WO2014041908A1 - Outil de test d'électrophorèse et son procédé de fabrication - Google Patents

Outil de test d'électrophorèse et son procédé de fabrication Download PDF

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Publication number
WO2014041908A1
WO2014041908A1 PCT/JP2013/070460 JP2013070460W WO2014041908A1 WO 2014041908 A1 WO2014041908 A1 WO 2014041908A1 JP 2013070460 W JP2013070460 W JP 2013070460W WO 2014041908 A1 WO2014041908 A1 WO 2014041908A1
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Prior art keywords
region
buffer
buffer solution
gel
main region
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English (en)
Japanese (ja)
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政俊 中川
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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
    • 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 an electrophoresis test device and a method for manufacturing the same.
  • Electrophoresis is a separation analysis method that utilizes 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 medium. It is.
  • electrophoresis using gel as a medium is a technique for separating biopolymers such as proteins and nucleic acids, in the fields of life science such as biochemistry and molecular biology, and in the field of clinical testing. 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 pHs 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 the gel sheet with a predetermined width in the pH gradient direction. By pasting on the plate, a gel plate for isoelectric focusing is obtained.
  • Patent Document 2 discloses a gel plate manufacturing method (inkjet method) in which a monomer solution is applied onto a plate as a technique capable of accurately managing a pH gradient. That is, after forming a liquid pool on the substrate and discharging the monomer solution into the liquid pool, a gel layer is formed on the substrate by applying a polymerization initiator to gel the coating film. In this case, a gel plate for isoelectric focusing having a predetermined pH gradient can be obtained by applying two types of monomer solutions having different pHs to the pool while changing the coating amount.
  • FIG. 9 (A) is a conceptual diagram illustrating the electrophoresis test device and the manufacturing method of Patent Document 2
  • FIG. 9 (B) is a state immediately after applying the pH buffer solution by the method shown in FIG. 9 (A). It is a conceptual diagram explaining a state.
  • FIGS. 9A and 9B are composed of an upper stage, a middle stage, and a lower stage, respectively.
  • the upper part of FIGS. 9A and 9B shows the change in application amount of the acidic monomer solution and the basic monomer solution, which are two types of pH buffer solutions used at the time of gel production described later, in terms of area.
  • FIGS. 9A and 9B is a line graph showing the change in the coating amount of the acidic monomer solution (thin line) and the basic monomer solution (thick line).
  • the pH at the position of the gel layer in the electrophoresis direction is shown by a line graph.
  • the pH value increases linearly from one end S 1 to the other end S 2 of the substrate S so as to have a pH gradient as shown in the lower line graph of FIG. 9A.
  • the acidic monomer solution and the basic monomer solution are applied to the liquid pool while changing the coating amount with the inkjet head (see the upper and middle stages of FIG. 9A).
  • the coating start end region I on the one end S 1 side and the coating end region on the other end S 2 side of the base material S that particularly increases the amount of solution.
  • the liquid film shape cannot be maintained at a right angle, and the amount of the solution decreases. Therefore, as shown in the middle part of FIG.
  • impurities in the gel are attracted to one electrode side (positive electrode side or negative electrode side) and collected.
  • the gel end where the impurities accumulate is a portion that is inherently unsuitable for protein separation.
  • the “impurities” include residual compounds at the time of gel chip production (unreacted monomer compounds, polymerization agents, etc.), impurities contained in the evaluation protein sample, impurities in electrophoresis reagents (electrophoresis buffer), and the like.
  • the present invention has been made in view of such problems, and provides an electrophoresis test device capable of performing electrophoresis with high accuracy and high reliability, and a manufacturing method capable of easily manufacturing the test device.
  • the purpose is to do.
  • the present invention comprising a gel having a pH gradient from the acidic side to the basic side,
  • the gel includes a main region and a buffer region adjacent to the main region;
  • an electrophoretic test device in which the pH value of the buffer region is substantially constant (the pH gradient of the buffer region is gentler than the pH gradient of the main region).
  • a gel material application step for applying a gel material on a base material to form a liquid pool, and a pH buffer solution is applied to the liquid pool after the gel material application step.
  • a pH buffer solution coating step and a gelation step of gelling the coating film after the pH buffer solution coating step
  • the application region on the substrate is divided into a main region and a region where a buffer region adjacent to the main region is to be formed, In the region where the main region is to be formed, the pH buffer solution is applied while continuously and gently changing the coating amount per unit area of the pH buffer solution, In the region where the buffer region is to be formed, manufacture of a test device for electrophoresis in which the pH buffer solution is applied so that the rate of change of the coating amount per unit area of the pH buffer solution is lower than the rate of change in the main region A method is provided.
  • the gel in the electrophoretic test device of the present invention includes a main region having a linear pH gradient, and a buffer region adjacent to (preferably both) at least one of the acidic side and basic side ends of the main region.
  • the pH gradient of the buffer region is gentler than the pH gradient of the main region, and the pH value of the buffer region is substantially constant.
  • the main region has a linear pH gradient of pH 3 to 10
  • a buffer region constant at pH 3 is provided on the acidic side of the main region
  • a buffer region constant at pH 10 is provided on the basic side of the main region. be able to.
  • the pH gradient in the coating start region I and the coating end region II (that is, the pH gradient in the main region) is reversed.
  • at least one (preferably both) of the application start region I and the application end region II is intentionally used as a buffer region, so that the pH gradient is reversed at the end of the main region in the electrophoresis direction.
  • a linear pH gradient can be formed without forming a gradient.
  • the electrophoresis test device of the present invention is advantageous in that the gel end is unsuitable for protein separation because impurities in the gel accumulate during electrophoresis, and the length of the main region in the electrophoresis direction is convenient. Is not substantially shortened. Therefore, the electrophoresis test device of the present invention can perform highly accurate separation and measurement of proteins in the main region of the gel during electrophoresis, and can obtain highly reliable analysis results.
  • the pH gradient in the main region is not affected even if the solution application amount changes in the application start region I and the application end region II.
  • an electrophoretic test device including a gel capable of performing highly accurate protein separation measurement.
  • FIG. 2 is a conceptual diagram illustrating an electrophoresis test device and a manufacturing method thereof according to Embodiment 1. It is a conceptual diagram explaining the state immediately after apply
  • FIG. It is the schematic bottom view which looked at the inkjet head in the manufacturing apparatus of FIG. 3 from the downward direction.
  • FIG. 6 is a conceptual diagram illustrating an electrophoresis test device and a manufacturing method thereof according to Embodiment 2.
  • the electrophoresis test device of the present invention comprises a gel having a pH gradient from the acidic side to the basic side,
  • the gel includes a main region and a buffer region adjacent to the main region.
  • the pH gradient of the buffer region is gentler than the pH gradient of the main region.
  • the test device for electrophoresis of the present invention may be configured as follows. (1) The number of the main regions is not particularly limited, and may be one or plural.
  • the buffer region may be disposed adjacent to the acidic side end and the basic side end in the main region.
  • buffer regions are disposed at both ends in the electrophoresis direction of the gel, and a main region having a single continuous pH gradient is disposed between the buffer regions at both ends.
  • main regions buffer regions are arranged at both ends in the electrophoresis direction of the gel, a plurality of main regions having a pH gradient are arranged between the buffer regions at both ends, and also between two adjacent main regions.
  • a buffer area is arranged.
  • the pH gradient in the buffer region may be a half or less of the pH gradient in the main region.
  • the pH gradient of the main region may be a linear gradient.
  • 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 gel layer is formed by forming a puddle of a gel material solution on a base material, and applying a pH buffer solution on the puddle to cause gelation.
  • a monomer solution a gel material solution containing a polymerization initiator
  • a monomer solution polymerization in which a crosslinking agent other than the polymerization initiator, a polymerization accelerator, and the like are mixed are used.
  • gel material liquid means all of a gel material liquid to which a polymerization initiator has been added in advance, a gel material liquid not containing a polymerization initiator, and a polymerization initiator, unless otherwise specified.
  • a gel material solution to which a polymerization initiator has been added in advance may be referred to as “a gel material solution containing a polymerization initiator”
  • a gel material solution that does not include a polymerization initiator may be referred to as a “monomer solution”.
  • the electrophoretic test device of the present invention comprises a gel material application step for applying a gel material on a substrate to form a liquid pool, and a pH buffer for applying a pH buffer solution onto the liquid pool after the gel material application step.
  • the pH buffer solution application step the application region on the substrate is divided into a main region and a region where a buffer region adjacent to the main region is to be formed.
  • the pH buffer solution is applied while continuously and gently changing the coating amount per unit area of the pH buffer solution.
  • the pH buffer solution is applied so that the change rate of the coating amount per unit area of the pH buffer solution is lower than the change rate in the main region.
  • the method for applying the gel material liquid onto the substrate is not particularly limited, and any method can be used as long as the gel material liquid can be applied to a predetermined region on the upper surface of the substrate.
  • a pipetter, a dispenser, an inkjet device, etc. 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.
  • 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, the film thickness, the pH gradient, the concentration gradient, etc. Can be controlled easily and with high accuracy.
  • FIG. 1 (A) is a perspective view showing a usable state of the isoelectric focusing test device of Embodiment 1 of the present invention
  • FIG. 1 (B) is the isoelectric focusing of FIG. 1 (A). It is a perspective view which shows the state which can be preserve
  • FIG. 2A is a conceptual diagram illustrating the electrophoresis test device and the manufacturing method thereof according to Embodiment 1
  • FIG. 2B is a pH buffer solution applied by the method shown in FIG. It is a conceptual diagram explaining the state immediately after.
  • An electrophoretic test device GP 1 shown in FIG. 1A is obtained by forming a bowl-shaped gel layer G 1 on a substrate S.
  • the gel layer G 1 has a gently convex curved upper surface (surface opposite to the substrate) and a pH gradient in the longitudinal direction (hereinafter referred to as “X direction”) that is the electrophoresis direction. ing.
  • the direction orthogonal to the X direction is the width direction of the substrate S (hereinafter referred to as “Y direction”).
  • the gel layer G 1 is dried to a moisture content of 5% or less, the gel layer G 1 is dried to form a dry film D 1.
  • the isoelectric focusing test device GPD 1 shown in FIG. can get.
  • the length and width of the gel layer G 1 are the same as the length and width of the substrate S.
  • the length and width of the substrate S are not particularly limited, but as an example, the length is about 50 to 250 mm and the width is about 0.5 to 5 mm.
  • the thickness of the gel layer G 1 is not particularly limited, but is, for example, about 195 to 1010 ⁇ m.
  • the thickness of the dry film D 1 obtained by drying the gel layer G 1 shrinks to 100 ⁇ m or less, but the length and width hardly change.
  • FIGS. 2 (A) and 2 (B) are composed of an upper stage, a middle stage and a lower stage, respectively.
  • the upper part of FIGS. 2 (A) and 2 (B) shows the change in application amount of the acidic monomer solution and the basic monomer solution, which are two types of pH buffer solutions used at the time of gel production described later, in terms of area.
  • the middle part of FIGS. 2 (A) and 2 (B) shows a change in the coating amount of the acidic monomer solution (thin line) and the basic monomer solution (thick line) in a line graph.
  • the pH at the position of the gel layer in the electrophoresis direction is shown by a line graph.
  • the application region is divided into regions A to C from one end S 1 to the other end S 2 of the substrate S, and the application amount of the acidic monomer solution and the basic monomer solution is controlled according to each region.
  • the region B corresponds to a region where a main region described later is to be formed
  • the regions A and C correspond to regions where a buffer region described later is to be formed.
  • the acidic side and the basic side of the region B where the main region is to be formed are the regions A and C where the buffer region is to be formed.
  • the gel layer G 1 has a pH gradient in a predetermined pH range in the X direction (electrophoresis direction).
  • the gel layer G 1 has a main region having a pH gradient of pH 3 to 10, a pH 3 buffer region adjacent to the acidic side end of the main region, and a pH 10 adjacent to the basic side end of the main region. The case of having a buffer region is illustrated.
  • the characteristic structure of the gel layer G 1 of Embodiment 1 is that buffer regions are arranged on the acidic side and basic side of the pH region (main region) to be analyzed in detail as described above.
  • the pH of the buffer region is set to be equal to the pH at the end of the adjacent main region.
  • FIG. 3 is a configuration diagram showing an apparatus capable of manufacturing the electrophoresis test device of the first embodiment.
  • This test device manufacturing apparatus 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).
  • 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 third inkjet heads 31b, 32b, and 33b, which will be described later.
  • the ink jet device 30 includes an acidic solution discharge unit 31, a basic solution discharge unit 32, a polymerization initiator discharge unit 33, and a negative pressure adjustment unit 34.
  • the acidic solution discharge unit 31 includes a first tank 31a that stores the acidic monomer solution A, a first inkjet head 31b, and a first pipe 31c that sends the acidic monomer solution A from the first tank 31a to the first inkjet head 31b. And the acidic monomer solution A is supplied from the first tank 31a to the first inkjet head 31b using the water head difference.
  • the acidic monomer solution A is set to a predetermined pH (for example, pH 2 to 7) by one or more pH buffers.
  • the basic solution discharge unit 32 includes a second tank 32a that stores the basic solution B, a second inkjet head 32b, and a second pipe 32c that sends the basic solution B from the second tank 32a to the second inkjet head 32b. And the basic monomer solution B is supplied from the second tank 32a to the second inkjet head 32b using the water head difference.
  • the basic monomer solution B is set to a predetermined pH (for example, pH 7 to 12) by one or more kinds of pH buffers.
  • the polymerization initiator discharge unit 33 includes a third tank 33a that stores the polymerization initiator C, a third inkjet head 33b, and a third pipe 33c that sends the polymerization initiator C from the third tank 33a to the third inkjet head 33b. And the polymerization initiator C is supplied from the third tank 33a to the third inkjet head 33b using the water head difference.
  • Examples of the first to third ink jet heads 31b to 33b include a thermal jet method, a piezo jet method, an electrostatic drive method, and the like, but each liquid (acidic monomer solution A, basic monomer solution B, polymerization in the ink jet device 30).
  • a thermal jet method a piezo jet method, an electrostatic drive method, and the like
  • each liquid acidic monomer solution A, basic monomer solution B, polymerization in the ink jet device 30.
  • the initiator C is cooled, it is desirable to use a piezo jet method or an electrostatic drive method without using a thermal jet method for applying heat to each liquid.
  • the negative pressure adjusting unit 34 is connected to the first to third tanks 31a to 33a by pipes 35 to 37, manages the atmospheric pressure in the first to third tanks, and controls the first to third inkjet heads 31b.
  • the insides of the first to third tanks 31a to 33a 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. 5).
  • the first to third ink jet heads 31b to 33b are integrated to form a set of discharge head units U, which are fixed by a fixing member (not shown). As shown in FIG. 4, the first to third ink jet heads 31b to 33b are arranged in a line on the movement locus E of the base material S, but the head arrangement order is not limited to this order. In the first embodiment, the first to third inkjet heads 31b to 33b 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 third inkjet heads 31b to 33b 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 liquid pool L0 is formed in advance.
  • a monomer solution obtained by diluting a monomer with pure water is used, and a crosslinking agent and a polymerization accelerator may be added to the monomer solution.
  • a coating process under normal temperature and atmospheric pressure based on a predetermined program is performed. That is, as shown in FIGS. 6A and 6B and FIGS. 7A and 7B, the support 40a is intermittently moved in the direction of the arrow M by the moving mechanism 40, and the first to third Minute droplets La, Lb, and Lc are intermittently ejected from the inkjet heads 31b to 33b, and a coating film L3 is formed on the liquid pool L0.
  • 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 coating amount per unit area of the fine droplets La discharged from the first inkjet head 31b onto the substrate S is changed every time the substrate S moves intermittently by a predetermined distance (at predetermined discharge intervals). The amount is controlled to a predetermined amount, which will be described in detail later.
  • the coating amount per unit area of the micro droplets Lb ejected from the second inkjet head 32b is a predetermined amount every time the substrate S is intermittently moved by a predetermined distance (at a predetermined ejection interval). This will be described in detail later.
  • the coating film L2 and the polymerization initiator mixture coating film with L3 (FIG. 7 (B) refer) is formed at one end S 1 side of the substrate S, one end S 1 of the thus substrate S continuously coated film L3 toward the other end S 2 side is gradually formed from the side.
  • the other end S 2 of the substrate S sequentially passes through the first to third inkjet heads 31b to 33b, as shown in FIG. 7B, the first to third inkjet heads 31b to 33b Droplet discharge stops sequentially.
  • the application amount control of the acidic monomer solution and the basic monomer solution will be described with reference to FIGS. 2 (A) and 2 (B).
  • the acidic monomer solution is controlled so that the coating amount decreases as it goes from one end S 1 of the substrate S to the other end S 2
  • the basic monomer solution is one end S 1 of the substrate S.
  • the coating amount is controlled to increase from the other end S 2 toward the other end S 2 . More specifically, in the region A on the substrate S, the application amount of the acidic monomer solution is constant at 100%, and the application amount of the basic monomer solution is constant at 0%.
  • the application amount of the acidic monomer solution continuously decreases in the range of 100 to 0%, and the application amount of the basic monomer solution continuously increases in the range of 0 to 100%.
  • the application amount of the acidic monomer solution is constant at 0%, and the application amount of the basic monomer solution is constant at 100%.
  • the pH buffer solution is applied while continuously and gently changing the coating amount per unit area of the pH buffer solution. Further, in the regions A and C where the buffer region is to be formed, the pH buffer solution is applied so that the change rate of the coating amount per unit area of the pH buffer solution is lower than the change rate in the main region.
  • the rate of change of the coating amount per unit area of the pH buffer solution means the degree of pH gradient.
  • the coating film immediately after the acidic and basic monomer solution is applied by such coating amount control is applied to the regions A and C in which the buffer regions are to be formed, as shown in the upper and middle parts of FIG. Although the shape collapses from the right-angle shape, the pH values of A and C in these regions are not changed.
  • coating process is performed when a control part controls each drive part based on a predetermined program. Thereafter, the support base 40a returns to the standby position, and the coating process is completed.
  • the door of the case 50 is opened, the base material S is taken out and stored in the case for the gelation process, and the gelation process of the coating film L3 is performed at room temperature in the case. Note that it takes about 3 to 5 hours to complete the gelation at room temperature.
  • the isoelectric focusing test device GP 1 in which a bowl-shaped gel layer G 1 having rounded corners on the four ends is formed on the substrate S as shown in FIG. 1 (A). Is obtained.
  • the isoelectric focusing test device GP 1 is an incomplete product.
  • the electrophoretic test device GPD 1 of the present invention as a finished product 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. 8A is a conceptual diagram illustrating the electrophoresis test device and the manufacturing method thereof according to Embodiment 2
  • FIG. 8B is a diagram immediately after applying a pH buffer solution by the method shown in FIG. It is a conceptual diagram explaining a state.
  • FIGS. 8A and 8B are composed of an upper stage, a middle stage, and a lower stage, respectively.
  • the upper part of FIGS. 8A and 8B shows, in terms of area, changes in the coating amounts of an acidic monomer solution and a basic monomer solution, which are two types of pH buffer solutions used during gel production described later.
  • FIGS. 8A and 8B shows a change in the coating amount of the acidic monomer solution (thin line) and the basic monomer solution (thick line) in a line graph.
  • the lower part of FIGS. 8A and 8B shows the pH at the position of the gel layer in the electrophoresis direction as a line graph.
  • the gel in the electrophoresis test device of the second embodiment is the same as that of the first embodiment except that the gel has a plurality of main regions and a buffer region disposed between two adjacent main regions.
  • the gel has an acidic buffer region (pH 3), a basic buffer region (pH 10), and a first main region (pH 3 to 5.5) disposed between the buffer regions on both ends. ), The second main region (pH 5.5 to 8.5) and the third main region (pH 8.5 to 10), and the buffer region (pH 5.5) disposed between the first main region and the second main region. ) And a buffer region (pH 8.5) disposed between the second main region and the third main region.
  • Embodiment 3 In Embodiments 1 and 2, the case where a monomer is thermally polymerized and gelled by using a thermal polymerization initiator such as ammonium persulfate (APS) or benzoyl peroxide as a polymerization initiator is exemplified.
  • APS ammonium persulfate
  • benzoyl peroxide As a polymerization initiator is exemplified.
  • photopolymerization initiators such as riboflavins, acetophenones such as 2,2-dimethoxy-2-phenylacetophenone, benzophenones, and azoisobutyronitrile are used as polymerization initiators. Polymerize to gel.
  • a light irradiator (not shown) is provided on the downstream side in the substrate transport direction (arrow M1 direction) of the ejection head unit U.
  • the coating film on the material S is gelled by irradiating light.
  • This light irradiator can irradiate light having a predetermined wavelength (for example, about 200 to 600 nm) with a predetermined light amount, and the irradiation light wavelength is appropriately set according to the type of the photopolymerization initiator to be used. Further, the light irradiator only needs to irradiate a part or the whole of the coating film on the substrate.
  • a coating film is formed on a substrate in the same manner as in the first embodiment (description in FIGS. 6A to 7B), and then the substrate is optically irradiated. Move to the irradiator side and irradiate the coating film with light to gel. At this time, when the light irradiator is of a type that irradiates a part of the coating film, the coating film is irradiated with light while moving the substrate from one end side to the other end side. When the light irradiator is of a type that irradiates light to the entire coating film, the substrate is moved directly below the light irradiator, and then the light is irradiated onto the coating film while the substrate is stationary.
  • (Other embodiments) 1 the case where a coating film in a room temperature state is formed on the base material in the coating step is exemplified. However, the coating film is formed on the base material under cooling using an apparatus including a Peltier element and a tank cooling unit. It may be formed. In the first embodiment, the case where the coating film is formed in the air in the coating process is illustrated, but the coating film may be formed in a nitrogen atmosphere.
  • the case where the monomer solution and the polymerization initiator are individually applied onto the substrate is exemplified, but the gel material solution containing the polymerization initiator may be applied onto the substrate to form the liquid pool L0. .
  • a gel material solution containing a polymerization initiator in a cooled state is used so that gelation of the gel material solution containing a polymerization initiator does not proceed during the coating process.
  • the substrate may be cooled.
  • the coating film L3 is formed by passing the substrate S once under the discharge head unit U is illustrated.
  • the coating film L3 is formed by moving the substrate S one or more times. May be.
  • the application timing of the polymerization initiator can be set, for example, at every movement, at a predetermined movement, or at the last movement.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Molecular Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Electrochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Application Of Or Painting With Fluid Materials (AREA)

Abstract

L'invention concerne un outil de test d'électrophorèse qui permet une électrophorèse à précision élevée et fiabilité élevée. L'outil de test d'électrophorèse est caractérisé en ce qu'il comprend un gel ayant un gradient de pH du côté acide vers le côté basique, et en ce que le gel comprend une région principale et des régions tampon adjacentes à la région principale, et les valeurs de pH des régions tampon sont sensiblement constantes.
PCT/JP2013/070460 2012-09-14 2013-07-29 Outil de test d'électrophorèse et son procédé de fabrication Ceased WO2014041908A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2012202816A JP2014059160A (ja) 2012-09-14 2012-09-14 電気泳動用試験具およびその製造方法
JP2012-202816 2012-09-14

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WO2014041908A1 true WO2014041908A1 (fr) 2014-03-20

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JP (1) JP2014059160A (fr)
WO (1) WO2014041908A1 (fr)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH01100448A (ja) * 1987-10-13 1989-04-18 Shimadzu Corp 等電点電気泳動用ゲル媒体
JP2003514829A (ja) * 1999-11-15 2003-04-22 プロテオム システムズ リミテッド 多室電気泳動
JP2003529762A (ja) * 2000-04-03 2003-10-07 ザ ウィスター インスティテュート 溶液等電点電気泳動による荷電分子解析の方法および装置
JP2009539101A (ja) * 2006-06-02 2009-11-12 コーニンクレッカ フィリップス エレクトロニクス エヌ ヴィ 等電点電気泳動による分離装置

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH01100448A (ja) * 1987-10-13 1989-04-18 Shimadzu Corp 等電点電気泳動用ゲル媒体
JP2003514829A (ja) * 1999-11-15 2003-04-22 プロテオム システムズ リミテッド 多室電気泳動
JP2003529762A (ja) * 2000-04-03 2003-10-07 ザ ウィスター インスティテュート 溶液等電点電気泳動による荷電分子解析の方法および装置
JP2009539101A (ja) * 2006-06-02 2009-11-12 コーニンクレッカ フィリップス エレクトロニクス エヌ ヴィ 等電点電気泳動による分離装置

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