Classifications

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  • B01D57/02—Separation, other than separation of solids, not fully covered by a single other group or subclass, e.g. B03C by electrophoresis
• • B—PERFORMING OPERATIONS; TRANSPORTING
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  • B32B27/00—Layered products comprising a layer of synthetic resin
  • B32B27/06—Layered products comprising a layer of synthetic resin as the main or only constituent of a layer, which is next to another layer of the same or of a different material
  • B32B27/08—Layered products comprising a layer of synthetic resin as the main or only constituent of a layer, which is next to another layer of the same or of a different material of synthetic resin
• • B—PERFORMING OPERATIONS; TRANSPORTING
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  • B01D67/0002—Organic membrane manufacture
  • B01D67/0006—Organic membrane manufacture by chemical reactions
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  • B01D67/0039—Inorganic membrane manufacture
  • B01D67/0053—Inorganic membrane manufacture by inducing porosity into non porous precursor membranes
  • B01D67/006—Inorganic membrane manufacture by inducing porosity into non porous precursor membranes by elimination of segments of the precursor, e.g. nucleation-track membranes, lithography or laser methods
  • B01D67/0062—Inorganic membrane manufacture by inducing porosity into non porous precursor membranes by elimination of segments of the precursor, e.g. nucleation-track membranes, lithography or laser methods by micromachining techniques, e.g. using masking and etching steps, photolithography
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  • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
  • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
  • B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
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  • B03C5/00—Separating dispersed particles from liquids by electrostatic effect
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  • B29C66/51—Joining tubular articles, profiled elements or bars; Joining single elements to tubular articles, hollow articles or bars; Joining several hollow-preforms to form hollow or tubular articles
  • B29C66/53—Joining single elements to tubular articles, hollow articles or bars
  • B29C66/534—Joining single elements to open ends of tubular or hollow articles or to the ends of bars
  • B29C66/5346—Joining single elements to open ends of tubular or hollow articles or to the ends of bars said single elements being substantially flat
  • B29C66/53461—Joining single elements to open ends of tubular or hollow articles or to the ends of bars said single elements being substantially flat joining substantially flat covers and/or substantially flat bottoms to open ends of container bodies
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  • B32B—LAYERED PRODUCTS, i.e. PRODUCTS BUILT-UP OF STRATA OF FLAT OR NON-FLAT, e.g. CELLULAR OR HONEYCOMB, FORM
  • B32B3/00—Layered products comprising a layer with external or internal discontinuities or unevennesses, or a layer of non-planar shape; Layered products comprising a layer having particular features of form
  • B32B3/26—Layered products comprising a layer with external or internal discontinuities or unevennesses, or a layer of non-planar shape; Layered products comprising a layer having particular features of form characterised by a particular shape of the outline of the cross-section of a continuous layer; characterised by a layer with cavities or internal voids ; characterised by an apertured layer
• • B—PERFORMING OPERATIONS; TRANSPORTING
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  • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
• • C—CHEMISTRY; METALLURGY
  • C23—COATING METALLIC MATERIAL; COATING MATERIAL WITH METALLIC MATERIAL; CHEMICAL SURFACE TREATMENT; DIFFUSION TREATMENT OF METALLIC MATERIAL; COATING BY VACUUM EVAPORATION, BY SPUTTERING, BY ION IMPLANTATION OR BY CHEMICAL VAPOUR DEPOSITION, IN GENERAL; INHIBITING CORROSION OF METALLIC MATERIAL OR INCRUSTATION IN GENERAL
  • C23C—COATING METALLIC MATERIAL; COATING MATERIAL WITH METALLIC MATERIAL; SURFACE TREATMENT OF METALLIC MATERIAL BY DIFFUSION INTO THE SURFACE, BY CHEMICAL CONVERSION OR SUBSTITUTION; COATING BY VACUUM EVAPORATION, BY SPUTTERING, BY ION IMPLANTATION OR BY CHEMICAL VAPOUR DEPOSITION, IN GENERAL
  • C23C14/00—Coating by vacuum evaporation, by sputtering or by ion implantation of the coating forming material
  • C23C14/06—Coating by vacuum evaporation, by sputtering or by ion implantation of the coating forming material characterised by the coating material
  • C23C14/12—Organic material
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
  • G01N21/03—Cuvette constructions
• • G—PHYSICS
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  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
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  • G01N21/05—Flow-through cuvettes
• • G—PHYSICS
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  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
  • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
  • G01N27/416—Systems
  • G01N27/447—Systems using electrophoresis
• • G—PHYSICS
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  • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
  • G01N27/416—Systems
  • G01N27/447—Systems using electrophoresis
  • G01N27/44756—Apparatus specially adapted therefor
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  • B01D2323/40—Details relating to membrane preparation in-situ membrane formation
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  • B01J2219/00781—Aspects relating to microreactors
  • B01J2219/00783—Laminate assemblies, i.e. the reactor comprising a stack of plates
• • B—PERFORMING OPERATIONS; TRANSPORTING
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• • B—PERFORMING OPERATIONS; TRANSPORTING
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• • B—PERFORMING OPERATIONS; TRANSPORTING
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  • B01L2300/0809—Geometry, shape and general structure rectangular shaped
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• • B—PERFORMING OPERATIONS; TRANSPORTING
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  • B01L2400/0421—Moving fluids with specific forces or mechanical means specific forces electrical forces, e.g. electrokinetic electrophoretic flow
• • B—PERFORMING OPERATIONS; TRANSPORTING
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  • B29C66/00—General aspects of processes or apparatus for joining preformed parts
  • B29C66/70—General aspects of processes or apparatus for joining preformed parts characterised by the composition, physical properties or the structure of the material of the parts to be joined; Joining with non-plastics material
  • B29C66/71—General aspects of processes or apparatus for joining preformed parts characterised by the composition, physical properties or the structure of the material of the parts to be joined; Joining with non-plastics material characterised by the composition of the plastics material of the parts to be joined
• • B—PERFORMING OPERATIONS; TRANSPORTING
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  • B32B2255/00—Coating on the layer surface
  • B32B2255/26—Polymeric coating
• • G—PHYSICS
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  • G01N2021/0346—Capillary cells; Microcells

Definitions

• microchannel chip Method for producing microchannel chip, microchannel chip, method for separating biomolecules using the microchannel chip, and electrophoresis apparatus having the microchannel chip
• the present invention relates to a method for manufacturing a microchannel chip.
• the present invention also relates to a microchannel chip, a method for separating biomolecules using the microchannel chip, and an electrophoresis apparatus.
• Non-Patent Document 1 Capillary electrophoresis or microchannel chip electrophoresis is a very excellent method for separating and analyzing minute amounts of biomolecules, and since automation and speeding up of analysis are possible, many studies have been conducted. Has been done.
• Glass is a typical material used for capillary electrophoresis or microchannel chip electrophoresis, but there are many problems to be solved in order to separate proteins.
• capillary electrophoresis made of glass and microchannel chip electrophoresis were affected by electroosmotic flow.
• Patent Documents 1, 2, and 3 As a coating method, a method of chemically bonding a compound to a surface or a method of physical adsorption have been tried.
• a method in which a coating agent is caused to flow in a flow channel to coat the flow channel is known.
• a method in which an electrophoresis buffer solution mixed with a coating agent is flowed to cover is flowed to cover.
• this method is a very simple method, there is a problem that the adsorption state is very weak because the adsorption is performed by electrostatic interaction or hydrophobic interaction, and the coating is easily peeled off.
• the electrostatic interaction there is a problem that the application range is narrow because the pH is easily affected.
• Non-Patent Document 2 Non-Patent Document 2
• Non-Patent Document 2 a method of bonding the base material and the cover material with an adhesive has been adopted (Non-Patent Document 2).
• the adhesive may ooze into the inside, and the manufacturing process may be complicated, for example, by controlling the amount of use or the location of application.
• Non-Patent Document 1 Journal of Chromatography (F.E.P.Mikkers, F.M.
• Non-Patent Document 2 Analyst, 2003, 128, 237-244
• Patent Document 1 Japanese Patent Publication No. 5-503989
• Patent Document 2 Japanese Patent Publication No. 7-506432
• Patent Document 3 Japanese Patent Publication No. 9-504375
• the microchannel chip is usually obtained by laminating a base material having a flow path on its surface and a cover material. Coating with a high molecular compound film such as It has been found that even if the cover material is attached to the base material, which tends to decrease, the medium flowing through the flow passage may leach out of the flow passage into the gap between the base material and the cover. Public knowledge
• the present invention provides a simple microchannel capable of improving the adhesive strength at the time of bonding a substrate and a cover material when the substrate surface is coated with a polymer compound film. It is an object to provide a method for manufacturing a chip.
• the present inventors have found that it is possible to improve the adhesive strength at the time of laminating the base material and the cover material, and to provide a simple method for producing a microchannel chip, and have completed the present invention. That is, the present invention includes the following.
• a method of manufacturing a microchannel chip comprising: attaching a cover material to a surface of the substrate on which a channel is formed.
• the surface of the cover material is shielded by a mask having the same shape as a part or all of the exposed portion of the mask of the base material, and a polymer compound film is formed on the exposed surface of the cover material.
• Both the base material and the cover material are thermoplastic resins
• the bonding step is a method of bonding the substrate and the cover material by thermocompression bonding.
• thermocompression bonding is performed at a temperature of 200 ° C or lower.
• One of the base material and the cover material is a silicone resin, and the other is a glass or a plastic,
• the bonding step is a method of bonding the base material and the cover material by pressure bonding.
• a surface of the base material having a flow path formed on the surface, the flow path side surface is bonded to a cover material, and a polymer is formed on a part or all of the flow path on the surface of the base material.
• a method for separating biomolecules including the following steps:
• a surface of the base material having a flow path formed on its surface, the flow path side surface is adhered to a cover material, and the flow path surface of the base material surface is coated with a polymer compound film. Adding a biomolecule to be analyzed to the microchannel chip; and
• FIG. 1 is an electron micrograph showing that a plasma-polymerized film was formed with a width of 200 ⁇ m.
• the acceleration voltage of the electron microscope is 5.00 kV, and the photographic magnification is 100 times.
• FIG. 2 shows the composition of the element mapping analysis of the film formation part by the electron probe microanalyzer It is a photograph.
• FIG. 3 is a schematic diagram showing how to apply a voltage during sample introduction and separation during chip introduction.
• FIG. 4 is a view showing the results of electrophoresis of Cy5-stained carbonic anhydrase between a chip having a plasma polymerized film and a chip without film formation.
• A shows the case of a chip having a plasma polymerized film (HMDS)
• B shows the case of a chip without film formation.
• the surface of a substrate having a groove-shaped channel formed on a surface thereof is shielded with a mask exposing the channel, and the exposed substrate surface is coated with a polymer.
• the method is characterized by including a step of forming a compound film and a step of bonding a cover material to the surface of the base material on the side where the flow path is formed.
• the mask that exposes the flow channel is preferably a mask that exposes the entire flow channel or the entire flow channel and the vicinity of the flow channel. Good.
• the type of the mask is not limited, and for example, a photoresist mask, a metal mask, or the like can be used.
• the surface of the substrate is covered with a polymer compound film on the surface of the substrate, and the other portions are not coated with the polymer compound film. Excellent adhesive strength when laminating and.
• a step of forming a polymer compound film on the surface of the cover material on the side to be bonded to the base material may be included. That is, the polymer compound film may be formed on both the surface of the base material and the surface of the cover material. If the polymer compound film is also formed on the surface of the cover material, the resolution of the sample to be separated using the microchannel chip can be further improved.
• the surface of the cover material is shielded by a mask having the same shape as a part or all of the exposed portion of the mask of the base material.
• a polymer compound film is formed on the exposed surface of the cover material.
• a polymer compound membrane can be formed in various patterns and gradients in the flow path provided on the base material surface or the cover material surface.
• a polymer compound film having a shape different from the mask shape of the base material may be formed on the cover material side.
• the bonding be performed so that the polymer compound films coated on the surface of the base material and the cover material just overlap with each other in an opposite shape.
• a plasma polymerized film is preferred. If it is a plasma polymerized film, a film that is more uniform and excellent in stability can be formed.
• a plasma polymerized film is preferred. If it is a plasma polymerized film, a film that is more uniform and excellent in stability can be formed.
• the combination of the types of polymer compound films to be used is not particularly limited, and the same polymer compound film or different polymer compound films may be used.
• the polymer compound film formed on the surface of the base material and the polymer compound film formed on the surface of the cover material are the same polymer compound film.
• the film be a plasma polymerized film having the same monomer raw material power.
• the material constituting the base material is arbitrary.
• at least the surface of the flow channel formed on the surface of the substrate is modified by a plasma polymerized film, a surface polymerized film, or a polymer binding film. Therefore, the material of the base material itself does not directly affect the results of separation such as electrophoresis. Therefore, for example, any material satisfying the following minimum conditions can be selected.
• a transparent material is generally used for the base material. By using transparent materials, optical observation of external forces is possible. Specifically, for example, glass or plastic can be used as a base material.
• plastic examples include thermoplastic resin and silicone resin.
• thermoplastic resin examples include, for example, poly (meth) acrylates such as polymethyl methacrylate (PMMA); polycarbonate (PC); polyethylene terephthalate (PET); polybutyl compounds such as polyethylene and polypropylene; Polystyrene and the like can be mentioned.
• PMMA polymethyl methacrylate
• PC polycarbonate
• PET polyethylene terephthalate
• PET polybutyl compounds
• polyethylene and polypropylene polystyrene and the like can be mentioned.
• the thermoplastic resin has a heat distortion temperature of preferably 200 ° C or lower, more preferably 150 ° C or lower, and particularly preferably 120 ° C or lower, although it depends on the type. In such a temperature range
• silicone resin examples include silicone rubber such as polydimethylsiloxane (PDMS).
• PDMS polydimethylsiloxane
• the shape of the substrate is preferably a plate-like planar substrate.
• the thickness of the substrate is not limited, If so, it is preferably in the range of about 120 mm.
• cover material the same material as the base material can be used.
• cover material covers the base material, it is preferable that its shape and size are the same as those of the base material.
• the thickness of the cover material is not limited, but is preferably, for example, in the range of about 120 mm.
• the combination of the materials of the base material and the cover material is not particularly limited, and the same material or different materials may be used.
• At least one of the base material and the cover material is preferably plastic.
• both the base material and the cover material are plastic.
• both of the base material and the cover material are thermoplastic resins.
• one of the base material and the cover material is a silicone resin
• the other may be glass or plastic, and the other is more preferably plastic.
• the method of bonding the base material and the cover material by thermocompression bonding can be adopted as the bonding method.
• the temperature at the time of thermocompression bonding depends on the type of plastic used, but is preferably 200 ° C or lower, more preferably 150 ° C or lower, and particularly preferably 120 ° C or lower.
• the base material and the cover material are made of a silicone resin and the other is made of an arbitrary plastic or glass
• a method of bonding the base material and the cover material is as follows. Can be adopted.
• Preferable combinations of the materials of the base material and the cover material include, for example, the following.
• PMMA PMMA ⁇ PDMS: PDMS ⁇ PDMS: PMMA, PDMS: Glass, PET: PET, PMMA: PET, PDMS: PET, PC: PC, PDMS: PC, PMMA: PC, PS: PS, PDMS: PS, PMMA : PS
• PMMA PMMA
• PDMS PDMS
• PDMS PMMA
• PMM A PET
• PDMS PET
• PDMS PC
• PMMA PC
• PDMS PS
• PMMA PS Any combination can be preferably used.
• bonding with excellent bonding strength can be performed at low temperature and without using an adhesive.
• the bonding of the base material and the cover material can be performed by pressure bonding or thermocompression bonding as described above.
• the flow path is a groove formed on the surface of the substrate.
• the width of the groove can be a minute space such as 1/100 / zm.
• the cross-section of the groove can be polygonal, such as a triangle or square, or U-shaped or semi-circular. Glass with such a fine structure groove
• the following method can be used to provide the base material such as plastic or the like.
• a fine structure having a free shape can be easily provided.
• a technique is known in which a groove having a width and a depth of 10-100 / zm is provided on a glass surface.
• the present inventors have succeeded in producing a microchannel using reactive ion etching. Utilizing different types of etching gas depending on the material of the base material, it is possible to perform etching with good selectivity and high etch rate.
• the groove formed on the surface of the base material can be made a closed system by overlapping a cover material. Further, a groove can be provided on the surface of the cover material. In this case, it is preferable to provide the groove so as to overlap with the groove provided in the base material.
• the cover material by providing a hole in the cover material at a position overlapping with the groove provided in the base material or the cover material, it is possible to form a communication channel for supplying a sample or a separation medium to the groove. Wear.
• the holes provided in the cover material can be used as a reservoir for holding a sample or a buffer solution.
• the surface of a substrate having a groove-shaped channel formed on a surface thereof is shielded by a mask that exposes the entire channel, and a high surface is provided on the exposed substrate surface.
• Forming a molecular compound film may include a step of forming a polymer compound film on the surface of the cover material to be bonded to the base material.
• the polymer compound film include a plasma-polymerized film, a surface-polymerized film, and a polymer-bonded film.
• the plasma polymerization it is possible to form a plasma polymerization film even on a fine groove surface. According to the plasma polymerization, the resulting film is extremely homogeneous. Therefore, the generation of pinholes on the surface of the substrate can be suppressed, and a highly reliable substrate for separation analysis can be prepared.
• a desired surface polymerization film in which peeling of the film is suppressed can be formed at a desired position on the substrate surface.
• a desired polymer compound film can be formed at a desired position on the substrate surface while controlling the film thickness.
• the base material or the cover material coated with the plasma polymerized film, the surface polymerized film, or the polymer binding film can be obtained by a known method. Hereinafter, each film will be described.
• plasma polymerization is a technique in which a monomer material is directly formed on a support surface by plasma excitation in a vacuum. By changing the components of the monomer material, A plasma polymerized film having various characteristics can be obtained. In principle, plasma polymerization can be carried out using any monomer. In order to obtain a normal polymer, it is necessary to cleave a double bond, whereas in plasma, monomer substances are separated and a polymerization reaction occurs via many active species.
• the monomer material for the plasma polymerized film in the present invention may be any as long as it can form a polymer film that gives suitable properties according to separation such as electrophoretic separation on the surface of a substrate or a cover material.
• suitable properties according to the electrophoretic separation include the following properties. Among these properties, a monomer substance capable of giving any property can be used in the present invention.
• the base material or the cover material is plastic
• the plasma polymerization even if the plastic surface is used, fine grooves are formed. It is also possible to form a plasma polymerized film on the surface.
• the membranes that are also pliable are extremely homogeneous and are particularly good for coating plastics.
• a microchannel chip in the method of manufacturing a microchannel chip according to the present invention, it is desirable to use a channel coated with a plasma polymerized membrane and to employ a combination of the plastics.
• a microchannel chip having an extremely uniformly coated channel and excellent adhesive strength between the substrate and the cover material can be manufactured easily and with high yield.
• the adsorption of the protein to the substrate can be controlled by a plasma polymerized membrane.
• a plasma polymerized membrane For example, it can be controlled by the degree of hydrophobicity and surface charge of the substrate.
• Examples of the monomer substance that provides a plasma-polymerized film satisfying the above conditions include the following ("Plasma Polymerization" edited by Yoshito Nagata ', edited by Mitsuo Tsunoda, Kaoru Nakajima, Masataka Takamura, Morita Shinzo, et al., Tokyo Chemical Dojin, 1986).
• the following compounds can be shown as alkanes or cycloalkanes.
• Alkenes, alkynes, and! / ⁇ represent the following compounds as cycloalkenes.
• Methanol ethanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, 2-methyl-1 propanol, 2-methyl-2 propanol, aryl alcohol, 1,3 butanediol, 2,3 butanediol, 2 , 3 Epoxy 1 propanol, formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, barrelaldehyde, isovaleraldehyde, acrylaldehyde, crotonaldehyde, glyoxal, acetone, 2-butanone, 2-pentanone, 3-methyl-2-butanone, 3 Pentanone, 2-Heki Sanone, 4-methyl-2-pentanone, 2-heptanone, cyclobutanone, cyclopentanone, cyclohexanone, cycloheptanone, cyclooctanone, 4-methyl-3-pentene 2-one, 2,
• the following halogenated compounds can be used as the monomer material.
• aromatic hydrocarbons can be used as monomer substances.
• Benzene toluene, ethylbenzene, propylbenzene, tamen, butylbenzene, s-butylbenzene, t-butylbenzene, 0-xylene, m-xylene, p-xylene, 0-diethylbenzene, m-getylbenzene, p-ethyl Benzene, mesitylene, 1,2,4,5-tetramethylbenzene, styrene, phenylacetylene, (E) -1 propenylbenzene, (E) -1 phenylbutadiene, 2-phenylbutadiene, biphenyl , Naphthalene, 1-methylnaphthalene, 2-methylnaphthalene, anthracene, phenanthrene, pyrene, naphthacene, Talycene and pentacene.
• heterocyclic compound can be used as a monomer substance.
• a troponoid compound such as tropone ditroborone, or an organometallic compound represented by tetramethylsilane, tetramethyltin, or tetramethyllead is used as a monomer substance.
• Hexamethyldisiloxane can be preferably used when the substrate surface has a negative charge under conditions where the pH is near neutrality.
• Hexylamine or aminoacetaldehyde dimethyl acetal can be preferably used when the substrate surface has a positive charge under conditions where the pH is near neutrality.
• Conditions for forming a plasma-polymerized film with these monomer substances are known. Specifically, the main factors affecting the reproducibility of the plasma polymerization reaction are, for example, flow rate, Conditions such as discharge power, discharge time, and pressure are considered important. In plasma polymerization, it is necessary to set optimal polymerization conditions according to the equipment and monomers. If the W / FM (where W is the discharge power, F is the flow rate, and M is the molecular weight of the monomer) are the same, it is reported that the film quality is almost the same (Yasuda, Plasma Polymerization, Academic Press, New York, 1985) There is.
• a plasma-polymerized membrane with hexamethyldisiloxane which is described below as an advantageous monomer substance for the purpose of immobilizing polynucleotides, for example, select the optimal conditions in the following range By doing so, it is possible to form a plasma polymerized film of approximately more than 0 and 240 A or less.
• the following conditions can be shown as more desirable conditions for forming a plasma polymerized film exceeding 0 and 240 A or less.
• various functional groups can be imparted to the surface of the base material by selecting a monomer substance, so that films having various properties can be easily formed.
• the zeta potential indicating the charge state of a force substance that varies depending on the pH can be controlled preferably within a range of 100 to 1 "hOOmV.
• the contact angle of the surface can be controlled preferably within a range of 1 degree to 140 degrees.
• the thickness of such a plasma polymerized film is preferably, for example, preferably in the range of 1200 nm.
• the plasma polymerized film obtained in this way is a very homogeneous film, and the generation of pinholes is significantly suppressed! Puru.
• a plasma-polymerized film can be formed on a substrate surface having an arbitrary shape.
• the surface of the substrate having a groove-shaped channel formed on its surface is shielded by a mask that exposes the entire channel, and the surface of the exposed substrate is exposed. Includes a step of forming a polymer compound film, but collectively transfers the photomask pattern with light.
• a device consisting of millions of components, such as a super LSI, can be fabricated as an integrated structure on a silicon substrate of several mm square. Further, in a photo application, a combination of a plurality of photo mask patterns can be used. By utilizing this feature, it is possible to combine different processing steps such as adhesion processing and surface modification processing.
• the technology for surface modification / thin film formation applied to photo applications is a dry process. Since the plasma polymerization method is a dry process, it is suitable for device fabrication by photofabrication. Furthermore, if a plasma polymerization method is used, a thin film having a functional group on the surface can be produced by selecting an appropriate monomer substance. In addition, the plasma polymerized film is a pinhole-free film with a highly cross-linked structure, and is optimal as a modified thin film inside the flow channel.
• the surface polymerized film is a polymerized film obtained by polymerizing a polymerizable monomer on the substrate surface.
• the polymerization is preferably carried out by polymerizing a polymerizable monomer on a hydrophobic functional group having a double bond at a terminal on the substrate surface.
• the hydrophobic functional group is preferably an alkyl group having a double bond at the terminal having 2 to 6 carbon atoms, more preferably 3 to 6 carbon atoms, and particularly preferably 4 to 16 terminals. No.
• hydrophobic functional group examples include a butyl group, an aryl group, a 1-butyl group, a 1-pentyl group, and an 11-hexynyl group.
• the surface-polymerized film is covalently bonded by a carbon-carbon single bond using the hydrophobic functional group as a spacer.
• the base material to which such a surface-polymerized film is bonded is prevented from approaching water molecules by a hydrophobic spacer, and therefore, the hydrophobic substrate due to hydrolysis due to the influence of pH or the like. Desorption of the pulse itself is suppressed.
• the hydrophobic spacer and the surface-polymerized film are bonded by carbon-carbon bonds, the surface-polymerized film does not peel off at the bonding position with the hydrophobic spacer.
• the substance to be analyzed is a protein, even if the analysis is performed in a water-soluble solvent, a highly reliable analysis in which the surface polymerized film is not peeled off due to the influence of pH can be performed.
• a polymerizable monomer is polymerized to form a polymer film on the surface.
• the polymer itself is bonded, there is no aggregation of the polymer.
• the coupling can be performed efficiently.
• the introduction of the hydrophobic functional group onto the substrate surface is performed by dissolving the compound that induces the hydrophobic functional group having a double bond at the terminal in a solvent such as toluene, methanol, or ethanol. It can be carried out by bringing materials into contact.
• the contact reaction is carried out, for example, at a temperature of about room temperature (about 25 ° C.) to about 100 ° C., for example, for about 1 hour to about 24 hours.
• Such a compound that induces a hydrophobic functional group having a double bond at the terminal preferably has one terminal capable of reacting with a silanol group on the glass surface.
• alkenyl silanes such as triethoxybutylsilane, triethoxyarylsilane, triethoxybutenylsilane, triethoxypentenylsilane, and triethoxyhexylsilane.
• triethoxyarylsilane, triethoxybutenylsilane, triethoxypentenylsilane, and triethoxyhexylsilane and particularly preferably, triethoxybuteninolesilane, triethoxypenten-nolesilane, and triethoxyhexylsilane. It is desirable to use These alkylsilanes can be commercially available or can be produced by a known method.
• the compound can be easily synthesized by reacting a Grignard reagent or an alkyllithium compound containing a desired alkyl group with a halogenated silane such as chlorosilane or an alkoxysilane in the presence of a solvent.
• a halogenated silane such as chlorosilane or an alkoxysilane
• the polymerizable monomer is not limited as long as it has a vinyl group, an aryl group, a diene or the like.
• Examples of such a polymerizable monomer include a non-on monomer, an aon monomer, and a cation monomer.
• Examples of the non-ionic monomer that forms a non-ionic (hydrophobic, hydrophilic, etc.) surface include amides such as acrylamide and methacrylamide;
• Methyl acrylate methyl methacrylate, butyl acetate, aryl acetate, aryl acetate acetate, butyl trimethyl acetate, burmic acid, hexyl hexate, laurate butyl, methacrylate, octanoate, palmitate, butyl pivalate , Vinyl propionate, butyl stearate, mono-2- (methacryloyloxy) ethyl hexahydrophthalate, mono-2- (methacryloyloxy) ethyl phthalate, butyl benzoate, P-butyl benzoate, BUYL BUTYLATE, BULL PROPYLATE, BURL CAPRONATE, BULL CROTONATE, BULL DECanoate, Vinyl Cinnamate, Aryl Butyrate Aryl Benzoate, Aryl n-butyrate, Aryl n-Pyrate Phenylate, n-Aryl Prop
• Ethers such as bininolebutinoleatenore, arinoreetenore, arinoleetinoreetenole, arinolebutinoleether, burethyl ether, and allyl n-decanoate;
• Alcohols such as bull alcohol and aryl alcohol
• Halides such as vinyl chloride, acrylyl chloride, methacryloyl chloride, butyl acetate, atariloyl chloride, aryl bromide, allyl iodide, arylacet acetate, allyl cloformate, and allyl chloroformate;
• Silanes such as 3-methacryloxypropyltrimethoxysilane, vinyltrichlorosilane, arylchlorodimethylsilane and arylchloromethyldimethylsilane;
• Cyanides such as metall-tolyl, buracetonitrile, acrylonitrile, allylic cyanoacetate, and allyl cyanide;
• Cycloalkane derivatives such as 2-arylcyclohexanone, 1-arylcyclohexanol, and arylcyclopentane;
• acrylamide / divinyl alcohol can be preferably used as the hydrophilic nonionic surface
• styrene / diarylbenzene or the like can be preferably used as the hydrophobic nonionic surface
• Examples of the a-on monomers for forming the a-on surface include, for example,
• Lipoxyl groups such as acrylic acid, methacrylic acid, mono-2- (atalyloyloxy) ethyl succinate;
• Sulfonic acid group-containing compounds such as arylsulfonic acid, vinylsulfonic acid, 2-acrylamide-2-methylpropanesulfonic acid, 3-aryloxy-2-hydroxy-1-propanesulfonic acid, and p-butylbenzenesulfonic acid.
• vinylsulfonic acid diarylsulfonic acid can be preferably used as the strong anionic property
• acrylic acid / methacrylic acid can be preferably used as the weak anionic property
• Examples of the cationic monomer that forms the cationic surface include:
• Primary amines such as arylamine, 3-acrylamide-, ⁇ -dimethylpropylamine, arylcyclohexylamine, 3-methacrylamide- ⁇ -dimethylpropylamine;
• Secondary amines such as methylarylamine
• Tertiary amines such as ⁇ -aryljetylamine, ⁇ -aryldimethylamine; aryltriethylammonium, (3-acrylamidopropyl) trimethylammonium-dimethyl chloride, butyltrimethylammonium-bromobromide, Quaternary ammonium salts such as 3- (methacryloylamino) propyltrimethylammonium chloride, ethylethyl methacrylatetrimethylammonium chloride, and diaryldimethylammonium chloride.
• a-ionic monomers, and cationic monomers for example, arylhydrazine, 2-bulpyrazine, 2-biphenyl having a heterocyclic compound in a side chain.
• Nylpyridine, 4-vinylpyridine, ⁇ -vinyl-2-pyrrolidone, 1-arylbenzotriazole And aryl-1-benzotriazole carbonate can also be used.
• diaryldimethylammonium salt as a strong thione and arylamine as a weak cation can be preferably used.
• Such a polymerizable monomer can be used alone or in combination of two or more.
• the radical polymerization of the polymerizable monomer on the surface of the base material a known method can be employed.
• the polymerization can be carried out in the presence or absence of a solvent, by adding a polymerization initiator as necessary, and polymerizing the polymerizable monomer on the surface of the base material into which the polymerizable functional group has been introduced.
• the solvent is not particularly limited as long as the solvent dissolves the polymerizable monomer.
• THF THF, methanol, DMF, DMSO and the like can be used.
• polymerization initiator examples include 2,2'-azobis (isobutyl-tolyl) (AIBN), 1,1'-azobis (cyclohexane-1 carbo-tolyl), and 2,2'-azobis ( 2-methylbutyro-tolyl) and the like can be used.
• AIBN isobutyl-tolyl
• 1,1'-azobis cyclohexane-1 carbo-tolyl
• 2-methylbutyro-tolyl 2-methylbutyro-tolyl
• peroxides, organometallic compounds and the like can also be used.
• a polymerizable monomer that does not dissolve in a solvent such as THF
• ultrapure water is used as a solvent
• the polymerization can be carried out using a polymerization initiator such as azobis cyanovaleric acid.
• the polymerization is different depending on the type of the polymerizable monomer and is not limited. Usually, for example, the polymerization can be carried out in a temperature range of about 100 ° C at room temperature for about 117 hours.
• the surface polymerized film obtained in this manner has a variety of polymerizable monomers to be used! / Can be formed into various ranges of charge, hydrophobic Z hydrophilic surface by a combination of a plurality of polymers.
• the zeta potential indicating the charge state of a force substance that differs depending on the pH can be controlled preferably in the range of 100 to 1 "hOOmV.
• the contact angle of the surface can be controlled preferably in the range of 11 to 140 degrees.
• a monomer-unmodified portion such as a pinhole may occur. is there. For this reason, a polymerizable monomer or polymer can be further bonded.
• another polymer or monomer may be further reacted with a functional group in a polymer side chain of the surface-polymerized film.
• a surface-polymerized film having a cationic functional group on the surface can be synthesized.
• the surface coated with such a surface polymerized film it is possible to perform protein electrophoresis while performing electrostatic interaction (positive charge of the film and negative charge of the protein).
• the polymer binding film is obtained by introducing a reactive functional group on the surface of a base material and covalently bonding a polymer to the functional reactive group.
• Examples of a reactive functional group serving as a site to which a polymer compound is bonded include an amino group, an epoxy group, a carboxyl group, and an aldehyde group. Among these, an amino group and an epoxy group can be preferably used.
• such a binding group having a reactive functional group further binds to the substrate surface via a hydrophobic spacer.
• the hydrophobic spacer preferably contains an alkyl group having 2 to 6 carbon atoms, more preferably 3 to 6 carbon atoms, and particularly preferably 4 to 6 carbon atoms. ,.
• a polymer compound was bonded to a reactive functional group via such a hydrophobic spacer. Since the approach of water molecules to the base material is suppressed by a hydrophobic spacer, peeling of the polymer-bound film due to hydrolysis due to the influence of pH or the like is suppressed.
• the introduction of the reactive functional group having a spacer onto the surface of the base material varies depending on the type of the base material.
• the base material is glass, it can be performed by a silane coupling method. If is a metal, it can be performed by a self-assembled monolayer method.
• a solvent such as toluene, methanol, or water is added to a solvent such as aminopropyltriethoxysilane, aminobutyltriethoxysilane, aminopentyltriethoxysilane, or aminohexyltriethoxysilane.
• an aminoalkyl-based silane coupling agent or an epoxyalkyl-based silane coupling agent can be used in the presence of a solvent in the presence of a Grignard reagent or an alkyllithium compound containing a desired alkyl group and functional group, and a chlorosilane or other silane.
• a Grignard reagent or an alkyllithium compound containing a desired alkyl group and functional group can be used in the presence of a Grignard reagent or an alkyllithium compound containing a desired alkyl group and functional group.
• a chlorosilane or other silane can be easily synthesized by reacting with silane or alkoxysilane
• the contact reaction is carried out, for example, at a temperature of about room temperature (about 25 ° C) to about 100 ° C for a time of, for example, about 124 hours.
• a metal thin film such as gold is formed on the surface of a base material by, for example, sputtering, and a spacer having a functional group and a thiol group is introduced on the metal thin film surface. Then, a polymer (or a polymerization initiator can be reacted with a functional group and polymerized using a monomer) can be reacted to form a polymer binding film. In addition, a polymer having a thiol group can be prepared in advance and then modified on the metal surface to form a polymer film.
• Examples of the metal include gold, silver, and copper.
• Examples of the spacer include aminoethanethiol having an amino group and thiotatoic acid having a carboxyl group.
• the solvent can be carried out by dissolving the spacer in a solvent such as DMSO or water and bringing the spacer into contact with the metal thin film.
• the contact reaction is performed at a temperature of, for example, room temperature and about 100 ° C, for a time of, for example, about 124 hours.
• polystyrene, polyallylbenzene, polybutyl alcohol, polyacrylamide, polybutylsulfonic acid, polyacrylic acid, polydiallyldimethylammonium salt, polyallylamine, polyethylene glycol and the like are preferably used. be able to.
• polyvinyl alcohol and polyallyl alcohol can be more preferably used as the nonionic surface.
• Polyacrylic acid or the like can be more preferably used as the strong anionic surface.
• Polyallylamine can be more preferably used as a strong thione surface.
• Such polymers can be used alone or in combination of two or more.
• the weight average molecular weight of such a polymer is, for example, preferably 5000 to 5000
• a reactive functional group such as a pinhole binds to a polymer to generate an unmodified portion of the polymer. For this reason, a polymer can be further bonded.
• Such a polymer binding film is not limited, as a known method can be employed.
• it can be produced by dissolving the polymer in a solvent and bringing a substrate having a reactive functional group introduced into the surface thereof into contact with a solution.
• the solvent is not limited as long as it dissolves the polymer.
• an activator may be used as necessary.
• an activator may be used as necessary.
• N-hydroxysuccinimide, 1-ethyl-3- (3-dimethylaminopropyl hydrochloride) are dissolved.
• Mcouth pill Carposimide is added and combined.
• the zeta potential indicating the charge state of the active substance can be controlled preferably in the range of 100-1 "hOOmV.
• the contact angle of the surface can be controlled preferably in the range of 11 to 140 degrees.
• the thickness of such a polymer-bound film can be easily controlled by preparing a polymer to be bound beforehand.
• a polymer binding film having an anionic functional group on the surface can be synthesized. As a result, it is an electrostatic interaction similar to that of an amino group, and enables electrophoretic separation and the like by interaction between the negative charge of the membrane and the positive charge of the protein.
• a polymer binding membrane having an extremely hydrophobic or hydrophilic surface can be synthesized, and thus separation based on hydrophobic interaction or hydrophilic interaction can be performed. Is possible.
• the anionic functional group may be, for example, a non-ionic polymer or a non-ionic polymer having a hydrophobic (or hydrophilic) functional group.
• a hydrophilic monomer By bonding a hydrophilic monomer, a substrate surface having both a ionic property and a hydrophobic (or hydrophilic) property can be formed. Further, by changing the modification ratio of the nonionic polymer or monomer, the balance of hydrophobicity (or hydrophilicity) can be controlled.
• the microchannel chip obtained in this manner a large area covered with the polymer compound film exists on the surface of the substrate, more preferably the surface of the substrate and the cover material. It has excellent adhesive strength when bonding the base material and the cover material.
• the microchannel chip according to the present invention is obtained by laminating a surface on the flow channel side of a base material having a flow channel formed on a surface thereof and a cover material. The entire surface is covered with a polymer compound film.
• the surface of the cover material on the substrate side is coated with a polymer compound film.
• the polymer compound film of the base material is formed in a region of the surface of the cover material on the base material side opposite to the region where the polymer compound film of the base material is formed. It is more preferable that a high molecular compound film having the same shape as part or all of the part is covered.
• a microchannel chip is preferably manufactured by the method for manufacturing a microchannel chip according to the present invention.
• the base material, the cover material, the flow path, and the polymer compound film have the same meanings as those described in the method for manufacturing a microchip.
• the method for separating biomolecules according to the present invention includes the following steps.
• a surface of the base material having a flow path formed on its surface, the flow path side surface is adhered to a cover material, and the flow path surface of the base material surface is coated with a polymer compound film. Adding a biomolecule to be analyzed to the microchannel chip; and
• the microchannel chip that can be used in the biomolecule separation method is the microchannel chip according to the present invention. Further, the base material, the cover material, the flow path, the height The molecular compound film has the same meaning as that shown in the method for manufacturing a microchip.
• the separation medium is not limited and may be a known medium for electrophoresis in electrophoresis or the like.
• the separation medium include organic solvents, gels such as polyacrylamide and agarose, and liquids such as buffer solutions.
• an electrophoretic medium is used.
• the electric swimming medium for example, it is preferable to use a gel, a buffer or the like.
• the separation medium to be used is not particularly limited.
• the separation pressure varies depending on the separation medium used and is not particularly limited, and electrophoresis, pumping, and the like can be employed. Of these, electrophoresis is preferred.
• biomolecules include proteins, DNA, viruses, bacteria, saccharides, amino acids, and other metabolites. Of these, the present invention is effective for separating proteins.
• the separation principle of the electrophoresis method is not limited!
• the electrophoretic separation using a base material having a surface covered with a polymer compound membrane enables separation based on various properties depending on the conditions of the separation medium. Separation conditions for electrophoretic separation can include pH gradient, molecular sieve, interaction with functional groups that come into contact in the separation medium, and the like. If electrophoresis in a separation medium with a pH gradient is used for proteins, isoelectric focusing will occur.
• molecular sieve electrophoresis under denaturing conditions can be established if a protein denaturant such as SDS, urea, or guanidine is present. Alternatively, if no denaturing agent is used, electrophoresis is performed under native conditions.
• nucleic acids are separated based on length.
• Analytical methods are also known in which the same nucleic acid is electrophoretically separated under non-denaturing conditions and denaturing conditions, such as PCR-SSCP, and the results of the two are compared to clarify differences in the three-dimensional structure.
• a separation medium having various functional groups. Specifically, it can show an electrostatic interaction, a hydrogen bond, a hydrophobic bond, or any combination of affinity substances.
• affinity substance include antigen-antibody, hybridization of a nucleic acid having a complementary base sequence, avidin-biotin, and a combination of affinity substances such as sugar-lectin.
• One of the principles of electrophoresis suitable for the present invention is capillary electrophoresis. When performing capillary electrophoresis according to the present invention, since the polymer compound film is applied, a flow path capable of controlling an electroosmotic flow can be formed.
• preferable monomer materials useful for capillary electrophoresis include, for example, in the case of a plasma-polymerized film, hexadene, hexamethyldisiloxane, acetonitrile, hexylamine, and aminoacetaldehyde dimethyl acetal. Can be.
• a surface polymerized film styrene, acrylamide, butylsulfonic acid, acrylic acid, diallyldimethylammonium salt, and arylamine are exemplified.
• examples include polybutyl alcohol, polyacrylic acid, and polyallylamine.
• the following is an example using a plasma polymerized film.
• the anolyte and catholyte are introduced at both ends and voltage is applied to both ends.
• the anolyte used is an acidic solution that gives a lower pH than the most acidic of the electrolytes.
• the catholyte an alkaline solution that gives a higher pH than the most basic one is used.
• Each ampholyte stops after moving to the isoelectric point.
• the protein component is concentrated at the position of the isoelectric point on the pH gradient formed in the channel, and is observed as a fine zone.
• capillaries one-zone electrophoresis an electric double layer is formed between the inner wall of the flow channel and the electrolyte solution in contact with the inner wall by introducing one type of electrolyte solution into the flow channel.
• electrolyte solution moves with the solvent, creating an electroosmotic flow.
• the electroosmotic flow serves as a driving force for moving the separated component ions.
• the sample components are attracted to the counter electrode by receiving the electrostatic force according to the charge and size of each sample, and the difference between the charge and size becomes a difference in mobility, and the components are separated.
• CZE capillary electrophoresis
• CGE capillary gel electrophoresis
• CIFE capillary isoelectric focusing
• chromatographic separation It is thought that biomolecules can be separated by ion exchange, reverse phase, normal phase, affinity chromatography, etc.).
• Channels coated with polymer compound membranes are very effective because they can control electroosmotic flow.
• the present invention relates to an electrophoresis analyzer comprising the following elements.
• a surface of the base material having a flow path formed on its surface, the surface on the flow path side, and a cover material are bonded together, and the surface of the flow path among the base material surfaces is coated with a polymer compound film.
• the microchannel chip that can be used in the electrophoresis analyzer is the microchannel chip according to the present invention. Further, the base material, the cover material, the flow path, and the polymer compound film have the same meanings as those described in the microchip manufacturing method.
• the support is not particularly limited as long as the microchannel chip is stably fixed. All prior art documents cited in this specification are incorporated herein by reference.
• an after glow method using an RF power supply and an external electrode method was used as a polymerization method for the plasma polymerization film.
• Various units were added based on the plasma basic research equipment BP-1 made of samcone earth, and a device capable of automatically controlling the flow rate, pressure, and power matching was fabricated. The configuration of the device is shown below.
• Reactor (chamber one): Pyrex (registered trademark) 210 mm ⁇ ,
• Sample stage SUS304, heater heating control stage installed at the bottom of the chamber
• Exhaust system Pfeiffer turbo molecular pump + Edwards rotary pump Power supply: Samco 13.56MHz, 300W, crystal oscillation
• a 200- ⁇ m-wide stainless steel mask is placed on a polymethyl methacrylate (PMMA) (Clarex 000 (trade name), manufactured by Nitto Toushi Co., Ltd., 3 mm thick x 70 mm long x 70 mm wide). It was placed in one chamber of the plasma polymerization apparatus. And the degree of vacuum in the chamber first and 3 X 10- 5 Torr. Hexamethyldisiloxane (HMDS) was filled in the chamber, discharge power (RF power) was 150 W, pressure was 0.1 lPa, flow rate was 100 sccm, and discharge was performed for 180 seconds to form a plasma polymerized film. The film thickness was 100 nm.
• PMMA polymethyl methacrylate
• HMDS Hexamethyldisiloxane
• An electrophoresis chip was prepared by attaching a molded chip (polymethyl methacrylate: PMMA) and polydimethylsiloxane: PDMS, and proteins were separated using this chip.
• PMMA (8 mm thick) was injection molded to provide a cross-shaped microchannel.
• a chip (base material) for plasma polymerization was produced (see FIG. 3 manufactured by Kobayashi Seiko Co., Ltd.).
• the depth and width of the microchannel are 100 / ⁇ , reservoir diameter: 4 mm, introduction channel: 10 mm, and separation channel: 50 mm.
• the cover material was produced by polymerizing polydimethylsiloxane (PDMS) (trade name: SYLGARD 184: manufactured by Shin-Etsu Silicone Co., Ltd.) in a polystyrene case.
• PDMS polydimethylsiloxane
• the monomer and the catalyst were mixed at a ratio of 10: 1, deaerated by a vacuum pump, cast in a polyethylene case, and reacted at 70 ° C. for 1 hour to obtain a lid material, PDMS.
• the metal mask was peeled off, the substrates were adhered while aligning the base material and the cover material, and an electrophoresis chip was produced.
• an electrophoresis buffer 0.1 M phosphate buffer (pH 8.5) containing 0.6% cellulose
• all plasma polymerized membranes and widths 150, 200, At all 1000 m it was confirmed that the attachment of the base material and the cover material was sufficient to prevent the migration buffer from leaking out of the channel.
• Example 2 a separation experiment was performed using HMDS, a microchannel chip prepared using a metal mask having a width of 1000 m, and using carbonic anhydrase as a protein. An uncoated tip was used for comparison.
• Cy5 a fluorescent dye
• Cy5 a fluorescent dye
• Electrophoresis was detected in the channel immediately before entering reservoir 4.
• the voltage at the time of introduction, the voltage at the time of separation, the introduction time, and the separation time are as follows.
• Figure 3 shows how to apply the voltage.
• Figure 4 shows the results of electrophoresis when using carbonic anhydrase as a sample.
• the first peak detected is considered to be due to unreacted Cy5 (no deposition: about 160 seconds, HMDS deposition: about 180 seconds). Subsequently, several peaks detected are likely to be due to carbonic anhydrase. Peaks detected on chips without film formation (approximately 170 to 1200 seconds) and peaks detected on chips with HMDS film formation (About 190-460 seconds), they were detected at about the same time (about 10 seconds later), based on the Cy5 peak. The resolution is judged to be better for the HMDS film-forming chip because many peaks were detected faster. The resolution here indicates the difference between the electrophoretic patterns and the number of peaks (the larger the number, the higher the resolution). The protein peak detected based on the Cy5 peak was considered.
• a region which is not covered with a polymer compound film is present on the surface of a substrate, more preferably the surface of a substrate and a cover material. Since the polymer compound film is formed as described above, the bonding strength between the base material and the cover material is excellent and the method is simple.

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