WO2004031757A1 - 物質の分離方法 - Google Patents
物質の分離方法 Download PDFInfo
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- WO2004031757A1 WO2004031757A1 PCT/JP2003/011352 JP0311352W WO2004031757A1 WO 2004031757 A1 WO2004031757 A1 WO 2004031757A1 JP 0311352 W JP0311352 W JP 0311352W WO 2004031757 A1 WO2004031757 A1 WO 2004031757A1
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
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- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N27/44704—Details; Accessories
- G01N27/44752—Controlling the zeta potential, e.g. by wall coatings
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- G—PHYSICS
- G01—MEASURING; TESTING
- 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
Definitions
- the present invention relates to a method for separating substances.
- Non-Patent Document 1 Various forms of electrophoresis have emerged in such a long history of electrophoresis. Furthermore, an electrophoresis (CE) method using an extremely thin capillary in a free solution has been developed (Non-Patent Document 2).
- gel electrophoresis has a problem in that the handling of the gel is complicated and the reproducibility is poor. In particular, in electrophoresis combining isoelectric focusing and gel electrophoresis, the separation operation is extremely complicated.
- the CE method can not only perform excellent separation, but also perform highly reproducible detection and quantification.
- CE is an on-cabillary detection. If detection is performed in a free solution, the background absorption will be uniform. As a result, the reproducibility of detection is overwhelmingly superior to conventional electrophoresis, and highly reliable quantification is possible.
- a thin capillary tube is used for CE.
- a two-dimensional structure such as a two-dimensional electrophoresis method using a slab gel, which has been used for general purposes and provides a certain level of performance, has been used for CE. It is technically difficult to produce and use it as it is.
- Electrophoretic separation is an important technique for separating components such as nucleic acids as well as proteins.
- nucleic acid separation as with proteins, if the separation can be achieved using a microscopic device, the effect of reducing the amount of sample required for analysis and shortening the time can be expected.
- Micromachining technology is a technology that builds a flow path, a structure that controls the flow of liquid in the flow path, or a control mechanism for temperature conditions in the flow path, on a micro chip.
- semiconductor processing Technology is a technology for building a fine structure on the surface of a substrate by photolithography or etching (Non-Patent Documents 6 and 7).
- Glass is one of the most common materials used to make depises using these technologies.
- electrophoresis is performed using a glass capillary, the inner wall in contact with the glass material and the solution is negatively charged, and the electroosmotic flow flows from the anode to the cathode. Under these conditions, only unidirectional zone electrophoresis is possible. Therefore, when the cation component is contained in the sample, the movement time of the cation becomes extremely short, and the separation of the cation component becomes difficult.
- devices manufactured using a glass substrate are expected to have the same problems as those pointed out in such glass capillaries.
- Isoelectric focusing is effective for separating proteins of neutral components. Isoelectric focusing requires modification of the inner wall of the capillary. However, there is no known surface modification method that can be easily implemented and gives a good modified surface.
- the conditions of electrophoresis can be adjusted as needed.
- Patent Document 1 attempts have been made to increase the separation efficiency by slowing down the electroosmotic flow generated inside the separation column by treating the inner wall of the separation column with a material that reduces the absolute value of the zeta potential.
- Patent Documents 2, 3, and 4 Attempts have also been made to adsorb the polymer to the inner wall of the capillary to suppress the electro-osmotic flow generated inside the capillary.
- the glass substrate modify the surface of the glass substrate by adsorbing or covalently bonding the polymer to the inner wall of the capillaries.
- a specific modification method for example, neutralization of the glass surface by chemical modification of acrylamide via silane coupling can be considered.
- the hydrophilic group introduced as a silane coupling agent was not stable, and there was a possibility that the modified surface would gradually peel off in a neutral to alkaline solution.
- Non-Patent Document 1 Journal of Biology and Chemistry (T. B. Coolidge, J. Biol. Chem..), 127, 551, 1939
- Non-Patent Document 2 Journal of Chromatography (F.E.P.Mikkers, F.M. Everaerts, Th.P.E.M.Veerheggen, J. Chromatogr.), 169, 11, 1979
- Non-Patent Document 3 Masayoshi Esashi, Micromachine, Applied Physics, 60, 1991
- Non-Patent Document 4 Neichia Biotechnology (P. N. Gilles, D. J. Wu, C. B. Foster, P. L. Dillon, S. J. Chanock, Nature Biotec.), 17, April, 1999
- Non-Patent Document 5 GeneChip systems, Affymetrix Inc. 3380 Central Expressway banta Shuara, CA 95051
- Non-Patent Document 6 Micromechanics (A. Heuberger (ed.), Micro-mechanics), Springer-Verlag, Berlin, 1989
- Non-Patent Document 7 Shizuhiro Furukawa, Taneo Asano, Introduction to Ultrafine Processing, Ohmsha, 1989
- Patent Document 1 Japanese Patent Application Publication No.
- Patent Document 2 Japanese Patent Application Laid-Open No. 5-503989
- Patent Document 3 Japanese Patent Application Publication No. 7-5046432
- Patent Document 4 Japanese Patent Publication No. 9 _5044375 Therefore, an object of the present invention is to provide a separation method and a device capable of controlling various properties of a substrate surface with which a separation medium comes into contact. Disclosure of the invention
- the present inventors have intensively studied to solve the above problems, and the separation medium comes into contact by coating the substrate surface with a polymer having various properties by plasma polymerization, chemical polymerization or chemical modification. It has been found that the substrate surface can be arbitrarily controlled under various conditions, for example, the potential of the substrate surface, the hydrophilicity and the hydrophobicity. In addition, they have found that plasma polymerization, surface polymerization, and polymer compound bonding are useful as a surface modification technique for a substrate with which a separation medium comes into contact, and completed the present invention.
- the plasma polymerization it is possible to obtain a very homogeneous polymer compound film in which pinholes are suppressed. Further, such a plasma polymerized film can be formed on the surface of a substrate having an arbitrary shape, and films having various properties can be easily formed by selecting a monomer material. Furthermore, a uniform film can be formed on many substrate surfaces at once. And it is possible to create a large number of devices while maintaining uniform quality.
- a desired polymer compound film in which the peeling of the film is suppressed can be formed at a desired position on the surface of the base material.
- the desired polymer compound film is formed on the base material surface while easily controlling the film thickness on the base material surface. Position can be formed.
- the present invention provides a method for separating and analyzing a large amount of samples simultaneously and easily on a miniaturized base material.
- the present invention relates to the following separation method, a method for manufacturing a device therefor, and a device.
- the method for separating a substance according to the present invention is characterized by comprising the following steps: a) adding a substance to be analyzed to a separation medium held on a substrate whose surface in contact with the separation medium is coated with a polymer compound membrane; and
- the polymer compound film is preferably a plasma polymerized film obtained by plasma polymerization.
- the plasma polymerized film is formed by plasma polymerizing any monomer selected from the group consisting of hexadiene, hexamethyldisiloxane, acetonitrile, hexylamine and aminoacetaldehyde dimethyl acetal. Is preferred.
- the polymer compound film is preferably a surface polymerized film obtained by polymerizing a polymerizable monomer on the substrate surface.
- the surface polymerized film is preferably bonded to the substrate surface via a hydrophobic spacer, and the surface polymerized film is preferably covalently bonded to the hydrophobic spacer by a carbon-carbon single bond. ,.
- the hydrophobic spacer is preferably an alkyl group having 2 to 6 carbon atoms.
- the polymer compound film is preferably a polymer binding film obtained by binding a polymer compound on the substrate.
- the polymer binding membrane is selected from the group consisting of polystyrene, polyallylbenzene, polyvinyl alcohol, polyacrylamide, polyvinyl sulfonic acid, polyatalylic acid, polydiallyl dimethyl ammonium salt, polyallylamine and polyethylene glycol. It is preferable that any one of the polymer compounds is covalently bonded to the substrate.
- the substrate is a planar substrate.
- the substrate is glass.
- the principle of the separation is preferably electrophoresis.
- the principle of the electrophoresis is preferably isoelectric focusing.
- the substance to be separated is a protein.
- the method for producing a substrate for separation analysis according to the present invention is characterized by including a step of forming a plasma polymerized film on the surface of the substrate by plasma polymerization.
- Hexadiene, hexamethyldisiloxane, acetonitrile Hexadiene, hexamethyldisiloxane, acetonitrile.
- a monomer selected from the group consisting of hexinoleamine and aminoacetaldehyde dimethyl acetal is plasma-polymerized on the surface of the base material to form a plasma polymerized film. It is preferable to form
- the method for producing a substrate for separation and analysis according to the present invention includes a step of forming a surface polymerized film by polymerizing a polymerizable monomer on the surface of the substrate.
- hydrophobic functional group having a terminal double bond exists on the surface of the base material, and the hydrophobic functional group and a polymerizable monomer be polymerized.
- the hydrophobic functional group is preferably an alkenyl group having 2 to 6 carbon atoms and having a double bond at a terminal.
- the method for producing a substrate for separation and analysis according to the present invention includes a step of forming a polymer-bound film by binding a polymer compound to the surface of the substrate.
- polystyrene polystyrene, polyallylbenzene, polyvinylalcohol, polyatarylamide, polyvinylsulfonic acid, polyatarylic acid, polydiallyldimethylammonium salt, polyallylamine, and polyethylenedaricol It is preferable to form a polymer binding film by covalent bonding to a material.
- the substrate is a planar substrate.
- the substrate is glass.
- the method for modifying the surface of a substrate for separation and analysis according to the present invention is characterized by including a step of forming a plasma polymerized film on the surface of the substrate.
- the method for modifying the surface of a separation and analysis substrate according to the present invention comprises the steps of: It is characterized by including a step of polymerizing the nomer to form a surface polymerized film.
- the method for modifying the surface of a substrate for separation and analysis according to the present invention is characterized by including a step of forming a polymer-bound film by bonding a polymer to the surface of the substrate.
- the surface in contact with the separation medium is coated with a polymer compound membrane.
- the polymer compound film is preferably a plasma polymerized film obtained by plasma polymerization.
- the polymer compound film is a surface polymerized film obtained by polymerizing a polymerizable monomer on the surface of the base material.
- the polymer compound film is a polymer binding film obtained by binding a polymer compound on the substrate.
- the electrophoresis analyzer according to the present invention comprises the following elements:
- FIG. 1 is a diagram showing the structure of a capillary electrophoresis chip used in Examples.
- FIG. 2 is a photograph showing the migration of spots over time when a sample having a protein concentration of 33 ⁇ g / ⁇ L was electrophoresed at 100 ° V using an acetonitrile-modified capillary. Each spot corresponds to phycocyanin, hemoglobin, and cytochrome c from the anode side (right side).
- Figure 3 is a photograph showing the state of protein enrichment when electrophoresis is performed using a chip (b) whose inner wall is unmodified (a) or chemically modified with acrylamide (b).
- FIG. 4 shows that the inner wall of the capillaries is acetonitrile (c), hexadiene (d) or FIG. 4 is a photograph showing the state of protein concentration when electrophoresis is performed using a capillary electrophoresis chip coated with HMDS (e).
- FIG. 5 is a graph showing the electrophoresis time when the applied voltage is set to 1000 V or 2000 V.
- the vertical axis indicates the migration time, and the horizontal axis indicates the modifying substance.
- Fig. 6 is a diagram showing the principle of capillary isoelectric focusing (CIEF).
- Figure 7 shows the principle of capillary one-zone electrophoresis (CZE). BEST MODE FOR CARRYING OUT THE INVENTION
- the present invention relates to a method for analyzing a substance including the following steps.
- the principle of the separation includes electrophoresis, pumping, etc. Among them, electrophoresis can be preferably employed.
- the separation medium a known medium for electrophoresis in electrophoresis or the like can be adopted, and it is not limited.
- the separation medium include organic solvents, gels such as polyacrylamide and agarose, and liquids such as buffer solutions.
- an electrophoretic medium is used.
- the electrophoresis medium for example, it is preferable to use a gel, a buffer or the like. In the case of pressure feeding, the separation medium to be used is not particularly limited.
- Separation pressure includes pressure, voltage, etc. In the case of electrophoresis, a voltage is applied.
- substrate refers to a support having any shape capable of holding a separation medium. Specifically, a support having a tubular, groove-like, or plate-like shape can be shown.
- a support having a plate-like shape can be preferably used. -1 o-OK.
- a plate-shaped support may be a flat substrate.
- a flat substrate By using a flat substrate, separation can be performed two-dimensionally. Also, various types of polymer compound films can be easily formed on the same flat substrate. For example, plasma polymerization, surface polymerization, or bonding of a polymer compound may be performed by covering a flat substrate with a mask for bonding a desired polymer compound film to a desired position at a desired position. A substrate coated with various polymer compound films can be obtained.
- a plate-shaped substrate can hold, for example, a liquid or gel electrophoretic medium.
- a liquid can be retained by capillary action in a fine space between two plates.
- planar shape of these supports is not limited. That is, the shape may be linear, circular, annular, polygonal, or curved.
- the material constituting the support is arbitrary.
- the surface in contact with the separation medium is modified by a plasma-polymerized membrane, a surface-polymerized membrane, or a polymer-bound membrane. Therefore, the material of the support itself does not directly affect the results of separation such as electrophoresis. Therefore, for example, any material that satisfies the following minimum conditions can be selected.
- a transparent material is generally used for the base material.
- the use of transparent materials enables external optical observations.
- a support made of a material such as glass or plastic can be used as a base material.
- the substrate may or may not have a groove, but preferably does not have a groove.
- the width of the groove holding the electrophoretic medium may be a fine space such as 1 to 100 m.
- the cross-section of the groove can be polygonal, such as a triangle or square, or U-shaped or semi-circular. The following method can be used to provide a groove having such a fine structure in a support such as glass.
- ⁇ Etching, dry etching, or laser drilling can easily provide a fine structure with a free shape.
- a technique is known in which a groove having a width and a depth of 100 to 100 jtm is provided on a glass surface.
- the groove formed on the surface of the base material may be an open system or a closed system.
- the groove can be made a closed system by superposing another flat substrate on the substrate on which the groove is formed.
- the substrate on which the groove is formed and the second substrate overlaid on the substrate may be the same material or different materials.
- a communication flow path for supplying a sample or a separation medium to the groove can be provided.
- the hole provided in the second substrate can be used as a reservoir for holding a sample or a buffer.
- a glass capillary can also be used as a substrate.
- a capillary ram holding a gel or a buffer in a glass capillary is generally used as a means for holding a medium for electrophoresis of a DNA protein.
- the present invention utilizes a substrate having a surface coated with a plasma polymerized film, a surface polymerized film, or a polymer binding film.
- a plasma polymerized film at least the surface of the substrate surface that comes into contact with the separation medium is covered with a plasma polymerized film, a surface polymerized film, or a polymer binding film.
- the plasma polymerization it is possible to form a plasma polymerization film even on a fine groove or a narrow surface inside the cavity. Moreover, according to the plasma polymerization, the obtained film is extremely homogeneous. For this reason, the generation of pinholes on the substrate surface can be suppressed, and a highly reliable substrate for separation analysis can be prepared.
- a desired surface polymerization film in which the film is suppressed from being peeled can be formed at a desired position on the surface of the base material.
- a desired polymer compound film can be formed at a desired position on the substrate surface while controlling the film thickness.
- 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.
- any type of monomer can be used for plasma polymerization.
- cleavage of the double bond is necessary, whereas in plasma, the monomeric material is separated and a polymerization reaction occurs via many active species.
- the monomer material for the plasma-polymerized film according to the present invention is formed by electrophoresis on the support surface. Any material can be used as long as it can form a polymer film giving appropriate properties according to separation such as dynamic separation.
- suitable properties according to the electrophoretic separation include the following properties. Among these properties, a monomer substance capable of giving any one of the properties can be used in the present invention.
- the glass used for capillary electrophoresis absorbs proteins on the surface and then dilutes them.
- the adsorption of the protein to the substrate can be controlled by a plasma polymerized membrane.
- it can be controlled by the degree of hydrophobicity of the substrate and the surface charge.
- alkanes or cycloalkanes The following compounds can be shown as alkanes or cycloalkanes. Methane, ethane, propane, butane, isobutane, pentane, isopentane, neopentane, hexane, isohexane, 3-methinolepentane, 2,2-dimethylbutane, 2,3-dimethy ⁇ / butane, heptane, 2,2 3-trimethylbutane, octane, nonane, decane, methane-dl, methane-d2, methane-d3, methane-d4, cyclopronocene, cyclobutane, cyclopentane, cyclohexane, methinolecyclohexane, cyclooctane, cis-decalin and trans-decalin.
- alkene alkyne, or cycloalkene
- Methanol ethanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, 2-methyl-11-propanol, 2-methyl-2-propanol, arylalkonole, 1,3-butanediol, 2,3-butanol 3-butanediol, 2,3-epoxy-11-propanol, formaldehyde, acetoethanol, propionaldehyde, butyraldehyde, valeraldehyde, isovaleraldehyde, acrylaldehyde, crotonaldehyde, glyoxal, acetone, 2-butanone , 2_pentanone, 3-methyl-2-butanone, 3-pentanone, 2-hexanone, 4-methinole-2-pentanone, 2-heptanone, cyclobutanone, cyclopentanone, cyclohexanone, cycloheptanone, cycloo
- Dimethinoleatenore Jetinoleatenole, Dipropinoleatenole, Diisopropinoleate ⁇ /, Dibutinoleateone ⁇ Ethylene oxide, 1,3-Dioxolan, 1,3-Dioxane, 1,4- Dioxane, Methivinyl, ⁇ Ethenole, Methiamine, Etya Min, propylamine, isopropylamine, butyramine, isobutylamine s-butylamine, t-butylamine, pentinoleamine, hexylamine, dimethylamine, trimethylamine, getylamine, triethylamine, dipropylamine, disopropylamine, tripropylamine Dibutylamine, arylamine, formamide, acetamide, N-methylacetamide, N, N-dimethylformamide, N, N-dimethylacetamide, methanethiol, ethane
- halides can be used as the monomer material.
- aromatic hydrocarbons can be used as monomer substances.
- Chlorotonolene p-chlorotonolene, o-bromotonolene, p-bromotonolene, 0-oddtonolene, m-oddomerene, p-oddtonolene, p-chlorofluorene, and 0-chloroiodobenzene.
- heterocyclic compounds can be used as monomer substances.
- a troponoid compound such as tropone ditroborone, or an organometallic compound represented by tetramethylsilane, tetramethyltin, and tetramethyllead can be used as the monomer material.
- the substrate surface with a charge of around neutrality in these of P H is near neutral conditions, it can be preferably used Kisajen Asetonitoriru to.
- Tildisiloxane can be preferably used.
- hexylamine-aminoacetaldehyde dimethyl acetal is preferably used.
- Conditions for forming a plasma-polymerized film with these monomer substances are known. Specifically, it is said that the main factors that affect the reproducibility of the plasma polymerization reaction are, for example, conditions such as flow rate, discharge power, discharge time, and pressure. 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
- Hexamethyldisiloxane which will be described later as an advantageous monomer substance for the purpose of immobilizing polynucleotides, is used to prepare plasma-polymerized membranes.For example, select the optimal conditions under the following ranges: By doing so, it is possible to form a plasma polymerized film of more than approximately 0 and not more than 24 OA.
- Discharge power 20 to: L 0 0 W
- various functional groups can be imparted to the surface of the base material by selecting the monomer substance, so that films having various properties can be easily formed. For example, it is possible to obtain a substrate surface having various ranges of surface charge and hydrophobicity.
- the zeta potential which varies depending on the pH, indicating the charge state of the substance, can be controlled preferably in the range of 100 to 110 OmV.
- the contact angle of the surface can be controlled preferably in the range of 1 to 140 degrees.
- the thickness of such a plasma polymerized film is, for example, preferably in the range of 1 to 200 nm.
- the plasma polymerized film obtained in this way is a very homogeneous film, and the generation of pinholes is significantly suppressed.
- a plasma polymerization film can be formed on the surface of a base material having an arbitrary shape.
- alkane-cycloalkane, aromatic hydrocarbon, or the like is used as a monomer material
- a plasma-polymerized film having an extremely hydrophobic surface is synthesized, so that separation based on hydrophobic interaction is possible. That is, in the above three examples, surfaces having actions similar to those of anion exchange chromatography, cation exchange chromatography, and hydrophobic chromatography can be realized.
- the technology for surface modification and thin film formation applied to photo applications be a dry process. Since the above-mentioned plasma polymerization method is a dry process, it is suitable for device fabrication by photo application. Furthermore, if the plasma polymerization method is used, a thin film having a functional group on the surface can be produced by selecting an appropriate monomer substance. Also, the plasma polymerized film is a pinhole-free film with a highly cross-linked structure, and is therefore 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 surface of the base material.
- 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 surface of the substrate.
- the hydrophobic functional group is preferably an alkyl group having 2 to 6 carbon atoms, more preferably 3 to 6 carbon atoms, and particularly preferably 4 to 6 having a double bond at a terminal.
- hydrophobic functional group examples include a vinyl group, an aryl group, an butyryl group, a 1-pentiel group, an hexynyl group and the like.
- the surface polymerized film is covalently bonded by a carbon-carbon single bond using the hydrophobic functional group as a spacer.
- the hydrophobic spacer itself by hydrolysis due to the influence of pH or the like is used. Desorption is suppressed. Further, since the hydrophobic spacer and the surface-polymerized film are bonded by a carbon-carbon bond, the surface-polymerized film does not peel off at the bonding position with the hydrophobic spacer.
- the substance to be analyzed is a protein
- the surface polymerized film is not peeled off due to the influence of pH, and the analysis can be performed with high reliability.
- the polymerizable monomer is polymerized to form a polymer film on the surface, so there is no aggregation of the polymer as compared with the case where the polymer itself is bonded, so that the bonding with the substrate surface is more efficient. Can be done.
- the introduction of the hydrophobic functional group onto the surface of the substrate is performed by dissolving a compound that induces a 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 contacting a substrate such as glass.
- the contact reaction is carried out, for example, at a room temperature (about 25 ° C.) to about 100 ° C. for, for example, about 1 to 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.
- Such compounds include, for example, alkenylsilanes such as triethoxybutylsilane, triethoxyarylsilane, triethoxybutenylsilane, triethoxypentenylsilane, and triethoxyhexylsilane.
- triethoxyarylsilane triethoxybutenylsilane, triethoxypentenylsilane, triethoxyhexylsilane, particularly preferably triethoxybutenylsilane, triethoxypentenylsilane, and triethoxyhexylsilane.
- alkenylsilanes 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 alkenyl group with a halogenated silane such as chlorosilane or alkoxysilane in the presence of a solvent.
- the polymerizable monomer is not limited as long as it has a bull group, an aryl group, a diene or the like.
- Examples of such a polymerizable monomer include a nonionic monomer, an anionic monomer, and a cationic monomer.
- Nonionic monomers that create a surface include, for example,
- Amides such as acrylamide and methacrylamide
- Methyl acrylate methyl methacrylate, butyl acetate, aryl acetate, arylacetate acetate, butyl trimethyl acetate, vinyl formic acid, hexyl hexate, laurate butyl, butyl methacrylate, octanoate, butyl palmitate, pipel Bivinilyl acid acid, Bibulyl proprolopionate, Vivinylyl steatea allylate, Hexoxashydodrolophphthalate momonono 22-((metamethacryloylylloyloxyloxy) )) Ethityl, momonono phthalate-22-- ((metatactic acryloylloyloxy)) ethityl, benzoyl benzoate, pp-bibininyl benzoate Acid acid, bibutyrate butyrate, bivinylyl capcaprylinate, bibieryl capcapronate, bibuleyl chloro
- Any ethers such as lulu ether, bibulul etytyl ether, and nn-arydecyl decanoate; ether; Any aralcohols, such as lyrylaalcohol;
- Chlorinated bibieryl chlorinated acrylylyl, chlorinated methacrylate acryloylyl, chlorinated oral vinegar acetate butylyl, chlorinated acrylinyl chloride, acrylylyl bromobromide, Any halologigen compounds, such as acrylyl, chloroacetic acid, acetic acid acetic acid, arylyl chloride, chlorochloroacetic acid, arylyl chloride, acryloyl chloroformate, and acryloyl chloroformate;
- Cycloalkyl derivatives such as 2-arylcyclohexanone, 1-arylcyclohexanol, and arylcyclopentane;
- acrylamide / vinyl alcohol is preferably used as the hydrophilic nonionic surface
- styrene / diarylbenzene is preferably used as the hydrophobic nonionic surface.
- anionic monomers that create an ayuonic surface include, for example,
- Examples include 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_bulbenzensulfonic acid. .
- vinyl sulfonic acid peryl sulfonic acid can be preferably used as strong anionic property
- acrylic acid / methacrylic acid can be preferably used as weak anionic property
- Cationic monomers that create a cationic surface include, for example,
- Primary amines such as rilamine, 3_acrylamide-N, N-dimethylpropylamine, arylsylhexylamine, 3-methacrylamide-N-dimethylpropylamine;
- Secondary amines such as methylarylamine
- Tertiary amines such as N-aryljetylamine and N-aryldimethylamine; , (3-acrylamidepropyl) trimethylammonium chloride, vinylinoletrimethinoleammonium bromide, 3- (methacryloylamino) propyltrimethylammonium chloride, ethyl methacrylate trimethylammonium-dimethyl chloride, diaryldimethyl Quaternary keys such as ammonia Monmonium.
- anionic monomers for example, arylhydrazine, 2-vinyl virazine, 2-vinyl pyridine, 4-butyl pyridine, Binole-2-pyrrolidone, 1-arylbenzotriazole, aryl-1-benzotriazole carbonate, and the like can also be used.
- diaryldimethylammonium salt as a strong thione, and arylamine as a weak cation may 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 needed, 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 limited as long as it can dissolve the polymerizable monomer.
- THF tetrahydrofuran
- methanol tetrahydrofuran
- DMF tetrahydrofuran
- DMSO DMSO
- polymerization initiator examples include 2,2'-azobis (isobutyronitrile) (AIBN), 1,1'-azobis (cyclohexane-11-carbonitrile), and 2,2'-azobis (2-methyl) (Ptyronitrile) can be used.
- AIBN 2,2'-azobis (isobutyronitrile)
- 1,1'-azobis 1,1'-azobis (cyclohexane-11-carbonitrile)
- 2,2'-azobis (2-methyl) Ptyronitrile
- peroxides, organometallic compounds, and the like can also be used.
- a polymerizable monomer that does not dissolve in a solvent such as THF for example, N, N, N ′, N′-tetramethylethylenediamine, 4,4′-azobiscyanone
- the polymerization can be carried out using a polymerization initiator such as herbic acid.
- the polymerization varies depending on the type of the polymerizable monomer and is not limited, but it is usually carried out, for example, in a temperature range from room temperature to about 100 ° C. for about 1 to 72 hours.
- the surface-polymerized film obtained in this manner can have various ranges of charge, hydrophobic Z-hydrophilic surface depending on the type of polymerizable monomer used or the combination of a plurality of polymers.
- the zeta potential which varies depending on the pH, indicating the charge state of the substance, can be controlled preferably in the range of 100 to 110 OmV.
- the contact angle of the surface can be controlled preferably in the range of 1 to 140 degrees.
- a monomer-unmodified portion such as a pinhole may be generated. Therefore, a polymerizable monomer or polymer can be further bonded.
- a functional group in a polymer side chain of the surface-polymerized film may be further reacted with another polymer or monomer.
- a surface polymerized film having a surface thione functional group 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).
- a surface polymerized film having an anionic functional group on the surface is synthesized.
- electrophoretic separation by interaction between the negative charge of the membrane and the positive charge of the protein becomes possible.
- nonionic polymerizable monomers a superficially hydrophobic or hydrophilic surface polymerized film is synthesized, so that hydrophobic interaction or Can be separated based on hydrophilic interaction.
- 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 the reactive functional group serving as a site for binding a polymer compound 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.
- the binding group having such a reactive functional group is further bound 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 substrate in which a high molecular compound is bonded to a reactive functional group via such a hydrophobic spacer has a hydrophobic spacer so that access of water molecules is suppressed. The peeling of the polymer binding film due to the hydrolysis due to the influence of is suppressed.
- the introduction of the reactive functional group having the spacer onto the surface of the base material varies depending on the type of the base material.
- the introduction can be performed by a silane coupling method. If is made of metal, it can be performed by a self-assembled monolayer method.
- silane coupling method for example, in a solvent such as toluene, methanol, or water, aminopropyltriethoxysilane, aminopentyltriethoxysilane, aminopentyltriethoxysilane, aminohexyltriethoxysilane, etc.
- Aminoalkyl silane coupling agent or 3-glycidoxy Melting epoxyalkyl-based silane coupling agents such as pilltriethoxysilane, 3-glycidoxybutyltriethoxysilane, 3-glycidoxypentyltriethoxysilane, and 3-glycidoxyhexyltriethoxysilane, and substrates such as glass Can be brought into contact with each other.
- 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 halogenated compound such as chlorosilane. It can be easily synthesized by reacting with silane or alkoxysilane.
- the contact reaction is carried out at a temperature of, for example, room temperature (about 25 ° C.) to about 100 ° C., for example, for about 1 to 24 hours.
- a metal thin film such as gold is formed on the surface of the base material by, for example, sputtering, and a spacer having a functional group and a thiol group is introduced on the surface of the metal thin film.
- a polymer or a polymerization initiator can be reacted with a functional group and polymerized using a monomer
- 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 spacer As a solvent for introducing a spacer or a polymer modified with a thiol group onto a substrate, the spacer can be dissolved in a solvent such as DMS0 or water, and the solution can be contacted with a metal thin film.
- a solvent such as DMS0 or water
- the contact reaction is carried out at a temperature of, for example, room temperature to about 100 ° C., for a time of, for example, about 1 to 24 hours.
- the polymer include a polymer obtained by previously polymerizing a polymerizable monomer used in the surface polymerization.
- polystyrene, polyallylbenzene, polybutyl alcohol, polyacrylamide, polyvinylsulfonic acid, polyacrylic acid, polydiaryldimethylammonium salt, polyallylamine, polyethylene glycol, etc. are preferably used. be able to.
- polybutyl 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 is 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 in the range of 5,000 to 500,000, more preferably 10,000 to 250,000.
- a polymer binding film obtained by binding a polymer to a substrate a polymer-unmodified portion where a reactive functional group is not bound to the polymer, such as a pinhole, may be generated. Therefore, a polymer can be further bonded.
- Such a polymer binding film is not limited because 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 the solution.
- the solvent is not limited as long as it dissolves the polymer.
- examples thereof include DMSO (dimethyl sulfoxide), HE PES (2- [4- (2-hydroxyethyl) 1-piperazinyl] ethanesulfonic acid) buffer, and the like. Is mentioned.
- an activating agent may be used as necessary.
- HEPES dimethyl sulfoxide
- HE PES HE PES (2- [4- (2-hydroxyethyl) 1-piperazinyl] ethanesulfonic acid
- an activating agent may be used as necessary.
- HEPES dimethyl sulfoxide
- HE PES 2- [4- (2-hydroxyethyl) 1-piperazinyl] ethanesulfonic acid
- an activating agent may be used as necessary.
- the polymer-bound membrane thus obtained may have an unmodified portion of the polymer.
- another polymer or monomer can react with the functional group in the attached polymer side chain.
- the zeta potential which varies depending on the pH, indicating the charge state of the substance, can be controlled preferably in the range of -100 to 110 OmV.
- the contact angle of the surface can be controlled preferably in the range of 1 to 140 degrees.
- the thickness of such a polymer binding film can be easily controlled by preparing a polymer to be bound in advance.
- a polymer derived from the cationic monomer by using a polymer derived from the cationic monomer, a polymer binding film having a cationic functional group on the surface can be synthesized. It is possible to perform protein electrophoresis and the like while performing electrostatic interaction (positive charge of the membrane and negative charge of the protein) using the surface covered with such a polymer binding film. .
- 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 the amino group, and the negative charge of the membrane and the positive charge of the protein Electrophoretic separation and the like due to the interaction between them can be performed.
- a polymer binding membrane having a very hydrophobic or hydrophilic surface can be synthesized, so that separation based on hydrophobic interaction or hydrophilic interaction can be achieved. It is possible.
- a nonionic polymer or a nonionic monomer having a hydrophobic (or hydrophilic) functional group is bonded to the anionic functional group.
- a substrate surface having both an anionic property and a hydrophobic (or hydrophilic) property can be formed.
- the modification ratio of the nonionic polymer or monomer the balance of hydrophobicity (or hydrophilicity) can be controlled.
- the separation pressure of the present invention differs depending on the separation medium to be used and is not particularly limited, and electrophoresis, pumping, and the like can be employed.
- the separation principle of the electrophoresis method is not limited.
- the electrophoretic separation using a substrate having a polymer compound membrane coated on the surface enables separation based on various properties depending on the conditions of the separation medium. Separation conditions for electrophoretic separation include pH gradient, molecular sieve, and interaction with functional groups that come into contact in the separation medium. If electrophoresis in a separation medium having a pH gradient is used for proteins, isoelectric focusing will be achieved.
- electrophoresis is performed in a medium having a molecular sieve effect such as polyacrylamide gel
- 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.
- an analysis method is also known in which the same nucleic acid is subjected to electrophoretic separation under non-denaturing conditions and denaturing conditions, and the results of both are compared to clarify the difference in three-dimensional structure.
- separation media with various functional groups can be used. Specifically, it can show an electrostatic interaction, a hydrogen bond, a hydrophobic bond, or any combination of affinity substances.
- affinity substance include antigen-antibody, nucleic acid hybridization comprising a complementary base sequence, avidin-biotin, and a combination of affinity substances such as sugar-lectin.
- Isoelectric focusing can be illustrated as one of the principles of electrophoresis suitable for the present invention.
- CIEF capillary isoelectric focusing
- a capillary that does not generate electroosmotic flow is produced by performing an inner surface treatment (coating).
- preferred monomer materials useful for CIEF include, for example, in the case of a plasma polymerized film, hexadene, hexamethyldisiloxane, acetonitrile, hexinoleamine, and aminoacetoaldehyde dimethyl acetal. .
- examples include polybutyl alcohol, polyataryl acid, and polyallylamine.
- the following is an example using a plasma polymerized film.
- Anolyte and catholyte are introduced at both ends and voltage is applied to both ends.
- the anolyte is an acidic solution that gives a lower pH than the most acidic of the electrolytes.
- 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 isoelectric point on the pH gradient formed in the capillary and is observed as a narrow zone (Fig. 6).
- capillary one-zone electrophoresis an electric double layer is formed between electrolyte solutions that are in contact with the inner wall of the capillary by introducing one type of electrolyte solution into the capillary.
- electrolyte solution When a voltage is applied, the electrolyte solution It moves and electroosmotic flow occurs.
- the electroosmotic flow is the driving force for moving the separated component ions.
- the sample components are attracted to the opposite electrode by receiving the electrostatic force according to each charge and size, and the difference in charge and size causes the difference in mobility, and the components are separated (Fig. 7).
- CIEF unlike CZE, causes electrophoresis, but the generation of electroosmotic flow should be minimized.
- the inner surface modification method of the capillaries, the size of the capillaries inner diameter, etc., and the composition of the amphoteric electrolyte as the running buffer solution greatly affect the effect of the electroosmosis phenomenon, and consequently the CIEF separation ability. give.
- the present invention relates to a method for producing a substrate for separation and analysis, comprising a step of forming a plasma polymerized film on the surface of a substrate.
- the method of coating the substrate with the plasma polymerized film is as described above.
- a substrate suitable for the above-described separation method can be obtained. Electrophoresis is preferred as the separation method.
- the present invention relates to a method for producing a separation and analysis substrate, which comprises a step of polymerizing a polymerizable monomer on a substrate surface to form a surface-polymerized film.
- the method of coating the substrate with the surface polymerized film is as described above.
- a substrate suitable for the above-described separation method can be obtained. Electrophoresis is preferred as a separation method.
- the present invention also relates to a method for producing a substrate for separation and analysis, comprising a step of forming a polymer-bound film by bonding a polymer compound to the surface of the substrate.
- the method of coating the substrate with the polymer binding film is as described above.
- a substrate suitable for the separation method described above can be obtained. Electrophoresis is preferred as a separation method.
- the present invention relates to a method for modifying the surface of a substrate for separation and analysis, comprising a step of forming a plasma polymerized film on the surface of the substrate.
- Plasma polymerized films have excellent characteristics as a surface modification method. In other words, a plasma polymerized film can easily form a uniform film on any complex surface structure. Further, by selecting the monomer substance, any property can be given to the substrate surface. Therefore, for example, when the surface of the base material may interfere with the separation, the interference can be prevented by coating the surface with a plasma polymerized film.
- properties required for separation can be positively imparted to the surface of the substrate. Electrophoresis is preferred as the separation method.
- the present invention relates to a method for modifying the surface of a separation / analysis substrate, which comprises a step of forming a surface-polymerized film by polymerizing a polymerizable monomer on the surface of the substrate.
- the surface polymerized film has an excellent feature as a surface modification method. According to the present invention, properties required for separation can be positively imparted to the surface of a substrate. Further, the surface polymerized film can be formed at a desired position on the surface of the base material by preventing the film from peeling off. Electrophoresis is preferred as a separation method.
- the present invention relates to a method for modifying the surface of a separation / analysis substrate, which comprises a step of forming a polymer-bound film obtained by binding a polymer compound to the substrate surface.
- the polymer binding film obtained by this method has an excellent feature as a surface modification method. That is, according to the present invention, properties required for separation can be positively imparted to the substrate surface.
- the polymer binding film can form a polymer compound film having desired performance at a desired position on the surface of the base material while controlling the film thickness. Electrophoresis is preferred as a separation method.
- the present invention also relates to a separation / analysis substrate having a surface in contact with a separation medium coated with a polymer compound membrane.
- the polymer compound film a brass obtained by plasma polymerization is used.
- examples thereof include a polymerized polymer film, a surface polymerized film obtained by polymerizing a polymerizable monomer on the surface of a substrate, and a polymer-bound film obtained by bonding a polymer compound on a substrate.
- a substrate having a surface in contact with a separation medium coated with these polymer compound membranes can be used in the separation method according to the present invention. Electrophoresis is preferred as a separation method.
- the present invention relates to an electrophoresis analyzer comprising the following elements.
- the polymer compound film examples include a plasma polymerized film obtained by plasma polymerization, a surface polymerized film obtained by polymerizing a polymerizable monomer on a substrate surface, and a polymer bonded film obtained by bonding a polymer compound on a substrate. Is mentioned.
- an electrophoresis apparatus using a substrate whose surface in contact with a separation medium is coated with these polymer compound membranes can be used in the electrophoresis method according to the present invention.
- 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 BP-1 basic plasma research equipment made by Samcone clay to create a device that can automatically control flow rate, pressure, and power matching. The configuration of the device is shown below.
- Reactor (first chamber): Pyrex (registered trademark) 21Omm 0, Sample stage: SUS304, heater heating control stage installed at the bottom of the chamber
- RF power supply Samco 13.56 MHz, 300 W, crystal oscillation
- Pressure control Automatically controls the pressure from the MKS Palato Gauge with a VAT automatic pressure control (APC) valve unit.
- APC VAT automatic pressure control
- DAD321 an automatic dicing machine manufactured by DISCO, was used.
- This dicing machine is equipped with an X and Y stage, and it is possible to manufacture a straight-through cab without changing the position of the base glass by hand.
- the position and velocity of the blade in the depth direction (Z direction) can be controlled arbitrarily, and a capillary structure with an arbitrary depth can be manufactured.
- the processing accuracy in the X, Y, and Z directions is on the order of micrometers. All of the above series of operations were controlled by a built-in computer using programming (recipe).
- a surface level difference measuring device manufactured by Veeco, DEKTAK3ST, was used.
- the measurement distance and speed are controlled by a computer, and the obtained results are digitized.
- the obtained data can be displayed on a monitor screen as a cross section curve, roughness curve, undulation curve, or the like.
- Various analyzes such as depth, height, and inclination were performed by the built-in software as needed.
- the data obtained at the end was printed on a printer and simultaneously recorded on a disk.
- the system for conducting an electrophoresis experiment using the fabricated capillary electrophoresis chip is a high-voltage power supply BP-3 manufactured by BIOCRAFT or Grassman Japan Heil Voltage Co., Ltd. (Yokohama, Japan) as a power supply for applying an electric field.
- BP-3 high-voltage power supply
- BIOCRAFT BIOCRAFT
- Grassman Japan Heil Voltage Co., Ltd. Yokohama, Japan
- PS / FC40R03CTZ10 supplies power up to 2000V.
- the substrate used was Tempax glass.
- the silicon wafer used was a silicon wafer manufactured by Shin-Etsu Silicon Co., Ltd. (P-type, 100 surfaces, diameter: 10 Omm, thickness: 525 mm, resistivity: 10 to 20 ⁇ ⁇ cm).
- UV-curable adhesive and Venix PC manufactured by Adel Co., Ltd., were used to bond the glass.
- Other reagents used were EL grade or special grade or higher.
- the device used in the experiment has a structure in which a lower glass substrate and an upper glass cover are overlapped as shown in Fig. 1.
- a groove is dug on the lower glass substrate with a dicing saw.
- the upper glass cover has a through hole.
- a solution reservoir consisting of a through hole is formed at one end of the cavities.
- thin films having various properties are formed on the upper glass substrate and the lower glass substrate surface.
- the glass substrate and the glass cover were overlaid with a light-hardening resin to form a capillary.
- the glass substrate and glass cover are 80 mm long, 10 mm wide and 1.1 mm thick, respectively, and the capillary is 70 thigh long, 0.9 mm wide and 10 mm deep. 0 ⁇ m It is.
- the through hole made in the upper glass part has a diameter of 4 mm and a penetration depth of 1.1.
- a silane coupling solution (8 0 L [3- (methacrloyloxy ) ropyl] trimethoxysilane + 20mLH 2 0), and adjusted to ⁇ 3 ⁇ 5 added Rooster ⁇ . Subsequently, the coupling solution was introduced into the capillary via a tube connected to the device, and reacted at room temperature for 1 hour. After the completion of the reaction, distilled water was poured to wash the inside of the capillary.
- the substrate was placed in the chamber one and the degree of vacuum in the 3 X 10- 5 Torr.
- the chamber material was filled with the monomer material and adjusted to a predetermined pressure and flow rate. Discharge was carried out for a certain time to form a plasma polymerized film, and then the substrate was taken out.
- Table 1 shows the film forming conditions, film quality, and film thickness of the plasma polymerized film of each monomer material.
- HDE HDE
- HMDS HMDS
- eGN Discharge power
- W Discharge power
- W Pressure
- nm Film quality Hydrophobic Hydrophilic Hydrophilic Thickness
- Isoelectric focusing was used for separation.
- the sample used was an IEF marker protein manufactured by Pharmacia. Since this sample contains three types of proteins that are visible to the naked eye, the band after electrophoresis can be seen with the naked eye. The isoelectric point and color of each protein are shown below.
- the experimental procedure is as follows. First, the sample was filled over the entire cavity. As shown in Table 2, the protein concentration in the sample was 20 / g / «L and 33 (ig / and 62 g / L). Next, 25 iL of 0.1 M NaOH was used as the catholyte on the cathode side. to Rizapa, also a 25 iL 0.2M 3 ⁇ 4P0 4 as anolyte was injected into the anode side reservoir one server. by applying a voltage across the Kiyabirari one was performed isoelectric focusing. protein concentration of the by electrophoresis (Focusing) is shown in Figs.
- electrophoresis completion times were compared between the unmodified, the conventional acrylamide modification method, and the capillary electrophoresis chip of the present invention using the plasma polymerization method.
- the applied voltage was 1000 V or 2000 V.
- Table 2 shows the results of electrophoresis of samples by each capillaries.
- Table 2 shows the migration position (Band position) and the band width (Band width) of each protein at an applied voltage of 1000 V in each of the capillaries.
- the migration position is the result of measuring the distance from the electrode to the center of the band with the electrode position set to 0.
- the electrophoresis results of the acetonitrile-modified capillaries showed a small migration error and excellent reproducibility. Therefore, acetonitrile-modified capillary is a preferable material as a substrate for isoelectric focusing of the protein according to the present invention. 4
- Table 2 shows the migration position (Band position) and the band width (Band width) of each protein at an applied voltage of 1000 V in each of the capillaries.
- the migration position is the result of measuring the distance from the electrode to the center of the band with the electrode position set to 0.
- the electrophoresis results of the acetonitrile-modified capillaries showed
- the substrate was placed in one of the chambers, and the degree of vacuum was set to 5 ⁇ 10 to 16 Torr. Each monomer was filled with the degree of vacuum shown in Table 3. The emitted power was all 200W. Discharge was performed for a certain time to form a plasma polymerized film, and then the substrate was taken out. The refractive index, film thickness and contact angle of the obtained plasma polymerized film were measured. Table 3 shows the results. The refractive index and the film thickness were measured using an ellipsometer (EMS-1T (trade name), manufactured by ULVA C).
- EMS-1T ellipsometer
- the contact angle was measured using a contact angle measuring device (CA-X (trade name), manufactured by Kyowa Interface Science Co., Ltd.). Ultrapure water was used for the measurement.
- Hexamethyldisiloxane, hexylamine The zeta potential on the surface was measured for a plasma polymerized film having a large contact angle derived from, a plasma polymerized film having a small contact angle derived from aminoacetaldehyde dimethyl acetal, and a glass substrate not subjected to plasma polymerization.
- zeta potential measuring device ELS-800 (trade name), manufactured by Otsuka Electronics Co., Ltd.
- TrisHCI Tris-Hydroxy-Aminomethane
- the plasma polymerized film derived from aminoacetaldehyde dimethyl acetal has a positive zeta potential and is highly hydrophilic.
- Hexylamine-derived plasma polymerized membranes have a positive zeta potential and are highly hydrophobic.
- the plasma polymerized film derived from hexamethyldisiloxane has a negative zeta potential and is highly hydrophobic.
- a monomer containing a large amount of benzene derivative or carbon in order to make the plasma polymerized film hydrophobic, and to use a monomer containing a large amount of oxygen to make it hydrophilic.
- the zeta potential is positive It is suggested that it is effective to use a monomer containing amine and to make the zeta potential negative, and to use one containing a large amount of hydroxyl group or carboxylic acid.
- Triethoxybiersilane (Silicon Chemicals LC_2300, manufactured by Shin-Etsu Chemical Co., Ltd.) was dissolved in toluene as a compound for inducing a hydrophobic functional group to a concentration of 3 mM.
- the washed glass substrate was immersed in this and reacted at 80 ° C for 8 hours. After the reaction, the glass substrate was washed with toluene and ethanol, and dried under vacuum to obtain a bullet-introduced substrate.
- Aminopropyltriethoxysilane (Silicon Chemicals LC-4480, manufactured by Shin-Etsu Chemical Co., Ltd.) was dissolved in toluene at a concentration of 3 mM. The washed glass substrate was immersed in this and reacted at 80 ° C for 8 hours. After the reaction, the glass substrate was washed with toluene and ethanol, and dried under vacuum to obtain an amino group-introduced substrate.
- polyacrylic acid polyacrylic acid 25000, manufactured by Wako Pure Chemical Industries, Ltd.
- polyacrylic acid 25000 manufactured by Wako Pure Chemical Industries, Ltd.
- amino group-introduced substrate was immersed.
- 0.1 mmol each of N-hydroxysuccinimide (NHS) and 1-ethyl-3- (3-dimethylaminopropyl) carboximide hydrochloride were added, and the mixture was shaken for 24 hours. After the reaction, the substrate was washed with ultrapure water and dried under vacuum to obtain a polyacrylic acid-modified substrate.
- NHS N-hydroxysuccinimide
- 1-ethyl-3- (3-dimethylaminopropyl) carboximide hydrochloride were added, and the mixture was shaken for 24 hours. After the reaction, the substrate was washed with ultrapure water and dried under vacuum to obtain a polyacrylic acid-modified substrate.
- the amino group-introduced substrate was immersed in a 3 mM glutaraldehyde solution (100 mM HEPES buffer (prepared at pH 7.0) and shaken for 8 hours. After the reaction, the substrate was washed with ultrapure water and dried under vacuum. Thus, an aldehyde group-introduced substrate was obtained.
- polyallylamine polyallylamine hydrochloride (trade name), manufactured by Aldrich) is dissolved in 20 mL of lO OmM-HEPES (pH 7.0), and the aldehyde-introduced substrate is immersed therein. And shaken for 24 hours. After the reaction, the substrate was washed with ultrapure water and dried under vacuum to obtain a polyallylamine-modified substrate.
- the contact angle and zeta potential of the surface of the obtained polyacrylic acid-modified substrate and polyallylamine-modified substrate on which the polymer was modified were measured in the same manner as in Example 2.
- Table 8 shows the results of the contact angle
- Table 9 shows the results of the zeta potential.
- Example 3 Immersed in the prepared butyl group-introduced substrate, and added ⁇ , ⁇ , ⁇ ', N, -tetramethylethylenediamine or 2,2-azobis (2-amidinopropane) diacetate to the polymerization initiator at 50 ⁇ mo 1 After sealing with a stopper, purging with nitrogen, 55. Shake at C for 24 hours. After the reaction, it was washed with water to obtain a surface-polymerized substrate.
- the contact angle and zeta potential of these surface-polymerized membranes were measured using buffer solutions such as tannic acid (0.0143M), potassium dihydrogen phosphate (0.0143M), and boric acid (0.0143M).
- buffer solutions such as tannic acid (0.0143M), potassium dihydrogen phosphate (0.0143M), and boric acid (0.0143M).
- the measurement was performed in the same manner as in Example 2 except that a mixed solution having a NaCl concentration of 10 (mM) was used and pH was adjusted as shown in Table 10 (pH was adjusted with an aqueous NaOH solution).
- Table 10 shows the measurement results of the contact angle and zeta potential.
- 3-glycidoxypropyl triethoxysilane (Silicon Chemicals LS-2940 (trade name), manufactured by Shin-Etsu Chemical Co., Ltd.) was dissolved to a concentration of 3 mM.
- the washed glass substrate was immersed therein and reacted at 80 ° C. for 8 hours. After the reaction, the glass substrate was washed with toluene and dried under vacuum to obtain a glycidyl group-introduced substrate.
- the contact angle and zeta potential of the obtained polymer-modified substrate were measured in the same manner as in Example 5.
- Table 10 shows the measurement results of the contact angle and the zeta potential.
- an electrophoresis separation method and device capable of controlling various properties of a substrate surface with which an electrophoresis medium comes into contact are provided.
- a substrate holding a migration separation medium such as glass sometimes interfered with the results of the migration separation.
- the glass surface was modified by coating with a plasma polymerized film, a surface polymerized film, or a polymer binding film, and as a result, a good substrate for electrophoretic separation could be obtained.
- plasma-polymerized films can be applied to fine structures, Materials can be processed together. In other words, it is an industrially useful technology that can easily mass-produce homogeneous electrophoresis substrates.
- the formation of the surface-polymerized film peeling of the film is suppressed, and a desired polymer compound film can be formed at a desired position on the substrate surface.
- a desired polymer compound film can be formed at a desired position while easily controlling the film thickness.
- the present invention can provide a method for separating and analyzing a large number of samples simultaneously and easily on a miniaturized substrate.
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CA002498040A CA2498040A1 (en) | 2002-09-05 | 2003-09-05 | Method for separating substances |
EP03799096A EP1548429A4 (en) | 2002-09-05 | 2003-09-05 | METHOD FOR DISCONNECTING SUBSTANCES |
AU2003261957A AU2003261957A1 (en) | 2002-09-05 | 2003-09-05 | Method for separating substances |
JP2004541218A JP4480081B2 (ja) | 2002-09-05 | 2003-09-05 | 物質の分離方法 |
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Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2006266895A (ja) * | 2005-03-24 | 2006-10-05 | National Institute Of Advanced Industrial & Technology | キャピラリー電気泳動方法並びにそのためのキャピラリー及び電気泳動装置 |
WO2008029684A1 (fr) * | 2006-09-04 | 2008-03-13 | National Institute Of Advanced Industrial Science And Technology | Procédé pour analyse d'échantillon par électrophorèse capillaire |
WO2008029685A1 (fr) * | 2006-09-04 | 2008-03-13 | National Institute Of Advanced Industrial Science And Technology | Procédé pour analyse d'échantillon par électrophorèse capillaire |
JP2008164382A (ja) * | 2006-12-27 | 2008-07-17 | Sekisui Chem Co Ltd | ヘモグロビン類の測定方法 |
WO2012176782A1 (ja) * | 2011-06-21 | 2012-12-27 | シャープ株式会社 | 電気泳動用チップおよびその製造方法 |
US9017536B2 (en) | 2006-12-26 | 2015-04-28 | Sekisui Chemical Co., Ltd. | Hemoglobin measurement method and electrophoresis apparatus |
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EP2081021A4 (en) * | 2006-10-16 | 2014-07-23 | Sekisui Chemical Co Ltd | HEMOGLOBIN DETERMINATION PROCESS |
WO2017095813A1 (en) * | 2015-11-30 | 2017-06-08 | Intabio, Inc. | Devices and methods for sample characterization |
CN110891671B (zh) * | 2017-04-24 | 2023-01-24 | 圣母大学 | 可调谐电渗流聚合物涂覆的毛细管 |
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- 2003-09-05 US US10/526,213 patent/US20060113189A1/en not_active Abandoned
- 2003-09-05 WO PCT/JP2003/011352 patent/WO2004031757A1/ja active Application Filing
- 2003-09-05 EP EP03799096A patent/EP1548429A4/en not_active Withdrawn
- 2003-09-05 CA CA002498040A patent/CA2498040A1/en not_active Abandoned
- 2003-09-05 JP JP2004541218A patent/JP4480081B2/ja not_active Expired - Lifetime
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US5322608A (en) * | 1992-12-23 | 1994-06-21 | Northeastern University | Siloxandediol coating for capillary electrophoresis and for general surface modification |
US5447617A (en) * | 1994-01-25 | 1995-09-05 | Beckman Instruments, Inc. | Coated capillary columns and electrophoretic separation methods for their use |
JP2002148236A (ja) * | 2000-11-08 | 2002-05-22 | Hitachi Ltd | 電気泳動装置 |
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Cited By (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2006266895A (ja) * | 2005-03-24 | 2006-10-05 | National Institute Of Advanced Industrial & Technology | キャピラリー電気泳動方法並びにそのためのキャピラリー及び電気泳動装置 |
JP4565204B2 (ja) * | 2005-03-24 | 2010-10-20 | 独立行政法人産業技術総合研究所 | キャピラリー電気泳動方法並びにそのためのキャピラリー及び電気泳動装置 |
WO2008029684A1 (fr) * | 2006-09-04 | 2008-03-13 | National Institute Of Advanced Industrial Science And Technology | Procédé pour analyse d'échantillon par électrophorèse capillaire |
WO2008029685A1 (fr) * | 2006-09-04 | 2008-03-13 | National Institute Of Advanced Industrial Science And Technology | Procédé pour analyse d'échantillon par électrophorèse capillaire |
JP4814944B2 (ja) * | 2006-09-04 | 2011-11-16 | 独立行政法人産業技術総合研究所 | キャピラリー電気泳動法による試料の分析方法 |
US8137512B2 (en) | 2006-09-04 | 2012-03-20 | National Institute Of Advanced Industrial Science And Technology | Process for analyzing sample by capillary electrophoresis method |
CN101501484B (zh) * | 2006-09-04 | 2012-12-26 | 独立行政法人产业技术综合研究所 | 通过毛细管电泳法分析试样的方法 |
US9121821B2 (en) | 2006-09-04 | 2015-09-01 | National Institute Of Advanced Industrial Science And Technology | Process for analyzing sample by capillary electrophoresis method |
US9017536B2 (en) | 2006-12-26 | 2015-04-28 | Sekisui Chemical Co., Ltd. | Hemoglobin measurement method and electrophoresis apparatus |
JP2008164382A (ja) * | 2006-12-27 | 2008-07-17 | Sekisui Chem Co Ltd | ヘモグロビン類の測定方法 |
WO2012176782A1 (ja) * | 2011-06-21 | 2012-12-27 | シャープ株式会社 | 電気泳動用チップおよびその製造方法 |
JPWO2012176782A1 (ja) * | 2011-06-21 | 2015-02-23 | シャープ株式会社 | 電気泳動用チップおよびその製造方法 |
Also Published As
Publication number | Publication date |
---|---|
EP1548429A1 (en) | 2005-06-29 |
EP1548429A4 (en) | 2009-06-17 |
AU2003261957A8 (en) | 2004-04-23 |
CA2498040A1 (en) | 2004-04-15 |
AU2003261957A1 (en) | 2004-04-23 |
JPWO2004031757A1 (ja) | 2006-02-02 |
US20060113189A1 (en) | 2006-06-01 |
JP4480081B2 (ja) | 2010-06-16 |
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