphate group, carboxyl group of the protein, DNA, sugar chain, etc. directly or via other functional groups By this, it can fix to the surface of the porous resin layer of a three-dimensional network structure by a covalent bond.

  What is necessary is just to select the thickness of the porous resin layer of a three-dimensional network structure suitably according to the intended purpose. For example, when used as affinity chromatography, the thickness of the porous resin layer is preferably 3 to 100 μm, and more preferably 5 to 50 μm.

  The obtained porous resin layer having a three-dimensional network structure may be subjected to surface treatment by the method described in the surface treatment of the support according to the use. For example, in order to suppress nonspecific adsorption of solutes such as proteins and DNA to the surface of the porous resin layer, for the purpose of introducing hydrophilic groups, hydrophobic groups and other functional groups to the surface of the porous resin layer. A composition containing at least one compound selected from the group of compounds listed as the polymerizable compound (a) (particularly a hydrophilic or amphiphilic polymerizable compound) is applied to the surface of the porous resin layer, and active Surface treatment can be performed by a method of curing by irradiating energy rays.

  In the step (2), the composition (X) is impregnated in the porous resin layer by applying the composition (X) to the porous resin layer having a three-dimensional network structure, and the porous resin layer An uncured coating film of the composition (X) is formed inside and on the porous body. Thereafter, the active energy ray is irradiated to the uncured coating film other than the portion to be formed with the flow path, and the uncured composition (X) in the non-irradiated portion is removed, so that the bottom surface is the porous resin layer and the wall surface is A recess made of a cured or semi-cured coating of the composition (X) is obtained, and the porous resin layer other than the portion that becomes the flow path is blocked by the cured or semi-cured product of the impregnated composition (X). Is done.

  The active energy ray-polymerizable compound (a) (hereinafter referred to as the polymerizable compound (a)) used in the step (2) is active energy rays in the presence or absence of a polymerization initiator. A compound that can be polymerized by an addition polymerization compound, and a compound having a polymerizable carbon-carbon double bond as an active energy ray polymerizable functional group are preferred. Of these, highly reactive (meth) acrylic compounds and vinyl ethers, and maleimide compounds that cure even in the absence of a photopolymerization initiator are preferred.

  Further, when the polymerizable compound (a) is a polyfunctional compound, it is polymerized to form a crosslinked structure, so that the strength after curing is also increased.

  As such a polymerizable compound (a), for example, the same compound as the polymerizable compound (b) that can be used in the reaction-induced phase separation method can be used.

The polymerizable compound (a) can be used alone or in admixture of two or more kinds. In addition, in order to adjust the viscosity or to provide functions such as adhesiveness, tackiness, hydrophilicity, etc. You may mix and use with a functional monomer.
As the monofunctional monomer that can be mixed, for example, the same compound as the monofunctional monomer that can be used in the reaction-induced phase separation method described above can be used.

  The composition (X) contains at least the polymerizable compound (a). The composition (X) contains, in addition to the polymerizable compound (a), an amphiphilic polymerizable compound copolymerizable with the polymerizable compound (a) (hereinafter referred to as amphiphilic polymerizable compound). (Referred to as a functional compound (c)). By containing the amphiphilic compound (c) in the composition (X), the obtained cured product can be hardly swelled in water, and the surface of the cured product is made hydrophilic with low adsorptivity to biological components. be able to.

  As the amphiphilic compound (c), the active energy ray-polymerizable compound (a) containing both a hydrophilic group and a hydrophobic group in the molecule and contained in the composition (X) by irradiation with active energy rays A compound having a polymerizable functional group capable of copolymerization can be used. The amphiphilic compound (c) does not have to be a crosslinked polymer, but may be a compound that becomes a crosslinked polymer. Moreover, what is necessary is just to be compatible with the polymeric compound (a) uniformly. Here, the term “compatible” means that the phases are not macroscopically separated, and includes a state where micelles are formed and stably dispersed.

  When the polymerizable compound (a) is a compound having two or more polymerizable carbon-carbon unsaturated bonds in one molecule, the amphiphilic compound (c) is one or more polymerized in one molecule. A compound having a carbon-carbon unsaturated bond is preferred.

  The amphiphilic compound (c) is a compound having a hydrophilic group and a hydrophobic group in the molecule and compatible with water or a hydrophobic solvent. Even in this case, “compatible” means that the phase does not macroscopically separate, and includes a state where micelles are formed and stably dispersed.

  The amphiphilic compound (c) has a solubility in water of 0.5% by mass or more at 0 ° C. and a solubility in a mixed solvent of cyclohexane: toluene = 5: 1 (mass ratio) at 25 ° C. of 25% by mass or more. Preferably there is. The term “solubility” used herein means, for example, that the solubility is 0.5% by mass or more means that at least 0.5% by mass of a compound can be dissolved. When at least one of the solubility in water or the solubility in cyclohexane: toluene = 5: 1 (mass ratio) mixed solvent is lower than these values, a cured product excellent in both surface hydrophilicity and water resistance can be obtained. It becomes difficult.

  When the amphiphilic compound (c) has a nonionic hydrophilic group, particularly a polyether-based hydrophilic group, the balance between hydrophilicity and hydrophobicity is 11 in terms of the HLB value of griffin. The thing in the range of -16 is preferable, and the thing in the range of 11-15 is still more preferable. Outside this range, it is difficult to obtain a molded product having high hydrophilicity and water resistance, or the combination and mixing ratio of the compounds for obtaining it are limited.

  The hydrophilic group possessed by the amphiphilic compound (c) is arbitrary, for example, a cationic group such as an amino group, a quaternary ammonium group or a phosphonium group; an anionic group such as a sulfone group, a phosphoric acid group or a carbonyl group; Nonionic groups such as polyethylene glycol chains and amide groups; zwitterionic groups such as amino acid residues may be used. The amphiphilic compound (c) is preferably a compound having a polyether group as a hydrophilic group, and particularly preferably a compound having a polyethylene glycol chain having 6 to 20 repeats.

  Examples of the hydrophobic group of the amphiphilic compound (c) include an alkyl group, an alkylene group, an alkylphenyl group, a long chain alkoxy group, a fluorine-substituted alkyl group, and a siloxane group. The amphiphilic compound (c) is preferably a compound having an alkyl group or alkylene group having 6 to 20 carbon atoms as a hydrophobic group. The alkyl group or alkylene group having 6 to 20 carbon atoms may be contained in the form of, for example, an alkylphenyl group, an alkylphenoxy group, an alkoxy group, or a phenylalkyl group.

  The amphiphilic compound (c) is preferably a compound having a polyethylene glycol chain having a repeating number of 6 to 20 as a hydrophilic group and an alkyl or alkylene group having 6 to 20 carbon atoms as a hydrophobic group. . Among these amphiphilic compounds (c), nonylphenoxypolyethylene glycol (n = 8 to 17) (meth) acrylate and nonylphenoxypolypropylene glycol (n = 8 to 17) (meth) acrylate are particularly preferable.

  The preferred ratio of the polymerizable compound (a) and the amphiphilic compound (c) contained in the composition (X) varies depending on the types and combinations of the polymerizable compound (a) and the amphiphilic compound (c). It is preferable that it is 0.1-5 mass parts with respect to 1 mass part of polymeric compounds (a), and it is still more preferable that it is 0.2-3 mass parts. When the amount of the amphiphilic compound (c) is less than 0.1 parts by mass relative to 1 part by mass of the polymerizable compound (a), it becomes difficult to form a highly hydrophilic surface, and from 5 parts by mass If it is too large, the polymer may swell with water and the polymer of the composition (X) may be gelled.

  Producing a cured product exhibiting high hydrophilicity and low adsorptivity without gelation in a wet state by appropriately selecting the mixing ratio of the polymerizable compound (a) and the amphiphilic compound (c). Can do. The more hydrophilic the amphiphilic compound (c), for example, the larger the HLB value of griffin, it is preferable to reduce the amount of the amphiphilic compound (c) added.

  It is also preferable to mix the composition (X) with a polymerizable compound (b) having a functional group introduced into the surface of the porous resin layer having a three-dimensional network structure. In particular, when a certain functional group is introduced to the surface of the porous resin layer having a three-dimensional network structure, a resin having the functional group is used as a resin for forming the porous resin layer having a three-dimensional network structure, and It is preferable to mix the polymerizable compound (b) having the functional group into the composition (X). As a result, in the step of removing the uncured composition (X) in the non-irradiated portion, which will be described later, even if the removal of the composition (X) is incomplete, Can be prevented from being exposed to the functional group.

  In addition, the composition (X) can be used by mixing a photopolymerization initiator, a polymerization retarder, a polymerization inhibitor, a solvent, a thickener, a modifier, a colorant and the like, if necessary. .

  Examples of the photopolymerization initiator, the polymerization retarder, and the polymerization inhibitor that can be added to the composition (X) include a photopolymerization initiator and a polymerization retarder for the film-forming solution (J) in the reaction-induced phase separation method described above. And the same compounds as the polymerization inhibitor can be preferably used.

  Although it does not specifically limit as a solvent, It is necessary to adjust the kind and addition amount of a solvent suitably with the additive added to the polymeric compound (a) and composition (X) to be used, or the required viscosity. Examples thereof include alcohols such as ethanol, ketones such as acetone, amide solvents such as N, N-dimethylformamide, and chlorine solvents such as methylene chloride.

  The viscosity of the composition (X) can vary depending on the pore size of the porous resin layer having a three-dimensional network structure, but when applied onto the porous resin layer having a three-dimensional network structure, When (X) penetrates rapidly into the porous resin layer having a three-dimensional network structure and the uncured composition (X) in the non-irradiated part is removed after irradiation with active energy rays, the composition (X) From the viewpoint of completely removing from the porous resin layer having a three-dimensional network structure, the viscosity of the composition (X) is preferably in the range of 30 to 3000 mPa · s at 25 ° C., and in the range of 100 to 1000 mPa · s. More preferably. When the viscosity is less than 30 mPa · s, it becomes difficult to control the depth of the concave portion. On the other hand, when the viscosity is more than 3000 mPa · s, the composition (X) penetrates into the porous resin layer having a three-dimensional network structure. It becomes difficult to remove the uncured composition (X) from the non-irradiated part.

  In the step (2), any coating method can be used as a method for coating the composition (X) on the porous resin layer having a three-dimensional network structure. For example, a spin coating method or a roller coating method can be used. , Casting method, dipping method, spray method, bar coater method, XY applicator method, screen printing method, letterpress printing method, gravure printing method, extrusion from nozzle and casting method. In addition, when the composition (X) has a high viscosity or is particularly thinly coated, the composition (X) is coated by adding a solvent and then volatilizing the solvent. You can also.

  The thickness for coating the composition (X) is not particularly limited as long as a cured or semi-cured coating is obtained on the upper part of the porous resin layer having a three-dimensional network structure after irradiation with active energy rays. When a specific substance is fixed to the porous resin layer and used as affinity chromatography, the thickness of the cured or semi-cured coating film formed on the porous resin layer after irradiation with active energy rays, that is, a concave portion The wall height is preferably in the range of 3 to 150 μm, more preferably in the range of 5 to 50 μm. If the thickness is less than 3 μm, another member that serves as a lid may be fixed to the recess to make the recess a hollow channel, and the channel may be blocked. On the other hand, when it is thicker than 150 μm, the aqueous solution passes through the channel, and the substance in the aqueous solution becomes difficult to adsorb and desorb (interact) with the porous resin layer having a three-dimensional network structure on the bottom surface. Is unsuitable.

  When a solvent is added to the active energy ray-curable resin composition (X), the solvent is removed by volatilization after coating. The removing method is arbitrary, and for example, air drying, hot air drying, infrared drying, vacuum drying, microwave drying, and the like can be used. The removal of the solvent may be performed after the irradiation with active energy rays, but is preferably performed before the irradiation with active energy rays in order to accurately control the size and shape of the flow path.

  Active energy rays to be irradiated include rays such as ultraviolet rays, visible rays, infrared rays, laser rays and radiant rays; ionizing radiations such as X-rays, gamma rays and radiant rays; particle rays such as electron rays, ion beams, beta rays and heavy particle rays Is mentioned. Among these, ultraviolet rays and visible light are preferable from the viewpoint of handleability and curing speed, and ultraviolet rays are particularly preferable. For the purpose of accelerating the curing and complete the curing, it is preferable to irradiate active energy rays in a low oxygen concentration atmosphere. The low oxygen concentration atmosphere is preferably a nitrogen stream, a carbon dioxide stream, an argon stream, a vacuum or a reduced pressure atmosphere.

  In order to form a recess formed on the entire bottom surface or a part of the bottom surface of the porous resin layer having a three-dimensional network structure, patterning irradiation is performed on the active energy beam when the active energy beam is irradiated. The patterning irradiation method is arbitrary, and for example, a photolithography technique such as masking and irradiating a portion that is not irradiated with active energy rays or scanning with a beam of active energy rays such as a laser can be used. In the case of using a photomask, the photomask may be a non-contact type or a contact type with the coating film.

  By semi-curing the uncured coating film of composition (X), it is possible to fix it to other members that become a lid without using an adhesive, and when using an adhesive. In addition, the adhesive strength is improved. When the cured state of the composition (X) is semi-cured, it is preferable to carry out post-curing in any step before forming the final microfluidic device, and to completely cure it. It is not always necessary to completely cure the fluid element as long as it does not interfere with the function of the fluid element. In the case of curing with active energy rays, the post-curing may be the same as or different from the active energy rays used for semi-curing. The post-curing may be cured by heat curing in addition to curing by active energy rays.

  Step (3) is a step of fixing another member serving as a lid to the concave portion of the member having the concave portion formed in step (2) to form the concave portion as a hollow flow path.

  As a member to be a lid, a member that can be appropriately selected according to the purpose of use, and a member that is not affected by the fluid flowing through the flow path may be used, and the member is an adhesive tape, sheet or plate. It may be.

  In order to cover the concave portion with the member to be the lid, the lid member and the member having the concave portion may be bonded together. As described above, if the member having the recess is a semi-cured coating film and has good adhesion to the lid member, it may be applied as it is. Moreover, what is necessary is just to bond both members using an adhesive agent etc., when the adhesiveness of the member which has a recessed part is low or it is a cured coating film.

  In addition, a composition containing an active energy ray-polymerizable compound is applied to a support such as a polymer film or sheet, irradiated with active energy rays, the coating film of the composition is semi-cured, and the concave portion There is also a method in which the material is bonded to the concave portion of the member having, and irradiated with active energy rays to be completely cured. The active energy ray polymerizable compound and the composition used here may be the same as the polymerizable compound (a) and the composition (X) used in the step (2), and can be used. One or more compounds selected from the compound group listed as the polymerizable compound (a) may be used. The coating method of the polymerizable compound may be performed according to the step (2).

  For example, an epoxy resin adhesive, a styrene butadiene resin adhesive, a (meth) acrylic adhesive, or the like can be used as the adhesive when the lid member and the member having the recess are bonded to each other.

  When the production method of the present invention is used, a microfluidic device having a porous resin layer having a uniform thickness and a three-dimensional network structure is easily obtained on the inner surface of a fine channel without blocking the channel. be able to. In addition, the manufacturing method allows a plurality of minute microfluidic elements to be easily formed on a single support (exposed and developed plate) without the need for alignment, so that excellent reproducibility and excellent A large number of microfluidic devices can be produced at once with dimensional stability.

  Among the microfluidic devices obtained by the above method, in a microfluidic device comprising a support, a porous resin layer, a flow path, and a lid, (I) a porous resin layer having a three-dimensional network structure above the support (II) filled with the cured resin of the active energy ray-curable resin composition (X) impregnated with the porous resin layer excluding the flow channel portion, and (III) the flow channel is cured with the active energy beam A porous resin layer having a three-dimensional network structure not filled with the cured resin of the resin composition (X), and a porous three-dimensional network structure filled with the cured resin of the active energy ray-curable resin composition (X) A microfluidic device characterized in that the cured resin layer of the active energy ray-curable resin composition (X) formed on the upper part of the resin layer and the lid part are used as wall surfaces to form a hollow shape. It can be particularly preferably used.

The size of the cross section of the flow path may be any size as long as the separation target substance contained in the fluid flowing through the flow path interacts with the porous resin layer having a three-dimensional network structure. In the flow path cross section in the portion having the porous resin layer having a network structure, x is an arbitrary point in the cross section, y is the portion of the porous resin layer having a three-dimensional network structure closest to the arbitrary point in a linear distance, It is preferable to design so that r _max is in the range of 1 to 50 μm, where r is the linear distance between xy and r _max is the maximum distance r can take in the cross section. When the r _max defined here is 50 μm or less, the separation target substance contained in the fluid flowing in the flow path can interact well with the porous resin layer having a three-dimensional network structure on the flow path inner wall. Can do. In addition, by setting _rmax to 1 μm or more, the pressure required to flow the fluid does not become excessively large.

In order to set r _max in the cross section of the flow path within the above range, it may be appropriately designed depending on the arrangement of the porous resin layer having a three-dimensional network structure and the cross sectional shape of the flow path. For example, in the case of a flow path having a rectangular or trapezoidal cross section having a porous resin layer having a three-dimensional network structure on the bottom and ceiling surfaces of the inner wall, the height of the flow path cross section may be 100 μm or less. In the case of a rectangular or trapezoidal flow path having a three-dimensional network porous resin layer only on one surface, the distance between the porous resin layer and the surface facing the porous resin layer is 1 to 50 μm. And it is sufficient. Similarly, when the cross-sectional shape of the flow path is circular or triangular, it may be appropriately designed according to the porous resin layer having a three-dimensional network structure on the inner wall. In the present invention, the rectangle or trapezoid includes a shape with rounded corners.

  On the other hand, the maximum width and the maximum height of the cross section of the flow path from the viewpoint of easy optical reading of the analyte spot flowing in the flow path, ease of control of the flow rate and temperature, and ease of manufacture. Is a ratio represented by (maximum width) / (maximum height), preferably in the range of 1/20 to 20/1, particularly preferably in the range of 1/10 to 10/1.

  The length of the flow path is arbitrary and may be a suitable length depending on the purpose of use, but is preferably 1 mm to 500 mm, and more preferably 5 mm to 200 mm. By setting it to the above lower limit or higher, sufficient separation performance can be obtained, and by setting it to the upper limit or lower, it is possible to reduce the required liquid feeding speed, shorten the separation time, and reduce the size of the flow path.

  Also, the form in a direction parallel to the flow direction of the fluid in the flow path is arbitrary, and may be a straight line, a curved line, a combination thereof, or a branch. The width of the flow path may not be constant. Further, the position and number of openings in the main channel are arbitrary, and a plurality of openings may be provided in one channel. Further, the number of independent flow paths existing in one member is also arbitrary.

  The flow path of the microfluidic device of the present invention has a porous resin layer having a three-dimensional network structure on one surface or two opposing surfaces of the inner wall surface.

  Forming a porous resin layer having a three-dimensional network structure on one surface in the flow path can be performed by using a non-porous member as a lid. In order to form a porous resin layer having a three-dimensional network structure on two opposite surfaces of the inner wall of the flow path, for example, the above-described member in which a porous resin layer having a three-dimensional network structure is formed on a support, It can be formed by using a member having a groove on the porous resin layer.

  The porous resin layer having the three-dimensional network structure is formed on one surface of the flow channel wall surface or on two opposite surfaces, and the analyte to be analyzed flowing inside the flow channel interacts with the porous resin layer in the flow channel. Separated by moving. Further, when a probe is fixed to the porous resin layer, the probe is separated by moving in the flow path while interacting with the probe.

  The formation site of the porous resin layer having a three-dimensional network structure in the direction parallel to the flow direction of the fluid in the flow path may be the entire flow path or may be interrupted. When the microfluidic device is a chromatography device or an electrophoretic analysis device, it is preferable that the microfluidic device is formed without interruption because the resolution is improved.

  The thickness of the three-dimensional network porous resin layer is preferably 0.5 μm to 30 μm, more preferably 1 μm to 20 μm, and most preferably 2 μm to 10 μm. By setting it as this lower limit or more, the surface area for interacting with the substance to be analyzed becomes sufficiently large, and the resolution becomes high. In addition, when a probe is fixed, a sufficiently large amount of probe can be fixed, and the resolution is increased. On the other hand, by making it below this upper limit, it is possible to prevent separation target substances in the solution from entering the deep pores and lowering the moving speed, and shortening the time for separation and analysis.

  Although the pore diameter of the pores of the three-dimensional network structure porous resin layer is also arbitrary, it is preferably 0.05 μm to 3 μm, more preferably 0.1 μm to 1 μm. By setting the lower limit or more, a sufficient amount of probe can be fixed. In addition, by setting below the upper limit, macromolecules such as proteins can be immobilized as probes, and the mass transfer rate between the deep part and the surface of the porous resin layer having a three-dimensional network structure is increased. Separation becomes possible. In addition, the said pore diameter is a hole diameter of the pore which exists in large quantities, and is not necessarily necessarily an average diameter. A narrow distribution of the pore diameter is preferable because the separation efficiency is high.

  The porosity of the three-dimensional network structure porous resin layer is arbitrary, but is preferably 30 to 90%, and more preferably 40 to 70%. By setting it within this range, a sufficient increase in surface area can be achieved without causing a decrease in mechanical strength.

  As the material of the porous resin layer having the three-dimensional network structure, the above-mentioned organic polymer can be used, and an active energy ray-curable resin is more preferable because it is easy to form. When fixing the probe, a material that can be easily fixed can be arbitrarily selected. However, an organic polymer is preferable because it is easy to manufacture, and an active energy ray-curable resin is easy to form. More preferred.

  Any functional group can be introduced into the porous resin layer having the three-dimensional network structure. Examples of hydrophilic functional groups include nonionic functional groups such as hydroxyl groups, polyethylene glycol groups, amide groups, and nitro groups, carboxyl groups, sulfone groups, phosphoric acid groups, phosphorous acid groups, (substituted) hydroxyphenyl groups, and silanol groups. And anionic functional groups such as (N-substituted) amino groups, quaternary ammonium groups, phosphonium compounds, sulfonium groups, and the like. Examples of the hydrophobic functional group include fluorine, chlorine group, siloxane structure, alkyl group, and phenyl group. Examples of amphoteric functional groups include amino acid residues. Other examples include amphiphilic groups. Other examples include photoreactive groups such as azide. In addition, these functional groups can be made to function by themselves, for example, imparting selective adsorptivity, or can be converted into functional functional groups by chemical reaction, (bio) chemical substances, biological substances, etc. It can also be used as an anchor for fixing.

  It is also preferable to immobilize other substances in the three-dimensional network porous resin layer. Examples of other substances include various catalysts, enzymes, antibodies, antigens and other proteins, oligonucleotides such as DNA and RNA, sugar-containing substances such as sugar chains and glycolipids, living bodies such as cell membranes, intracellular tissues, and cells. Examples include tissues and organisms such as bacteria. Of course, these may be chemically modified. In particular, when an oligonucleotide is used as an immobilized probe, the gene can be separated and detected, and can be used effectively for detecting a single base mutation of a gene.

  When an oligonucleotide is used as the probe, the length of the oligonucleotide is preferably 5 to 30, more preferably 5 to 20, and most preferably 5 to 10 in terms of the number of bases. By setting the length of the oligonucleotide within this range, sufficient reliability and a high separation rate can be obtained at a temperature at which room temperature to 60 ° C. is easy to carry out. For the same purpose, it is also preferable to use an oligonucleotide having a base sequence that intentionally has a mismatch with the polynucleotide or oligonucleotide to be analyzed.

  When the microfluidic device of the present invention is used as a column for liquid chromatography, the amount of the probe (including the functional group) immobilized on the surface of the flow path is the amount of the analyte to be analyzed having affinity with the probe. It is preferable that the amount is excessive. When the amount of the probe is excessive, the amount of the analyte to be involved in the interaction with the probe increases and the concentration of the analyte is locally increased compared to when the amount is too small. It becomes possible to detect a substance to be analyzed with high sensitivity. For these reasons, it is preferable that the amount of the probe fixed to the porous resin layer having a three-dimensional network structure is large.

  In addition, when the microfluidic device of the present invention is used as a member for affinity electrophoresis, it is preferable that the amount of immobilized probes is large as in the case of the liquid chromatography.

  EXAMPLES Hereinafter, although this invention is demonstrated further in detail using an Example, this invention is not limited to the range of these Examples. In the following examples, “part” and “%” represent “part by mass” and “% by mass”, respectively, unless otherwise specified.

Viscosity measurement and UV lamp irradiation in the examples are performed by the following methods.
[Viscosity measurement]
The viscosity of the composition at 25 ° C. was measured using a VDH-K viscometer manufactured by Shibaura System Co., Ltd.

[Irradiation by ultraviolet lamp 1]
Using a UE031-353CHC type UV irradiation device manufactured by Eye Graphics Co., Ltd. using a 3000 W metal halide lamp as a light source, ultraviolet rays having an ultraviolet intensity at 365 nm of 40 mW / cm ² were irradiated in a nitrogen atmosphere at room temperature unless otherwise specified.

[Irradiation by ultraviolet lamp 2]
Using a light source unit for multi-light 200 type exposure apparatus manufactured by USHIO INC. With a 200W metal halide lamp as the light source, UV light with an ultraviolet intensity at 365 nm of 100 mW / cm ² is irradiated in a nitrogen atmosphere at room temperature unless otherwise specified. did.

(Example 1)
Example 1 is an example in which a porous resin layer having a three-dimensional network structure was produced by a “reaction-induced phase separation method”.

[Preparation of film-forming solution (J)]
Average molecular weight 2000 trifunctional urethane acrylate oligomer "Unidic V-4263" (Dainippon Ink Chemical Co., Ltd.) 72 parts, dicyclopentanyl diacrylate "R-684" (Nippon Kayaku Co., Ltd.) 18 parts , 10 parts of glycidyl methacrylate (manufactured by Wako Pure Chemical Industries, Ltd.), 150 parts of methyl decanoate (manufactured by Wako Pure Chemical Industries, Ltd.), 10 parts of acetone as a volatile good solvent, 1- A film-forming solution (J1) was prepared by uniformly mixing 3 parts of hydroxycyclohexyl phenyl ketone “Irgacure 184” (Ciba Geigy).

[Preparation of Composition (X)]
50 parts of a trifunctional urethane acrylate oligomer with an average molecular weight of 2000 “Unidic V-4263” (Dainippon Ink Chemical Co., Ltd.), 40 parts of hexanediol diacrylate “New Frontier HDDA” (Daiichi Kogyo Seiyaku Co., Ltd.), 10 parts of glycidyl methacrylate (manufactured by Wako Pure Chemical Industries, Ltd.), 5 parts of 1-hydroxycyclohexyl phenyl ketone “Irgacure 184” (manufactured by Ciba Geigy) as a photopolymerization initiator, and 2,4-diphenyl- as a polymerization retarder A composition (X1) was prepared by mixing 0.5 parts of 4-methyl-1-pentene (manufactured by Kanto Chemical Co., Inc.). The viscosity of the composition (X1) was 192 mPa · s.

[Step 1: Formation of porous resin layer having three-dimensional network structure]
Using a 1 mm thick acrylic plate as a support, a spin coater (manufactured by Mikasa Co., Ltd.) was used to apply the film forming solution (J1) at a rotation of 600 rpm, and the film forming solution ( Porous resin layer (1) having a three-dimensional network structure by irradiating J1) with ultraviolet lamp 40 for 40 seconds to cure the film-forming solution (J1), washing and removing the poor solvent (R) with n-hexane Formed.

[Step 2: Formation of a recess (flow path) in which a porous resin layer having a three-dimensional network structure is exposed on the bottom surface]
On the porous resin layer (1) having the three-dimensional network structure, the composition (X1) was applied at a rotation speed of 800 rpm using a spin coater (manufactured by Mikasa Co., Ltd.). An uncured coating film is formed, and the uncured coating film other than the portion to be formed with the flow path is irradiated with ultraviolet rays by an ultraviolet lamp 2 through a photomask for 120 seconds to form a semi-cured coating film of the composition (X1). The uncured composition (X1) in the non-irradiated portion is removed with ethanol to form a recess (flow path 1) on the support with the porous resin layer (1) having a three-dimensional network structure exposed on the bottom surface. did.

[Immobilization of DNA]
(Introduction of amino group)
A 5% by mass polyallylamine (molecular weight: 15000, manufactured by Nittobo Co., Ltd.) aqueous solution was brought into contact with the concave portion (channel 1) prepared in the above step 2 and reacted at 50 ° C. for 2 hours (part of amino in polyallylamine). The group was reacted with the epoxy group in the porous resin layer having a three-dimensional network structure), and then washed with running water for 15 minutes to introduce amino groups into the porous resin layer.

(Introduction of aldehyde group)
The support having the concave portion (channel 1) into which the amino group was introduced was placed in a 5% by mass aqueous solution of glutaraldehyde (manufactured by Wako Pure Chemical Industries, Ltd.) and reacted at 50 ° C. for 2 hours (in polyallylamine). Almost all amino groups were reacted with one aldehyde group in glutaraldehyde) and then washed with running water for 10 minutes to introduce aldehyde groups into the porous resin layer having a three-dimensional network structure.

(Immobilization of DNA)
DNA modified with amino modification at the 5 'end and fluorescein isothiocyanate-isomer I type (FITC-I) at the 3' end (length 25 bases, ESPEC oligo service stock) After 1 μL of an aqueous solution (concentration: 50 μM) was dropped and reacted at 100% humidity and 50 ° C. for 15 hours (reacting the terminal amino group of DNA with the aldehyde group of the porous resin layer having a three-dimensional network structure) In a 0.2% by weight sodium tetrahydroborate aqueous solution, the reaction is reduced for 5 minutes, followed by rinsing with 0.2 × SSC / 0.1% SDS solution, followed by rinsing with 0.2 × SSC. The DNA was further rinsed with distilled water and naturally dried to immobilize the DNA on the porous resin layer having a three-dimensional network structure on the bottom of the recess (channel 1). (Here, 0.2 × SSC is a 0.03 M NaCl, 3 mM sodium citrate aqueous solution, and 0.1% SDS is a 0.1 mass% sodium dodecyl sulfate aqueous solution.)

[Step 3: Adhering the lid]
72 parts of trifunctional urethane acrylate oligomer with an average molecular weight of about 2000 “Unidic V-4263” (Dainippon Ink Chemical Co., Ltd.), 18 parts of hexanediol diacrylate “New Frontier HDDA” (Daiichi Kogyo Seiyaku Co., Ltd.) A composition obtained by uniformly mixing 10 parts of glycidyl methacrylate (manufactured by Wako Pure Chemical Industries, Ltd.) and 2 parts of 1-hydroxycyclohexyl phenyl ketone “Irgacure 184” (manufactured by Ciba Geigy) as a photopolymerization initiator, Was applied on a biaxially stretched polypropylene film (manufactured by Nimura Chemical Co., Ltd.) having a thickness of 30 μm subjected to corona discharge treatment using a spin coater (manufactured by Mikasa Co., Ltd.) at 800 rpm. The uncured coating film is irradiated with ultraviolet rays by an ultraviolet lamp 1 for 1 second to form a semi-cured coating film of the composition, which is bonded to the concave portion (channel 1) prepared in the above step 2, and again by the ultraviolet lamp 1 Then, the microfluidic device (1) having a capillary channel (channel 1) in which a porous resin layer having a three-dimensional network structure was exposed on the bottom surface was manufactured by irradiation with ultraviolet rays for 40 seconds.

[Structural observation of porous resin layer with three-dimensional network structure]
When the surface of the porous resin layer (1) having a three-dimensional network structure produced in the above step 1 was observed with a scanning electron microscope, the pore diameter was about 0.4 μm as a gap between aggregated particles having a diameter of about 0.5 μm. Pore was observed. When the cross section of the porous resin layer having a three-dimensional network structure was observed, the thickness of the porous resin layer having the three-dimensional network structure was about 10 μm. A scanning electron micrograph of the porous resin layer having the three-dimensional network structure is shown in FIG.

[Observation of recess (channel) structure]
When the cross section of the concave portion (channel 1) produced in the above step 2 was observed with a scanning electron microscope, the concave cross section had a width of about 250 μm and a depth of about 30 μm excluding the porous resin layer having a three-dimensional network structure. It was a rectangle.

[Quantification of DNA]
As a result of measuring the fluorescence intensity emitted by FITC-I of the porous resin layer having a three-dimensional network structure on the bottom surface on which the DNA is fixed, the fluorescence intensity is measured with a fluoro imaging scanner “FLA-3000G” (manufactured by Fuji Photo Film Co., Ltd.). The value was 1069 LAU (unit of fluorescence intensity displayed on the measuring apparatus) / mm ² .

(Example 2)
Example 2 is an example in which a porous resin layer having a three-dimensional network structure was manufactured by the “surface swelling method”.
[Preparation of Composition (X)]
In place of 40 parts of hexanediol diacrylate “New Frontier HDDA” (Daiichi Kogyo Seiyaku Co., Ltd.), 40 parts of 1,6-hexanediol ethoxylate diacrylate “Photomer 4361” (Cognis Japan Co., Ltd.) was mixed. Except for mixing 10 parts of nonylphenoxypolyethylene glycol (n = 17) acrylate “N-177E” (Daiichi Kogyo Seiyaku Co., Ltd.) instead of 10 parts of glycidyl methacrylate (manufactured by Wako Pure Chemical Industries, Ltd.) The composition (X2) was prepared in the same manner as the preparation of the composition (X1) in Example 1. The viscosity of the composition (X2) was 220 mPa · s.

[Step 1: Formation of porous resin layer having three-dimensional network structure]
(Formation of three-dimensional network structure)
Using a 150 μm-thick polystyrene sheet (Dainippon Ink Chemical Co., Ltd.) as a support, the support is immersed in N, N-dimethylacetamide (Wako Pure Chemical Industries, Ltd.) at room temperature for 5 seconds. After that, it was poured into water and further washed with running water for about 5 minutes to obtain a polystyrene sheet in which the porous resin layer having a three-dimensional network structure and the support were integrated.

(Introduction of epoxy group)
Furthermore, 2.5 parts of trifunctional urethane acrylate oligomer “Unidic V-4263” (manufactured by Dainippon Ink & Chemicals, Inc.) having an average molecular weight of about 2000 is formed on the porous resin layer having a three-dimensional network structure. 6 parts of 6-hexanediol ethoxylate diacrylate “Photomer 4361” (manufactured by Cognis Japan), 0.5 part of glycidyl methacrylate (manufactured by Wako Pure Chemical Industries, Ltd.), 1-hydroxycyclohexyl phenyl ketone as a photopolymerization initiator A composition in which 0.25 part of “Irgacure 184” (manufactured by Ciba Geigy) and 95 parts of ethanol as a solvent were mixed was applied at a rotation speed of 1500 rpm using a spin coater, and ultraviolet rays were irradiated with an ultraviolet lamp 1 for 40 seconds. A porous resin layer having a three-dimensional network structure in which epoxy groups are introduced on the surface of the pores (2) Formed.

[Step 2: Formation of recess (flow path) with porous resin layer exposed on bottom surface]
Instead of the composition (X1), a recess (flow path 2) in which the porous resin layer having a three-dimensional network structure was exposed on the bottom surface was formed in the same manner as in Example 1 except that the composition (X2) was used. .
(Immobilization of DNA)
In the same manner as in Example 1, amino group introduction, aldehyde group introduction, and DNA fixation were performed.

[Step 3: Adhering the lid]
The same composition and the same method as in Example 1 have a capillary channel (channel 2) in which the lid is fixed to the recess (channel 2) and the porous resin layer having a three-dimensional network structure is exposed on the bottom surface. A microfluidic device (2) was produced.

[Structural observation of porous resin layer with three-dimensional network structure]
When the surface of the porous resin layer (2) having a three-dimensional network structure produced in the above step 1 was observed with a scanning electron microscope, sponge-like pores having a pore diameter of about 0.8 μm were observed. Further, when the cross section of the porous resin layer was observed, the thickness of the porous resin layer was about 2 μm.

[Observation of recess (channel) structure]
When the cross section of the recess (channel 2) prepared in the above step 2 was observed with a scanning electron microscope, the cross section of the recess had a width of about 250 μm and a depth of about 30 μm excluding the porous resin layer having a three-dimensional network structure. It was a rectangle.
[Quantification of DNA]

The DNA was immobilized in the recess (channel 2) formed in step 2 by the same method as the DNA immobilization method described in Example 1 above. As a result of measuring the fluorescence intensity emitted from FITC-I fixed in the recess (flow path 2) by the same method as the quantification of DNA in Example 1, the fluorescence intensity value was 954 LAU / mm ² .

(Example 3)
Example 3 is an example in which a porous resin layer having a three-dimensional network structure was manufactured by a “wet method”.
[Preparation of film-forming solution (K)]
5 parts of aromatic polyamide ("Conex" manufactured by Teijin Ltd.) as the chain polymer (P), 90 parts of N, N-dimethylacetamide (manufactured by Wako Pure Chemical Industries, Ltd.) as the solvent (U), additive As a result, 5 parts of ethylene glycol was uniformly mixed to obtain a film-forming solution (K) 3.

[Preparation of Composition (X)]
50 parts of tritetraethylene glycol bismaleimide “LumiCure MIA200” (Dainippon Ink Chemical Co., Ltd.), 40 parts of 1,6-hexanediol ethoxylate diacrylate “Photomer 4361” (manufactured by Cognis Japan Co., Ltd.), N, N -10 parts of dimethylacrylamide "DMAA" (manufactured by Kojin Co., Ltd.) and 0.5 part of 2,4-diphenyl-4-methyl-1-pentene (manufactured by Kanto Chemical Co., Ltd.) as a polymerization retarder are mixed. A product (X3) was prepared. The viscosity of the composition (X3) was 100 mPa · s.

[Step 1: Formation of porous resin layer having three-dimensional network structure]
(Formation of three-dimensional network structure)
A film-forming solution (K) 3 was applied onto a support of an acrylic plate having a thickness of 1 mm using a 50 μm bar coater, and the support was poured into water to obtain a milky white solidified coating film. The obtained solidified coating film was further washed with running water for 10 minutes and dried in a vacuum at 40 ° C. for 1 hour to obtain a porous resin layer (3) having a three-dimensional network structure.

[Step 2: Formation of a recess (flow path) in which a porous resin layer having a three-dimensional network structure is exposed on the bottom surface]
A recess (channel 3) in which the porous resin layer (3) having a three-dimensional network structure is exposed on the bottom surface in the same manner as in Example 1 except that the composition (X3) is used instead of the composition (X1). Formed.
(Immobilization of DNA)
In the same manner as in Example 1, amino group introduction, aldehyde group introduction, and DNA fixation were performed.

[Step 3: Adhering the lid]
The same composition and the same method as in Example 1 are used to have a capillary channel (channel 3) in which the lid is fixed to the recess (channel 3) and the porous resin layer having a three-dimensional network structure is exposed on the bottom surface. A microfluidic device (3) was produced.

[Structural observation of porous resin layer with three-dimensional network structure]
When the surface of the porous resin layer (3) having a three-dimensional network structure produced in the above step 1 was observed with a scanning electron microscope, sponge-like pores having a pore diameter of about 0.6 μm were observed. When the cross section of the porous resin layer was observed, the thickness of the porous resin layer was about 35 μm.

[Observation of recess (channel) structure]
When the cross section of the concave portion (channel 3) prepared in the above step 2 was observed with a scanning electron microscope, the cross sectional shape of the concave portion was about 250 μm in width and about 30 μm in depth excluding the porous resin layer having a three-dimensional network structure. It was a rectangle.

(Comparative Example 1)
This comparative example relates to a microfluidic device in which the surface of a known amino group-fixed glass substrate is the bottom surface of the flow path, and shows that the DNA immobilization density is low.
[Formation of recess (flow path)]
Composition prepared in Example 1 on a slide glass “Amine Silane” (manufactured by Matsunami Glass Industrial Co., Ltd.) having an amino group introduced on the surface thereof at a rotation speed of 800 rpm using a spin coater (manufactured by Mikasa Co., Ltd.). The product (X1) is applied to form an uncured coating film of the composition (X1), and the UV lamp 2 irradiates the uncured coating film other than the portion to be the flow path with a UV ray for 120 seconds. Then, a semi-cured coating film of the composition (X1) is formed, the uncured composition (X1) in the non-irradiated part is removed with ethanol, and the concave portion (channel 4) where the glass is exposed on the bottom surface is formed. Formed.

[Immobilization of DNA]
The recessed part (flow path 4) which has the said amino group in the bottom face was glutaraldehyde-treated by the method similar to Example 1, and the aldehyde group was introduce | transduced into the bottom face.

  Subsequently, the concave portion (channel 4) into which the aldehyde group was introduced was treated in the same manner as in Example 1 to immobilize DNA.

[Quantification of DNA]
As a result of measuring the concave portion (channel 4) in which DNA was fixed to the bottom glass by the same method as in Example 1, the fluorescence intensity value was 73 LAU / mm ² .

(Comparative Example 2)
This comparative example shows that the amount of DNA immobilized on the silicon substrate surface is low. However, it measured in the state which does not form a flow path.
[Introduction of amino group]
After pre-processing the silicon wafer by vacuum ultraviolet irradiation (manufactured by Senko Machine Co., Ltd.), a silane coupling agent having an amino group, 3-aminopropyltriethoxysilane (LS-3150 manufactured by Shin-Etsu Silicone), in a 1 mM isopropanol solution, It was immersed for 3 hours at 25 ° C., washed with ethanol and dried with hot air at 80 ° C. for 30 minutes to introduce amino groups on the surface of the silicon wafer.

[Immobilization of DNA]
The surface of the silicon wafer into which the amino group had been introduced was treated with glutaraldehyde and amino group-modified DNA in the same manner as in Example 1 to immobilize DNA on the silicon wafer surface.
[Quantification of DNA]
As a result of measuring the surface of the silicon wafer on which the DNA was fixed in the same manner as in Example 1, the fluorescence intensity value was about 78 LAU / mm ² .

(Comparative Example 3)
This comparative example shows that the amount of DNA immobilized on the silicon porous layer is low. However, it measured in the state which does not form a flow path.
[Formation of porous flow path]
A porous layer having a width of 250 μm, a depth from the surface of the silicon wafer, and a thickness of about 30 μm was formed using a silicon wafer according to the method of Prior Art Document 1.
[Introduction of amino group]
In the same manner as in Comparative Example 2, amino groups were introduced into this porous layer.
[Immobilization of DNA]
In the same manner as in Comparative Example 2, DNA was fixed to this porous layer.

[Quantification of DNA]
As a result of measuring the porous layer in which DNA was fixed to the above-mentioned bottom porous silicon by the same method as in Example 1, the fluorescence intensity value was about 114 LAU / mm ² . That is, it increased only about 1.46 times compared with the surface of the smooth silicon wafer.

(Comparative Example 4)
This comparative example shows that the amount of DNA immobilized on the surface having unevenness formed by electron beam etching is low.
[Formation of uneven surface]
Using an E1030 type ion sputter manufactured by Hitachi, Ltd., the acrylic plate was subjected to electron beam etching at 15 mA for 10 minutes to provide irregularities with a depth of about 0.4 μm on the surface.
[Production of microfluidic devices]
Instead of using an acrylic plate with a porous layer having a three-dimensional network structure, the introduction of an epoxy group, the flow path Formation, introduction of an amino group, introduction of an aldehyde group, fixation of DNA, and fixation of a lid were carried out to produce a microfluidic device.

[Quantification of DNA]
As a result of measurement in the same manner as in Example 2, the fluorescence intensity value was about 288 LAU / mm ² .

  From the quantification results of DNA in Examples 1 to 3 and Comparative Examples 1 to 4, the flow path without the porous resin layer of Comparative Example 1, the flow path with a silicon porous bottom surface, or three-dimensional The specific surface area of the flow path having the porous resin layer of the three-dimensional network structure of the embodiment is much larger than that of the flow path having a concavo-convex surface that is not a porous flow path of a network structure, and can immobilize many substances. It was clear.

2 is a scanning electron micrograph of a porous resin layer having a three-dimensional network structure created in Example 1. FIG.

Claims (16)

In a microfluidic device comprising a support, a porous resin layer having a three-dimensional network structure, a flow path, and a lid, (I) having the porous resin layer above the support, and (II) the porous resin The layer is filled with the cured resin of the active energy ray curable resin composition (X) impregnated except for the flow path portion, and (III) the flow path is filled with the cured resin of the active energy ray curable resin composition (X). Active energy rays formed on the upper part of the porous resin layer having a three-dimensional network structure and a porous resin layer having a three-dimensional network structure filled with a cured resin of the active energy ray-curable resin composition (X) A microfluidic device comprising a cured resin layer of the curable resin composition (X) and a lid portion as wall surfaces to form a hollow shape. The microfluidic device according to claim 1, wherein the porous resin layer having a three-dimensional network structure is made of an active energy ray-curable resin composition. In the cross section in the direction perpendicular to the flow direction of the fluid in the portion having the porous resin layer having the three-dimensional network structure of the flow path, x is an arbitrary point in the cross section, and the porosity closest to the arbitrary point by a linear distance the portion of the quality resin layer y, the linear distance between the xy and r, the maximum distance r can take in the cross section upon a r _max, claim 1 or the r _max is in the range of 1~50μm 3. The microfluidic device according to 2. The microfluidic device according to any one of claims 1 to 3, wherein a cross-sectional shape in a direction perpendicular to a fluid flow direction of the flow path is a rectangle or a trapezoid. The porous resin layer having the three-dimensional network structure is formed only on one surface of the inner wall of the flow path, and the average distance to the inner wall facing the porous resin layer is in the range of 1 to 50 µm. Microfluidic device. The microfluidic device according to any one of claims 1 to 5, wherein a layer thickness of the porous resin layer having the three-dimensional network structure is in a range of 0.5 to 30 µm. The microfluidic device according to any one of claims 1 to 6, wherein an average pore diameter of the porous resin layer having a three-dimensional network structure is in a range of 0.05 to 3 µm. The microfluidic device according to any one of claims 1 to 7, wherein the porous resin layer having a three-dimensional network structure is a porous resin layer to which a probe having affinity for an analysis target substance is fixed. The microfluidic device according to claim 8, wherein the probe is an oligonucleotide. A member for liquid chromatography comprising the microfluidic device according to claim 1. An electrophoretic member comprising the microfluidic device according to claim 1. (1) A step of forming a porous resin layer having a three-dimensional network structure having a large number of pores on the surface of the support, (2) containing an active energy ray polymerizable compound (a) on the porous resin layer Coating the active energy ray-curable composition (X), forming an uncured coating film of the composition (X), and irradiating the uncured coating film other than the portion to be formed with the flow path with active energy rays Then, a cured or semi-cured coating film of the composition (X) is formed, and the uncured portion of the uncured composition (X) is removed, and the porous resin layer having a three-dimensional network structure is exposed on the bottom surface. A step of forming a concave portion, and (3) a step of fixing another member serving as a lid to the concave portion of the member having the concave portion to form the concave portion as a hollow flow path. Device manufacturing method. The method for producing a microfluidic device according to claim 12, further comprising a step of surface-treating the surface of the resin layer after the step of forming a porous resin layer having a three-dimensional network structure on the surface of the support. The active energy ray-curable composition (X) contains an active energy ray-polymerizable compound (a) and an amphiphilic polymerizable compound copolymerizable with the active energy ray-polymerizable compound (a). The method for producing a microfluidic device according to claim 12 or 13, which is a composition. The method for producing a microfluidic device according to claim 12, wherein the viscosity of the composition (X) is 30 to 3000 mPa · s at 25 ° C. The step of forming a porous resin layer having a three-dimensional network structure on the surface of the support is compatible with the active energy ray-polymerizable compound (b) and the compound (b) on the support. After applying an active energy ray-curable film-forming solution (J) containing a poor solvent (R) that is incompatible with the polymer produced from (b), active energy rays are applied to the film-forming solution (J). The method for producing a microfluidic device according to claim 12, wherein the step of forming a porous resin layer having a three-dimensional network structure on the surface of the support is performed.

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