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WO2013161368A1 - Outil de test de focalisation isoélectrique et son procédé de production - Google Patents

Outil de test de focalisation isoélectrique et son procédé de production Download PDF

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Publication number
WO2013161368A1
WO2013161368A1 PCT/JP2013/054478 JP2013054478W WO2013161368A1 WO 2013161368 A1 WO2013161368 A1 WO 2013161368A1 JP 2013054478 W JP2013054478 W JP 2013054478W WO 2013161368 A1 WO2013161368 A1 WO 2013161368A1
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Prior art keywords
test device
substrate
electrophoresis
gel layer
gel
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PCT/JP2013/054478
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English (en)
Japanese (ja)
Inventor
政俊 中川
真也 上柿
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Sharp Corp
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Sharp Corp
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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
    • G01N27/44756—Apparatus specially adapted therefor
    • G01N27/44795—Isoelectric focusing

Definitions

  • the present invention relates to a test device for isoelectric focusing and a method for producing the same.
  • Electrophoresis is a separation analysis method using a phenomenon in which a charged substance in a medium moves in an electric field according to the electric charge when a voltage is applied to the medium such as a solution or a hydrophilic support immersed in the solution. It is.
  • electrophoresis using gel as a medium is a technique for separating biopolymers such as proteins and nucleic acids, in bioscience, molecular biology and other life science fields and clinical laboratory fields. Widely used.
  • electrophoresis isoelectric focusing method
  • proteins are separated by gathering at a pH position equal to their isoelectric point in a pH gradient.
  • an amphoteric carrier has been used in the past, but in recent years, an immobilized pH gradient (Immobilized pH Gradient: IPG) gel that does not collapse during energization is often used. ing.
  • IPG immobilized pH Gradient
  • gel electrophoresis is an indispensable technique for separating and analyzing biopolymers such as proteins.
  • the accuracy and reproducibility of analysis largely depend on the quality of the gel used. Therefore, in this field, it is desired to develop a technique capable of stably producing an electrophoretic test device equipped with a high-resolution gel.
  • Patent Document 1 discloses a gel sheet having a concentration gradient by mixing two types of gel stock solutions having different concentrations in a stirring tank, and introducing the mixed solution into a gel container from the bottom to cause gelation (polymerization).
  • a method of making is disclosed.
  • an SDS-PAGE gel sheet having a predetermined concentration gradient can be obtained by changing the ratio of each gel stock solution in the mixed solution to be introduced into the gel container.
  • two types of gel stock solutions having different pH are mixed in a stirring tank, and the mixture is introduced into the gel container from the bottom to cause gelation, thereby adjusting the pH gradient.
  • the gel sheet which has can be produced.
  • a gel sheet having a predetermined pH gradient is obtained by changing the ratio of each gel stock solution in the mixed solution to be introduced into the gel container, and the gel sheet is elongated by cutting it at a predetermined width in the pH gradient direction.
  • a gel plate for isoelectric focusing is obtained.
  • Patent Document 2 discloses a gel plate manufacturing method in which a monomer solution is applied onto a plate as a technique capable of accurately managing a concentration gradient or pH gradient. That is, after forming a puddle on the plate and discharging a monomer solution into the puddle, a polymerization initiator is applied to gel the coating film, thereby forming a gel layer on the substrate.
  • an SDS-PAGE gel plate having a predetermined concentration gradient or a predetermined pH gradient is prepared by mixing two types of monomer solutions having different concentrations or pHs and applying them to the pool while changing the mixing ratio.
  • a gel plate for isoelectric focusing is obtained.
  • the gel layer In order to be able to store the gel layer produced as described in Patent Documents 1 and 2 for a long period of time, the gel layer is usually dried. And a gel layer is decompress
  • a pH gradient is formed in the first direction which is the longitudinal direction of the gel layer, and protein separation by isoelectric focusing is performed in the first direction.
  • the central portion Gc extending in the length direction of the gel layer G and the both sides of the central portion arranged in the width direction orthogonal to the length direction can be shown.
  • the same band B is formed at positions shifted from each other in the length direction.
  • each band B is formed in a shape called “smileing” that is curved or refracted from the central portion Gc toward both end portions Ge. There is a problem that decreases.
  • an electrophoretic test device in which a gel layer is formed on a substrate, and the gel layer includes a highly permeable portion having a high permeability of an evaluation object in a sample solution.
  • an electrophoretic test device comprising a low permeability portion having a lower permeability than the high permeability portion.
  • a first step of forming a puddle containing a monomer on a substrate, a second step of applying a crosslinking agent on the substrate, and polymerization on the substrate A third step of applying an initiator, In the second step, a test for electrophoresis in which a coating amount distribution in which the crosslinking agent coating amount is different in the first direction is formed and the crosslinking agent coating amount in the second direction orthogonal to the first direction is uniform.
  • Manufacturing method of ingredients or Including a first step of applying a monomer containing a cross-linking agent on a base material, and a second step of applying a polymerization initiator on the base material, In the first step, a test for electrophoresis in which a coating amount distribution having a different crosslinking agent coating amount in the first direction is formed and the crosslinking agent coating amount in the second direction orthogonal to the first direction is uniform.
  • a method of manufacturing the tool is provided.
  • a crosslinking agent coating part for coating a crosslinking agent on a liquid pool containing monomers on a substrate, and a polymerization initiator coating part for coating a polymerization initiator on the liquid pool
  • the apparatus for producing an electrophoretic test device in which the cross-linking agent application part comprises an inkjet head, or a mixed solution application part for applying a monomer containing a cross-linking agent on a base material, and polymerization on the base material
  • a polymerization initiator application unit for applying the agent, and the mixed solution application unit includes an inkjet head.
  • the test device for electrophoresis of the present invention includes a highly permeable portion having a high permeability of an evaluation object (evaluation protein) in a sample solution and a low permeable portion having a lower permeability than the highly permeable portion. Since the gel layer is provided, it is possible to prevent the evaluation target in the sample liquid from penetrating into the low permeability portion by setting the permeability of the low permeability portion to be extremely low.
  • the permeability of the gel layer can be adjusted by, for example, the density of the monomer cross-linking structure.
  • the monomer cross-linking structure is set so dense that the evaluation protein cannot enter the low-permeability portion (high density), and the high-permeability portion Then, the monomer cross-linked structure may be set to be coarse (low density) to the extent that the evaluation protein can enter and move freely.
  • an electrophoresis test device of the present invention it is possible to manufacture an electrophoresis test device capable of performing a highly accurate and reliable protein separation test.
  • FIG. 1 (A) It is a perspective view which shows the state which can use the test device for electrophoresis of this invention. It is a perspective view which shows the state which can be preserve
  • FIG. 1 is a configuration diagram illustrating an apparatus for manufacturing an electrophoretic test device according to Embodiment 1.
  • FIG. It is the schematic bottom view which looked at the inkjet head in the manufacturing apparatus of FIG. 4 from the downward direction. It is an enlarged view which shows the nozzle hole group of the inkjet head in the manufacturing apparatus of FIG. It is explanatory drawing which shows the state which apply
  • FIG. 6 is a configuration diagram illustrating an apparatus for manufacturing an electrophoretic test device according to a second embodiment. It is explanatory drawing which shows the state which set the base material with which the puddle containing a monomer was apply
  • FIG. 5 is a configuration diagram illustrating an apparatus for manufacturing an electrophoretic test device according to a third embodiment. It is explanatory drawing which shows the state which apply
  • the electrophoretic test device of the present invention is an electrophoretic test device in which a gel layer is formed on a substrate, and the gel layer is highly permeable with high permeability of an evaluation object in a sample solution. A portion and a low permeability portion that is less permeable than the high permeability portion. At this time, from the viewpoint of obtaining a high-resolution gel layer, the permeability of the low-permeability portion is set low enough that the solvent in the sample solution permeates the low-permeability portion but the evaluation object cannot permeate (enter). It is preferable to do.
  • the high permeability portion and the low permeability portion are disposed adjacent to each other, and the high permeability portion and the low permeability portion are adjacent to each other.
  • the dimension of the first direction arranged in a row may be shorter than the dimension of the second direction orthogonal to the first direction.
  • the low permeability portion may be disposed on both sides of the high permeability portion in the first direction.
  • a long gel layer can be obtained in the second direction in which the low-permeability portions are arranged on both sides in the first direction (width direction) of the high-permeability portion, and one-dimensional in two-dimensional electrophoresis
  • An elongated electrophoresis test device suitable for eye electrophoresis can be obtained.
  • both ends in the width direction of the gel layer which is likely to cause electrolysis abnormalities, are composed of low-permeability portions, in addition to preventing smiles, the electrophoretic test device is slimmed to the minimum necessary. The necessary amount of the material, the material of the gel layer, and the sample solution can be reduced and waste can be eliminated.
  • the highly permeable portion of the gel layer may have a pH gradient in the second direction, whereby isoelectric focusing can be performed with high accuracy and high reliability.
  • a test device can be obtained.
  • the form of the base material of the electrophoresis test device is not particularly limited, and examples thereof include an elongated plate and a chip molded into a predetermined shape.
  • the material of the base material is not particularly limited as long as it can function as a base material for a test device for electrophoresis.
  • glass such as quartz glass and non-alkali glass, polyethylene terephthalate (PET), polymethacryl
  • resins such as acid methyl resin (PMMA), ceramics such as alumina, and low-temperature co-fired ceramic.
  • the surface of the substrate on which the gel layer is formed may be subjected to a hydrophilic treatment, thereby improving the wettability of the monomer solution described below with respect to the substrate, and the monomer solution Adhesion between the gelled gel layer and the substrate is improved.
  • a hydrophilic treatment include nitration using sulfuric acid, sulfonation using nitric acid, oxygen plasma treatment and the like.
  • the material of the gel layer of the electrophoresis test device is not particularly limited as long as it can function as the gel layer of the electrophoresis test device.
  • acrylamide Monomer
  • bisacrylamide crosslinking agent
  • pH adjusting material pH buffer
  • TEMED polymerization accelerator
  • ammonium persulfate APS
  • the electrophoretic test device of the present invention can be manufactured by the following first or second manufacturing method.
  • a first step of forming a puddle containing a monomer on a substrate, a second step of applying a cross-linking agent on the substrate, and a polymerization initiator are applied on the substrate.
  • a third step wherein in the second step, a coating amount distribution having a different crosslinking agent coating amount in the first direction is formed, and a crosslinking agent coating amount in a second direction orthogonal to the first direction is set. Make it uniform.
  • the first manufacturing method corresponds to Embodiments 1 and 2 described later.
  • the second production method includes a first step of applying a monomer containing a crosslinking agent on a substrate and a second step of applying a polymerization initiator on the substrate.
  • the first step the first step
  • the coating amount distribution in which the coating amount of the crosslinking agent is different in the direction is formed, and the coating amount of the crosslinking agent in the second direction orthogonal to the first direction is uniform.
  • the second manufacturing method corresponds to Embodiment 3 to be described later.
  • the terms “first”, “second”, and “third” in the first to third steps do not mean the order of the steps, and the respective steps are distinguished. It is only a display to do.
  • the gel layer having a pore size distribution with different gel pore sizes in the first direction is formed on the substrate by the first or second manufacturing method. That is, a gel layer in which the density of the monomer crosslinked structure is different in the first direction can be obtained.
  • “gel pore size” means the density of the monomer cross-linked structure, the level of the gel concentration, etc., and when the gel pore size is large, the monomer cross-linked structure is coarse (low density), Gel concentration is low. On the contrary, when the gel pore size is small, the monomer cross-linked structure is dense (high density) and the gel concentration is high.
  • the gel pore size can be adjusted by adjusting the coating amount of the crosslinking agent. The method for adjusting the gel pore size will be described later in detail.
  • the amount of the cross-linking agent applied to both side regions of the central region in the first direction is larger than the amount of the cross-linking agent applied to the central region in the first direction on the substrate.
  • the gel pore size in the both side regions of the gel layer can be made smaller than the gel pore size in the central region. That is, the highly permeable portion can be formed in the central region of the gel layer, and the low permeable portion can be formed in both side regions.
  • an elongated electrophoresis test device suitable for the first-dimensional electrophoresis in the two-dimensional electrophoresis can be obtained.
  • the method of applying a gel material solution such as a monomer, a crosslinking agent, a polymerization initiator on the substrate is not particularly limited, as long as the gel material solution can be applied to a predetermined region on the upper surface of the substrate,
  • a pipetter, a dispenser, an ink jet device and the like can be mentioned.
  • an ink jet apparatus provided with an ink jet head that discharges fine droplets with high accuracy and adheres them to a substrate. If an inkjet head is used, minute droplets can be applied to a predetermined area of an elongated base material with high accuracy and quantitatively. Therefore, the formation area of the gel layer to be obtained, film thickness, pH gradient, concentration gradient, etc. Can be controlled easily and with high accuracy.
  • FIG. 1 (A) is a perspective view showing a usable state of the electrophoresis test device of Embodiment 1 of the present invention
  • FIG. 1 (B) is a gel layer in the electrophoresis test device of FIG. 1 (A). It is a perspective view which shows the state which can be preserve
  • 2A is a view showing a highly permeable portion and a low permeable portion when the gel layer of FIG. 1A is viewed from the length direction
  • FIG. 2B is a view showing FIG. 2) shows a highly permeable portion and a low permeable portion when the dried membrane is viewed from the length direction
  • FIG. 2C is a view when the gel layer of FIG. It is a figure which shows a highly permeable part and a low permeable part.
  • FIG. 3 is a diagram illustrating a state after performing an isoelectric focusing using the electrophoresis test device of FIG.
  • the electrophoresis test device GP 1 shown in FIG. 1 (A) is obtained by forming a gel layer G 1 on a rectangular base material S that is long in the X direction.
  • the gel layer G 1 has a width indicated by an arrow Y.
  • Gel pore size of the high permeability portion G 11 is sized to penetrate water or sample liquid (Evaluation protein and solvent).
  • the gel pore size of the low permeability portion G 12 is a solvent of water or the sample solution is infiltrated, evaluated proteins in the sample solution is very small size not to penetrate (enter).
  • the gel pore sizes of the high permeability portion G 11 and the low permeability portion G 12 are reduced, but the gel pore size of the relatively high permeability portion G 11 is relatively low. no different to that larger than the gel pore size portions G 12.
  • the width W 2 of the low permeability portion G 12 (one side) is about 5 to 20% of the width W of the substrate S.
  • fine mesh of coarse mesh and the low permeability portion G 12 of high permeability portions G 11 gel layer G 1 is to express the magnitude of the gel pore size of each portion ing.
  • the highly permeable portion G 11 of the gel layer G 1 has a pH gradient of, for example, about pH 3 to 10 in the length direction (X direction). That is, the electrophoretic test devices GP 1 and GPD 1 of Embodiment 1 are isoelectric focusing test devices that can be used for the first-dimensional electrophoresis in the two-dimensional electrophoresis.
  • pH gradient are not required for low permeability portion G 12, a problem with pH gradient is formed by pH buffer in the low permeability portion G 12 when the gel layer formed is mixed intentionally or unintentionally There is no.
  • the isoelectric focusing test tool GPD 1 shown in FIG. 1B is immersed in the sample liquid, and the dry film D 1 absorbs the sample liquid to a saturated state. Swell.
  • the gel layer G 1 swollen as a whole is restored (see FIG. 1A).
  • evaluation protein in the sample solution from the surface of the high permeability portion D 11 of the dry film D 1 enters.
  • evaluation protein in the sample solution from the low permeability portion D 12 surface of the dried film D 1 does not enter.
  • the evaluation protein When the sample solution is dropped on the gel layer G 1 , the evaluation protein can enter the highly permeable part G 11 but cannot enter the low permeable part G 12 . Furthermore, evaluation proteins in high permeability portions G 11 can not enter the low permeability portion G 12 in.
  • adjustment of the gel pore size of the gel layer G 1 may be carried out by forming a coating weight distribution of the crosslinking agent in the width direction (Y-direction) at the time of the gel layer formed.
  • the amount of the crosslinking agent applied to both side regions (regions with a width W 1 ) in the width direction on the substrate S is set larger than the amount applied to the central region (regions with a width W 2 ).
  • the coating amount is adjusted in consideration of the total monomer concentration (% T) and the degree of crosslinking (% C).
  • the total monomer concentration (% T) is the weight percent of the total monomer including the crosslinking agent (monomer amount g / 100 ml), and the crosslinking degree (% C) is the ratio of the crosslinking agent to the total monomer.
  • Total monomer concentration (% T) (total weight of monomer + total weight of crosslinking agent / total liquid amount) ⁇ 100
  • Crosslinking degree (% C) (total weight of crosslinking agent / total weight of monomer + total weight of crosslinking agent) ⁇ 100
  • the gel pore size of the highly permeable portion G 11 and the low permeable portion G 12 of the gel layer G 1 may be determined according to the type of protein to be evaluated. Specifically, for the highly permeable part G 11 , the general monomer concentration (% T) is set to about 4 to 6 and the degree of crosslinking (% C) is set to about 2 to 3, While allowing the protein (mass: several tens to several hundred kDa) to permeate, for example, the total monomer concentration (% T) of the low-permeability portion G 12 is about 4 to 6, and the degree of crosslinking (% C) May be set to 5 or more so that the protein hardly penetrates.
  • FIG. 4 is a configuration diagram showing an apparatus that can manufacture the electrophoresis test device of the first embodiment.
  • the test tool manufacturing apparatus T1 includes a stage 10 on which a base material S is set, an ink jet apparatus 30 as an application unit, a moving mechanism 40 that moves the stage 10 in a linear direction, and a sealable case 50 that houses these. And a control unit (not shown).
  • the case 50 is provided with an opening / closing door (not shown).
  • the moving mechanism 40 includes a support base 40a that supports the stage 10, and the support base 40a can be reciprocated in a linear direction by a linear guide mechanism (not shown).
  • the support base 40a indicated by the solid line is in the standby position, and the support base 40a, the stage 10 and the substrate S travel straight to the position indicated by the two-dot chain line in the coating process.
  • the substrate S set on the stage 10 passes directly below first to fifth inkjet heads 31b, 32b, 33b, 34b, and 35b, which will be described later.
  • the ink jet device 30 includes a monomer discharge unit 31, a crosslinking agent discharge unit 32, an acidic buffer discharge unit 33, a basic buffer discharge unit 34, a polymerization initiator discharge unit 35, and a negative pressure adjustment unit 36. Yes.
  • the configurations of the monomer discharge unit 31, the crosslinking agent discharge unit 32, the acidic buffer discharge unit 33, the basic buffer discharge unit 34, and the polymerization initiator discharge unit 35 are basically the same.
  • the monomer discharge unit 31 includes a first tank 31a that stores the monomer solution A, a first inkjet head 31b, and a first pipe 31c that sends the monomer solution A from the first tank 31a to the first inkjet head 31b.
  • the monomer solution A is supplied from the first tank 31a to the first inkjet head 31b using the water head difference.
  • the monomer solution A for example, a solution in which acrylamide and a thickener are dissolved in water at a predetermined concentration can be used.
  • the cross-linking agent discharge unit 32 includes a second tank 32a that stores the cross-linking agent solution B, a second ink jet head 32b, and a second pipe 32c that sends the cross-linking agent solution B from the second tank 32a to the second ink jet head 32b.
  • the crosslinking agent solution B for example, a solution in which bisacrylamide and a thickener are dissolved in water at a predetermined concentration can be used.
  • the acidic buffer discharge unit 33 includes a third tank 33a that stores the acidic buffer solution C, a third inkjet head 33b, and a third pipe 33c that sends the acidic buffer solution C from the third tank 33a to the third inkjet head 33b.
  • the acidic buffer solution C for example, a solution in which one or more acidic buffers and a thickener are dissolved in water at a predetermined concentration can be used.
  • the basic buffer discharge unit 34 stores a basic buffer solution D, a fourth tank 34a, a fourth inkjet head 34b, and a fourth pipe that sends the basic buffer solution D from the fourth tank 34a to the fourth inkjet head 34b. 34c.
  • the basic buffer solution D for example, a solution in which one or more basic buffers and a thickener are dissolved in water at a predetermined concentration can be used.
  • the polymerization initiator discharge unit 35 includes a fifth tank 35a that stores the polymerization initiator solution E, a fifth inkjet head 35b, and a fifth pipe 35c that sends the polymerization initiator E from the fifth tank 35a to the fifth inkjet head 35b. And have.
  • the polymerization initiator solution E for example, a solution in which ammonium persulfate and a thickener are dissolved in water at a predetermined concentration can be used.
  • Examples of the first to fifth ink jet heads 31b to 35b include a thermal jet method, a piezo jet method, an electrostatic drive method, and the like, but each liquid (monomer solution A, crosslinker solution B, acidic buffer solution) in the ink jet device 30 is used.
  • a thermal jet method a piezo jet method, an electrostatic drive method, and the like
  • each liquid monomer solution A, crosslinker solution B, acidic buffer solution
  • the negative pressure adjusting unit 36 is connected to the first to fifth tanks 31a to 35a by pipes 31d to 35d, manages the atmospheric pressure in the first to fifth tanks, and controls the first to fifth inkjet heads 31b.
  • the insides of the first to fifth tanks 31a to 35a are adjusted to be constant at a predetermined pressure lower than the atmospheric pressure so that the liquid does not drip from the nozzle holes H (see FIG. 6) of .about.35b.
  • the first to fifth ink jet heads 31b to 35b are integrated to form a set of discharge head units U1, and the discharge head units U1 are fixed by a fixing member (not shown). As shown in FIG. 5, the first to fifth ink jet heads 31b to 35b are arranged in a line on the movement locus E of the substrate S, but the head arrangement order is not limited to this order. In the first embodiment, the first to fifth inkjet heads 31b to 35b are arranged in this order from the upstream side in the moving direction of the substrate S.
  • a plurality of nozzles are formed on the lower surfaces of the first to fifth inkjet heads 31 b to 35 b facing the movement locus E of the substrate S in a direction orthogonal to the direction of the movement locus E.
  • the holes H are provided in one row. That is, the nozzle hole group HG in one row extends in a direction orthogonal to the direction of the movement locus E and with a length exceeding the width of the movement locus E.
  • the nozzle hole diameter D and the nozzle hole interval P are not particularly limited, but the diameter of the nozzle hole H is suitably about 10 to 100 ⁇ m, and the nozzle hole interval P is suitably about 100 to 200 ⁇ m.
  • the nozzle hole group HG may be provided in a plurality of rows of two or more.
  • an elongated rectangular base material S is set on the stage 10 in the standby position.
  • a coating process under normal temperature and atmospheric pressure based on a predetermined program is performed. That is, as shown in FIGS. 7A to 7D, the support base 40a is intermittently moved in the direction of the arrow M by the moving mechanism 40, and the micro droplets La from the first to fifth inkjet heads 31b to 35b. ⁇ Le is discharged intermittently, and a coating film is formed on the substrate S.
  • the nozzle hole H that discharges the micro droplet La is selected from the nozzle hole group HG in the first inkjet head 31b so that the micro droplet La is not discharged onto the stage 10.
  • the second to fifth ink jet heads 32b to 35b the coating amount per unit area of the fine droplets La discharged from the first inkjet head 31b onto the substrate S is constant.
  • the other end S 2 of the substrate S is moved to a position directly below the nozzle hole group HG of the second ink jet heads 32b, FIG.
  • microdroplets Lb of the crosslinking agent solution are ejected from the second inkjet head 31b and applied onto the coating liquid L1.
  • the coating film L2 in which the crosslinking agent is mixed in the coating liquid L1 is formed on the entire surface of the substrate S.
  • the application amount to the both side regions is larger than the application amount of the micro droplet Lb to the central region (width W 2 region) of the substrate S described in FIGS.
  • the ejection of the minute droplets Lb from each nozzle hole H of the second inkjet head 32b is controlled so that the coating amount is increased.
  • the total monomer concentration (% T) is set to about 4 to 6, and the degree of crosslinking (% C) is set to about 2 to 3.
  • the total monomer concentration (% T) is set to about 4 to 6, and the degree of crosslinking (% C) is set to about 5.
  • the support table 40a is moved back to the direction M, at one end S 1 of the substrate S is moved to a position directly below the third and fourth inkjet head 33b, 34b of the nozzle hole group HG, FIG 7 (C As shown in FIG. 5, the acidic buffer solution micro droplets Lc are discharged from the third inkjet head 33b, and the basic buffer solution micro droplets Ld are discharged from the fourth inkjet head 34b. Thereby, the coating film L3 in which the acidic and basic buffers are mixed in the coating film L2 is formed on the entire surface of the substrate S.
  • the coating amount of the micro droplet Lc (acidic buffer solution) gradually decreases from one end S 1 to the other end S 2 of the substrate S, and the micro droplet Ld (basic buffer solution) is applied.
  • the application amount is controlled every time the base material S is intermittently moved by a predetermined distance so that the amount gradually increases (every predetermined discharge interval).
  • the support table 40a is moved in the opposite direction (N direction) again, where the other end S 2 of the substrate S is moved to a position directly below the nozzle hole group HG of the fifth ink jet head 35b, in FIG. 7 (D) As shown, a minute droplet Le of the polymerization initiator solution is ejected from the fifth inkjet head 35b. Thereby, the coating film L4 in which the polymerization initiator is mixed in the coating film L3 is formed on the entire surface of the substrate S. At this time, the coating amount per unit area of the fine droplet Le discharged from the fifth inkjet head 35b onto the substrate S is constant.
  • the coating process by the test device manufacturing apparatus T1 is performed in four processes by the ink jet apparatus 30 having a five-head configuration and is completed.
  • coating process is performed because a control part controls each drive part based on a predetermined program.
  • an isoelectric focusing test device GP 1 in which a bowl-shaped gel layer G 1 having roundness at four ends is formed on the substrate S by the gelation process. can get.
  • the gel layer G1 as shown in FIG. 2 (A) and (C), with the density of the monomer crosslinked structure in the central region has a height less permeable portions G 11, a large density of the monomer crosslinked structure side regions having low permeability portion G 12.
  • a storable isoelectric focusing test device GPD 1 shown in FIG. 1B is obtained.
  • the method of drying the gel layer G 1 is not particularly limited, and examples thereof include a method of heating the gel layer G 1 with a heater or blowing hot air to the gel layer G 1 for drying.
  • a cooling step for cooling the dry film D 1 to ⁇ 20 ° C. or less may be performed. Or you may perform a freeze-dry process instead of a drying process and a cooling process.
  • FIG. 8 is a configuration diagram illustrating an apparatus for manufacturing an electrophoretic test device according to the second embodiment.
  • the same elements as those in FIG. 4 are denoted by the same reference numerals.
  • a manufacturing apparatus and a manufacturing method capable of manufacturing the isoelectric focusing test device GP 1 substantially the same as in the first embodiment. Will be explained.
  • points of the second embodiment different from the first embodiment will be mainly described.
  • the test device manufacturing apparatus T2 of the second embodiment is the same as that of the first embodiment except that the monomer discharge unit 31 in the test device manufacturing apparatus T1 (see FIG. 4) of the first embodiment is omitted. That is, the inkjet device 130 of the test device manufacturing apparatus T2 of Embodiment 2 includes a cross-linking agent discharge unit 32, an acidic buffer discharge unit 33, a basic buffer discharge unit 34, a polymerization initiator discharge unit 35, and a negative pressure adjustment. Part 36. Then, the second to fifth ink jet heads 32b to 35b of the crosslinking agent discharge unit 32, the acidic buffer discharge unit 33, the basic buffer discharge unit 34, and the polymerization initiator discharge unit 35 are integrated to form a set of discharge head units. U2 is configured.
  • a liquid pool containing a monomer is formed on the base material S before the base material S is set in the test device manufacturing apparatus T2. At this time, a water film is formed on the substrate S, and a monomer is dropped on the water film, or the monomer solution A used in Embodiment 1 is dropped on the substrate S to form a liquid pool L1. can do.
  • coating process is performed similarly to Embodiment 1.
  • FIG. 9B first, the coating film L2 is formed by applying the microdroplet Lb of the crosslinking agent solution on the liquid pool L1 with the second inkjet head 32b.
  • the microdroplet Lc of the acidic buffer solution and the microdroplet Ld of the basic buffer solution are applied onto the coating film L2 by the third and fourth inkjet heads 33b and 34b.
  • the coating film L3 is formed.
  • the coating film L4 is formed by applying the minute droplets Le of the polymerization initiator solution on the coating film L3 with the fifth inkjet head 35b.
  • the coating process by the test device manufacturing apparatus T2 is performed in four processes by the inkjet apparatus 130 having a four-head configuration. Thereafter, the coating film L4 on the substrate S is gelled in the same manner as in the first embodiment, whereby the isoelectric focusing test shown in FIGS. 1 (A), 2 (A) and 2 (C) is performed. Tool GP 1 can be obtained. The process is completed in four steps by the five-head inkjet device 30.
  • FIG. 10 is a configuration diagram illustrating an apparatus for manufacturing an electrophoretic test device according to the third embodiment.
  • the same elements as those in FIG. 4 are denoted by the same reference numerals.
  • a manufacturing apparatus and a manufacturing method capable of manufacturing the isoelectric focusing test device GP 1 that is substantially the same as the first embodiment. Will be explained.
  • differences from the first and second embodiments in the third embodiment will be mainly described.
  • the test device manufacturing apparatus T3 of Embodiment 3 is substantially the same as the test device manufacturing apparatus T2 of Embodiment 2 (see FIG. 8). However, in the case of the third embodiment, the liquid ejected from the second inkjet head 32b is different from that of the second embodiment. That is, the inkjet device 230 of the test device manufacturing apparatus T3 of Embodiment 3 includes a mixed solution discharge unit 232, an acidic buffer discharge unit 33, a basic buffer discharge unit 34, a polymerization initiator discharge unit 35, and a negative pressure adjustment. Part 36. In the tank 32a of the mixed solution discharge unit 232, a mixed solution F in which a monomer and a crosslinking agent are dissolved in water at a predetermined concentration is stored.
  • the second ink jet head 32b is mixed on the base material S.
  • a coating film L1 ab is formed by applying a fine droplet Lab of the solution.
  • the application amount of the both side regions is larger than the application amount of the microdroplet Lab to the central region of the substrate S. Adjust so that there are more.
  • acidic buffer solution microdroplets Lc and basic buffer solution microdroplets Ld are applied onto the coating film L1 ab by the third and fourth inkjet heads 33b and 34b. Coating is performed to form a coating film L3. Subsequently, as shown in FIG. 11C, the coating film L4 is formed by applying the minute droplets Le of the polymerization initiator solution onto the coating film L3 with the fifth inkjet head 35b.
  • the coating process by the test tool manufacturing apparatus T3 is performed in three processes by the ink jet apparatus 130 having a four-head configuration. Thereafter, the coating film L4 on the substrate S is gelled in the same manner as in the first embodiment, whereby the isoelectric focusing test shown in FIGS. 1 (A), 2 (A) and 2 (C) is performed. Tool GP 1 can be obtained.
  • (Other embodiments) 1 the case where a coating film in a room temperature state is formed on the substrate in the coating process is illustrated. However, the coating is performed on the substrate under cooling using an apparatus including a Peltier element and a tank cooling unit. A film may be formed. In the first to third embodiments, the case where the coating film is formed in the air in the coating process is exemplified. However, the coating film may be formed in a nitrogen atmosphere.
  • each solution in the application process is not limited to the application order of the embodiment.
  • the monomer solution may be applied after the crosslinking material solution is applied.
  • the liquid pool of a crosslinking agent solution may be formed on the base material S, and a monomer solution may be apply
  • Embodiments 1 to 3 exemplify a case where the application of the monomer solution, the crosslinking agent solution or the mixed solution of the monomer and the crosslinking agent is performed only when the substrate S passes once under the ejection head units U1 and U2. However, the substrate S may be applied by moving it one or more times.

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PCT/JP2013/054478 2012-04-26 2013-02-22 Outil de test de focalisation isoélectrique et son procédé de production Ceased WO2013161368A1 (fr)

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JP2012-101351 2012-04-26
JP2012101351A JP2013228317A (ja) 2012-04-26 2012-04-26 等電点電気泳動用試験具およびその製造方法

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WO2020066860A1 (fr) * 2018-09-28 2020-04-02 パナソニックＩｐマネジメント株式会社 Dispositif de séparation d'échantillon et procédé de séparation d'échantillon

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0552808A (ja) * 1991-08-23 1993-03-02 Hitachi Ltd キヤピラリーゲル電気泳動用カラムとその製造方法及びそれを用いた電気泳動装置
JP2012002801A (ja) * 2011-05-12 2012-01-05 Sharp Corp ゲル固定用基材、電気泳動用反応器具、電気泳動用反応器具の製造方法及び電気泳動用キット

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0552808A (ja) * 1991-08-23 1993-03-02 Hitachi Ltd キヤピラリーゲル電気泳動用カラムとその製造方法及びそれを用いた電気泳動装置
JP2012002801A (ja) * 2011-05-12 2012-01-05 Sharp Corp ゲル固定用基材、電気泳動用反応器具、電気泳動用反応器具の製造方法及び電気泳動用キット

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