JP2012002801A - Substrate for fixing gel, reaction instrument for electrophoresis, method for manufacturing reaction instrument for electrophoresis, and kit for electrophoresis - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a substrate for fixing gel that can form gel with high reliability and in a good yield.SOLUTION: A substrate 1 for fixing gel in this invention is a substrate for fixing gel which fixes gel 3 for electrophoresis to the substrate. And the substrate 1 for fixing the gel has a gel attachment region 2 treated so that the gel 3 is attached on at least a part of a surface where the gel 3 is to be fixed.

Description

  The present invention relates to a gel-fixing substrate, an electrophoresis reaction instrument, a method for producing an electrophoresis reaction instrument, and an electrophoresis kit, and more specifically, for electrophoresis for separating a biological sample. The present invention relates to a gel fixing substrate for fixing a gel, an electrophoresis reaction instrument, a method for producing an electrophoresis reaction instrument, and an electrophoresis kit.

  Electrophoresis is a phenomenon in which charged particles or molecules move an electric field, and is particularly important as a technique for separating DNA or proteins in the fields of molecular biology and biochemistry.

  In recent years, proteosome analysis has attracted attention as a post-genome. This proteosome analysis refers to a large-scale study on the structure and function of a protein, and in order to analyze the proteosome, usually a protein sample is first separated into individual proteins. At this time, two-dimensional electrophoresis is widely used as one method for separating proteins.

  Two-dimensional electrophoresis is a technique for two-dimensionally separating proteins by two-stage electrophoresis. For example, in the first dimension, proteins are separated by isoelectric focusing (IEF), and in the second dimension, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Is done. Such two-dimensional electrophoresis has a very high resolution and can separate several thousand kinds of proteins into spots.

  In the first-dimensional IEF, for example, an immobilized pH gradient (IPG) method having excellent reproducibility and resolution is used. In the immobilized pH gradient method, an immobilized pH gradient gel (IPG gel) is used.

  In the second-dimensional SDS-PAGE, for example, an agarose gel or a polyacrylamide gel is used as the SDS-PAGE gel. In particular, as a polyacrylamide gel, a homogeneous gel with a uniform acrylamide solution is used in many cases, but when observing a wide range of molecular weight, the concentration of the acrylamide solution is gradient from higher to lower. Diendogel is used.

  These IPG gels and SDS-PAGE gels are, for example, coated on plastic or glass, or by casting the gel solution into a mold (for example, a mold between glass substrates opposed via a spacer). Formed and used for first-dimensional electrophoresis and second-dimensional electrophoresis.

  Recently, the frequency of use of gel electrophoresis in animal and plant genome analysis and the like has increased dramatically, and a technique for producing a highly productive and homogeneous gel plate is desired. In response to such demands, an electrophoresis gel plate using an inkjet method and a method for producing the same have been developed (Patent Document 1).

JP 2004-77393 A (published March 11, 2004)

  However, the gel plate of Patent Document 1 has poor wettability between the plate and the gel solution. For this reason, even if the gel solution is ejected from the inkjet head onto the plate, the gel may not be formed in a desired region, resulting in a decrease in yield.

  Further, when a gel having a concentration gradient is produced by the method of Patent Document 1, since the wettability between the plate and the gel solution is poor, mixing of a plurality of minute droplets ejected from the inkjet head is sufficiently performed. Absent.

  This invention is made | formed in view of said subject, The objective is to provide the base material for gel fixation which can form a gel with high reliability and sufficient yield.

  The gel fixing substrate according to the present invention is a gel fixing substrate for fixing a gel for electrophoresis in order to solve the above-described problems, and is provided on at least a part of a surface for fixing the gel. , And having a gel adhesion region that has been subjected to a treatment for adhering the gel.

  In order to solve the above-described problems, the electrophoresis reaction instrument according to the present invention is characterized in that an electrophoresis gel is fixed to the gel fixing base material according to the present invention.

  According to said structure, the process for making a gel adhere is given to the base material for gel fixation of this invention. This treatment can be applied to a desired region of the fixing substrate in a desired shape, and the gel solution discharged to the fixing substrate is preferentially fixed to the gel adhesion region subjected to the treatment. Is done.

  As described above, since the gel can be aligned with the fixing substrate with high accuracy, the gel can be formed with high reliability and high yield.

  In particular, according to the method for producing an electrophoresis reaction instrument according to the present invention described later, a liquid pool can be formed in the gel adhesion region by discharging the liquid to the gel adhesion region of the gel fixing base material. . By further discharging the gel solution into the liquid reservoir, the gel solutions discharged as droplets can be sufficiently mixed.

  Moreover, in the base material for gel fixation which concerns on this invention, it is preferable that the said gel adhesion area | region is formed in concave shape or convex shape.

  According to said structure, the gel adhesion area | region has the structure formed in concave shape or convex shape with respect to the surface of the base material for gel fixation. By forming these structures, it is possible to control the formation position of the gel and improve the alignment of the gel with respect to the fixing substrate.

  Moreover, in the base material for gel fixation which concerns on this invention, it is preferable that the some uneven structure is formed in the said gel adhesion area | region.

  Thus, wettability can be controlled by forming a plurality of concavo-convex structures in the gel adhesion region on the gel fixing substrate. In particular, by forming a concavo-convex structure in a minute region, it is possible to efficiently form a gel in the region.

  Moreover, in the base material for gel fixation which concerns on this invention, at least one part of the said gel adhesion area | region has hydrophilicity, and at least one part of areas other than the said surface and said gel adhesion area | region has hydrophobicity. It is preferable.

  For example, a region that becomes a gel adhesion region on the surface of a hydrophobic material such as a plastic substrate is made hydrophilic by hydrophilic treatment such as oxygen plasma treatment, or a region other than the gel adhesion region on the surface of a hydrophilic material such as a glass substrate is treated with a silane cup. It is made hydrophobic by a hydrophobic treatment such as a ring treatment.

  In this way, by patterning the surface of the gel fixing substrate to have a hydrophilic region and a hydrophobic region, the wettability between the contact surface of the gel fixing substrate and the gel in the gel adhesion region is improved. Well, a gel with a desired shape can be formed.

  Moreover, in the base material for gel fixation which concerns on this invention, it is preferable that the area | region which has the hydrophilic property of the said gel adhesion area | region is a composition containing an oxygen containing functional group.

  According to said structure, the base material for gel fixation with the better wettability of the contact surface of a base material for gel fixation and a gel can be provided.

  Moreover, in the electrophoresis reaction instrument according to the present invention, the gel is preferably formed so as to have a gradient of gel concentration or pH.

  For example, when preparing a gel having a pH gradient such as an immobilized pH gradient gel, or a gel having a gel concentration gradient such as a gradient gel, the composition, concentration and the like must be sufficiently controlled. According to the present invention, since the gel adhesion region is subjected to a surface modification treatment, the surface of the gel adhesion region of the substrate and the gel solution have good wettability when the gel is produced, and the position for fixing the gel is easy. Can be controlled.

  Therefore, it is possible to form a gel having an arbitrary composition and concentration in an arbitrary size at an arbitrary place, and suitably forming a gel having a pH or gel concentration gradient such as an IPG gel or a gradient gel. can do.

  The method for producing an electrophoresis reaction instrument according to the present invention is a method for producing an electrophoresis reaction instrument according to the present invention, wherein the first ejection step of ejecting a liquid to the gel adhesion region and the first After the discharging step, the method includes a second discharging step of discharging the gel solution to the gel adhesion region.

  For example, when a gel solution is discharged onto a gel fixing substrate, the wettability between the substrate and the gel solution may be poor, and the discharged gel solution droplets may not be sufficiently mixed. Therefore, the liquid droplets are preliminarily ejected to the gel adhesion region to form a liquid pool, so that the coupling between the micro droplets is likely to occur. Therefore, the gel solution can be sufficiently mixed.

  For example, if the liquid is a gel solution containing a reagent related to gel formation, the gel solution is discharged in multiple stages. In this case, it is possible to control the gelation time, and for example, it is possible to prevent the malfunction of the apparatus such as the gelation reaction proceeding unnecessarily and the piping clogging.

  In the method for producing a reaction device for electrophoresis according to the present invention, it is preferable that the gel solution is discharged using an inkjet head in the second discharge step.

  According to said structure, it is easy to control a gel density | concentration and a formation area by using the inkjet head which can discharge a gel solution with a micro droplet.

  For example, when producing an IPG gel or a gradient gel, a high-definition gray scale (gradient) can be produced by discharging a gel solution using an inkjet head. Therefore, a high-performance IPG gel or SDS-PAGE gradient gel can be provided.

  Moreover, it is preferable that the manufacturing method of the electrophoresis reaction instrument according to the present invention includes a gel adhesion region forming step of forming the gel adhesion region by oxygen plasma treatment before the first discharge step.

  According to said structure, since the wettability of a gel adhesion area | region can be improved before discharging a liquid, the discharged liquid can form a liquid pool in a desired area | region.

  In addition, for example, by performing oxygen plasma treatment using a plasma shielding mask, it is possible to form a region having a composition containing a large amount of oxygen-containing functional groups at a desired location, which greatly contributes to improvement in gel position reproducibility. To do.

  Moreover, it is preferable that the manufacturing method of the reaction instrument for electrophoresis according to the present invention includes a third discharge step of discharging a polymerization initiator to the gel adhesion region after the second discharge step.

  According to said structure, discharge of the polymerization initiator which starts superposition | polymerization of gel is performed after discharge of a gel solution. This suppresses gelation of the gel solution in the preparation jig or gel preparation apparatus, and prevents problems such as clogging of pipes that are unnecessarily gelled in the gel preparation jig or gel preparation apparatus. Can do.

  In order to solve the above-described problems, the electrophoresis kit according to the present invention includes the gel fixing base material according to the present invention.

  The base material for gel fixation of the present invention is a base material for gel fixing for fixing a gel for electrophoresis, and a treatment for attaching the gel to at least a part of a surface for fixing the gel is performed. Since the applied gel adhesion region is provided, the gel can be formed with high reliability and high yield.

  Moreover, the manufacturing method of the reaction instrument for electrophoresis of this invention discharges a gel solution to the said gel adhesion area | region after the 1st discharge process which discharges a liquid to the said gel adhesion area | region, and the said 1st discharge process. Since the second ejection step is included, a gel that is suitably used for electrophoresis can be formed.

FIG. 1A is a perspective view showing a configuration of an electrophoresis reaction instrument according to an embodiment of the present invention, and FIG. 1B is an electrophoresis reaction according to an embodiment of the present invention. It is sectional drawing which shows the structure of an instrument. It is a perspective view which shows the other structure of the gel adhesion area | region in the reaction instrument for electrophoresis shown in FIG. It is a perspective view which shows the other structure of the gel adhesion area | region in the reaction instrument for electrophoresis shown in FIG. It is a perspective view which shows the other structure of the gel adhesion area | region in the reaction instrument for electrophoresis shown in FIG. It is a perspective view which shows the other structure of the gel adhesion area | region in the reaction instrument for electrophoresis shown in FIG. It is sectional drawing which shows the flow at the time of manufacturing the reaction instrument for electrophoresis which concerns on one Embodiment of this invention. It is a perspective view which shows the flow at the time of manufacturing the reaction instrument for electrophoresis which concerns on one Embodiment of this invention. It is a perspective view which shows the flow at the time of manufacturing the reaction instrument for electrophoresis which concerns on one Embodiment of this invention.

  Hereinafter, an embodiment of the present invention will be described with reference to FIGS.

  First, the structure of the gel plate 10 (electrophoretic reaction instrument) according to the present embodiment will be described with reference to FIGS.

(Configuration of gel plate 10)
FIG. 1A is a perspective view showing a configuration of a gel plate 10 according to an embodiment of the present invention, and FIG. 1B shows a configuration of the gel plate 10 according to an embodiment of the present invention. It is sectional drawing shown.

  The gel plate 10 of the present embodiment is a plate on which a gel, which is a support for separating each component of a sample in electrophoresis, is fixed. As shown in FIG. 1, the gel plate 10 is used for fixing an electrophoresis gel. An electrophoresis gel 3 is fixed to a substrate 1 (gel fixing base material).

  Electrophoresis is a method for separating biopolymers such as proteins, DNA, and RNA using differences in moving speed in an electric field due to differences in size or charge. Electrophoresis includes a method of moving a biopolymer in a support such as a gel, and includes, for example, polyacrylamide gel electrophoresis or agarose gel electrophoresis.

  The electrophoresis reaction instrument of the present invention is obtained by, for example, further isolating the protein separated by isoelectric focusing (IEF) from sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). ) Can be suitably used for two-dimensional electrophoresis.

  The shape of the electrophoresis reaction tool is not limited to a flat plate as shown in the present embodiment, and may be, for example, a chip molded into a desired shape.

  The substrate 1 has a gel adhesion region 2 on which treatment for attaching the gel 3 is performed on at least a part of the surface on which the gel 3 is fixed.

  In the present embodiment, the gel adhesion region 2 is provided in a frame shape in the vicinity of the outer periphery of the upper surface of the substrate 1. Have By providing such a gel adhesion region 2 on the substrate 1, the gel 3 can be suitably controlled so that the gel 3 is formed above the gel adhesion region 2.

  However, the configuration of the gel adhesion region 2 is not limited to this. For example, a concave or convex structure may be formed in an arbitrary region on the upper surface of the substrate 1, and the properties of the surface of the substrate 1 may be changed. A chemically modifying treatment may be performed. Moreover, these structures can be combined.

  Although details of the gel adhesion region 2 will be described later, since the gel solution is fixed to the gel adhesion region 2 when the gel 3 is formed by forming the gel adhesion region 2 on the substrate 1, the gel 3 The region to be formed can be controlled.

  Further, when the surface modification treatment or the like is performed, the wettability between the surface of the gel adhesion region 2 and the gel solution in the substrate 1 is improved, so that the gel solutions discharged to the gel adhesion region 2 are suitably mixed and desired. The gel 3 having the position and size of can be formed.

  Further, according to the method of manufacturing the gel plate 10 described later, since a liquid pool can be formed by discharging a liquid to the gel adhesion region 2, even if a gel solution is discharged into the liquid pool with a minute droplet. The gel solutions can be sufficiently mixed to form a high quality gel.

  Examples of the substrate 1 include a substrate made of glass, resin, ceramics, or the like. Examples of the glass substrate include a quartz glass substrate and a non-alkali glass substrate. Examples of the resin substrate include a polyethylene terephthalate (PET) substrate, a polymethyl methacrylate resin (PMMA) substrate, and the like. Examples of the ceramic substrate include an alumina substrate and a low-temperature co-fired ceramic substrate.

  The gel 3 is a support that separates biopolymers such as proteins by electrophoresis, and examples thereof include polyacrylamide gel and agarose gel. According to the gel plate 10 of this embodiment, since the formation region of the gel 3 is controlled by the gel adhesion region 2, the gel 3 can be formed at a desired position with good reproducibility.

  The thickness of the gel 3 is not particularly limited. For example, the gel adhesion region 2 shown in FIG. 1B has a thickness of several nanometers to several hundred micrometers with respect to the surface of the substrate 1. In this case, it is desirable to be about several hundred millimeters to several millimeters. If the thickness of the gel 3 formed with respect to the thickness of the gel adhesion region 2 is within this range, it can be optimally used for electrophoresis experiments.

  In the gel plate 10, it is easy to form a gel 3 having a gel concentration or pH gradient, such as an immobilized pH gradient (IPG) gel or a gradient gel.

  An IPG gel is a gel used for isoelectric focusing and has a gradient with respect to pH. The gradient gel is a gel used for SDS-PAGE and has a gradient with respect to the acrylamide concentration.

  When producing these gels, it is necessary to sufficiently control the pH or the gel concentration. When the gel solution is discharged onto the substrate 1, it is often discharged as fine droplets. Therefore, by performing a desired process for improving the wettability with respect to the gel adhesion region 2, minute droplets of the gel solution discharged onto the substrate 1 are suitably mixed.

  Therefore, a gel having a pH gradient or a gel concentration gradient such as an IPG gel or a gradient gel can be suitably formed.

(Configuration of gel adhesion region 2)
Here, the structure of the gel adhesion area | region 2 formed in the board | substrate 1 is further demonstrated with reference to FIGS. FIGS. 2-5 is a perspective view which shows the other structure of the gel adhesion area | region 2 in the gel plate 10 shown in FIG.

  As described above, the gel adhesion region 2 may have, for example, a concave or convex structure formed in an arbitrary region on the upper surface of the substrate 1, or a process for chemically modifying the properties of the surface of the substrate 1. May be given.

  For example, the substrate 1 shown in FIG. 2 is provided with a concave structure (hollow structure) having a desired pattern at the center of the upper surface. This hollow structure may have a depth of several micrometers to several hundred micrometers, for example, but may be appropriately set according to the thickness of the substrate 1.

  What is necessary is just to select according to the material of the board | substrate 1 as a manufacturing method of a hollow structure. For example, in the case of a glass substrate, photolithography, that is, a region other than the desired region to be the gel adhesion region 2 is masked with a photoresist mask, and the desired region can be etched to produce a punched structure. For example, if it is a resin substrate, a hollow structure can be produced by cutting or injection molding.

  As another configuration of the gel adhesion region 2, for example, the substrate 1 shown in FIG. 3 is provided with a convex structure (protruding structure) having a desired pattern at the center of the upper surface opposite to the substrate 1 shown in FIG. . The protruding structure may have a depth of several micrometers to several hundred micrometers, for example, but may be appropriately set according to the thickness of the substrate 1.

  As a method for producing the protruding structure, it can be produced by a method similar to the above-described hollow structure. For example, when the substrate 1 is a glass substrate, a desired region that becomes the gel adhesion region 2 is masked with a photoresist mask, and a protruding structure can be manufactured by etching other than the desired region.

  If a concave structure or a convex structure is formed in the gel adhesion region 2, a gel or a liquid pool described later can be successfully formed in the gel adhesion region 2 by the action of surface tension.

  Further, the gel adhesion region 2 may have a plurality of concavo-convex structures formed therein. By forming a plurality of concavo-convex structures, the surface area of the gel adhesion region 2 can be increased and wettability can be improved. The uneven structure is preferably fine. For example, in the substrate 1 shown in FIG. 4, a fine uneven structure 4 is formed inside the gel adhesion region 2.

  For example, when the gel solution is ejected to the substrate 1 with fine droplets, if the wettability between the gel solution and the substrate 1 is poor, the ejected droplets are not sufficiently mixed together, resulting in a gel with poor electrophoretic properties. Become. On the other hand, in the region where the fine uneven structure 4 shown in FIG. 4 is formed, the wettability can be controlled by this structure.

  That is, since the wettability is good in the minute region where the uneven structure 4 is formed, the droplets discharged to the region are likely to be combined. Therefore, the gel 3 having a desired shape can be efficiently formed.

  The concavo-convex structure 4 may have a depth or thickness of several nanometers to several tens of nanometers, for example, and can be suitably produced by using a generally known nanoimprint technique. .

  In the example shown in FIG. 4, the concavo-convex structure 4 is formed directly on the plane of the substrate 1, but the present invention is not limited to this. For example, the gel adhesion region 2 having the configuration shown in FIG. 2 or FIG. The concavo-convex structure 4 may be formed in the inside.

  On the other hand, in the substrate 1, it is preferable that at least a part of the gel adhesion region 2 has hydrophilicity and at least a part of the region other than the gel adhesion region 2 has hydrophobicity. For example, it is desirable that the gel adhesion region 2 of the substrate 1 shown in FIG. 5 includes a hydrophilic region having hydrophilicity and the other surfaces of the substrate 1 have hydrophobicity. Thereby, since the wettability of the gel adhesion area | region 2 improves, the gel 3 can be formed with sufficient position reproducibility.

  As a method of forming a hydrophilic region in the gel adhesion region 2, a different process is performed depending on whether at least the surface of the substrate 1 on which the gel adhesion region 2 is formed is made of a hydrophilic material or a hydrophobic material. Is possible.

  For example, if the gel-forming surface of the substrate 1 is made of a hydrophobic material, the gel adheres by hydrophilic treatment such as nitration using sulfuric acid, sulfonation using nitric acid, introduction of oxygen-containing functional groups using oxygen plasma treatment, etc. A hydrophilic region can be formed inside the region 2.

  In particular, oxygen plasma treatment is preferably used as the hydrophilic treatment. By using this method, it is possible to easily form a hydrophilic region on the substrate 1 having at least the formation surface of the gel adhesion region 2 made of a hydrophobic material.

  For example, when the gel forming surface of the substrate 1 is made of a hydrophilic material, an appropriate hydrophobizing process may be performed according to the material of the substrate 1. For example, when the substrate 1 is a glass substrate, a region that becomes a hydrophilic region is masked with a Kapton tape or the like, and a region other than the region is hydrophobized by treatment with a silane coupling agent. Further, for example, the hydrophilic region can be obtained by subjecting the glass substrate to a hydrophobization treatment with a photodegradable silane coupling agent and then irradiating the portion that becomes the hydrophilic region with ultraviolet light. Thereby, a hydrophilic region and a hydrophobic region are formed on the substrate 1.

  For example, when the substrate 1 is a silicone substrate, a region to be a hydrophilic region may be masked with a natural oxide film, and a region other than the region may be hydrophobized by wet etching with dilute hydrofluoric acid. After washing with dilute hydrofluoric acid, the region other than the part may be masked and then oxidized. Also by this method, a hydrophilic region and a hydrophobic region are formed on the substrate 1.

  Moreover, it is preferable that a hydrophilic area | region is a composition containing many oxygen containing functional groups. In this case, for example, an organic resin having an oxygen-containing functional group may be used as the substrate 1 or a commercially available organic resin may be hydrophilized and used as the substrate 1. If the hydrophilic region is a composition containing many oxygen-containing functional groups, the wettability is even better.

  In this manner, by forming the hydrophilic region and the hydrophobic region on the substrate 1 by the chemical surface modification treatment, the gel solution is patterned with good position reproducibility using the wettability related to the hydrophilicity / hydrophobicity. be able to.

  In addition, you may perform the surface modification process of the board | substrate 1 with respect to the board | substrate 1 of the structure shown to FIGS.

(Manufacturing method of the gel plate 10)
Next, the manufacturing method of the gel plate 10 of this embodiment is demonstrated with reference to FIG. FIG. 6 is a cross-sectional view showing a flow when manufacturing the gel plate 10 according to one embodiment of the present invention.

  In the present embodiment, a method for producing a gel plate 10 on which 4% polyacrylamide gel, which is one of typical electrophoresis gels, is formed will be described.

  As a reagent for forming a 4% polyacrylamide gel, for example, a 30% acrylamide mixed solution (acrylamide + N, N′-methylenebisacrylamide), 1M Tris-HCl buffer (Tris-HCl), ammonium persulfate (APS; ammoniumpresulfate) ), Tetramethylethylenediamine (TEMED; N, N, N ′, N′-tetramethylethylenediamine), and pure water.

  The acrylamide mixed solution is a gel solution in which acrylamide that forms the main skeleton of the gel and N, N′-methylenebisacrylamide that crosslinks the main skeleton of the gel are mixed, Tris-HCl buffer is a buffer, and APS is It is a polymerization initiator and TEMED is a polymerization accelerator.

  In the present embodiment, a configuration in which the reagents for forming these gels are discharged in three stages will be described, but the method for manufacturing the gel plate 10 is not limited to this. For example, the number of times of discharging the solution is not limited to this, and it may be two stages of a first discharge process for discharging a liquid and a second discharge process for discharging a gel solution. As described in 1, it may be formed in one step.

  First, as shown in FIG. 6A, the gel adhesion region 2 is formed on the upper surface of the substrate 1. As the substrate 1, for example, polyethylene terephthalate (PET) of 70 mm × 13 mm can be used, and the gel adhesion region 2 is formed by performing a surface modification process on the substrate 1.

  In the present embodiment, before the first discharge step, a portion other than the gel adhesion region 2 is masked, and oxygen plasma treatment is performed to form the gel adhesion region 2 having a hydrophilic region (gel adhesion region formation step). ). At this time, the area of the gel adhesion region 2 can be set to, for example, 50 millimeters × 2.4 millimeters.

  The surface modification treatment such as oxygen plasma treatment is more preferable because it is easy to pattern and has high productivity. However, the structure of the gel adhesion region 2 is not limited to this, and the above-described hollow structure or A protruding structure may be formed, or a fine uneven structure 4 may be formed. Moreover, it is possible to combine these.

  Next, the first solution (liquid) is discharged to the gel adhesion region 2 of the substrate 1 (first discharge process). At this time, since the gel adhesion region 2 is made hydrophilic by oxygen plasma, the first solution stays in the gel adhesion region 2 with good position reproducibility to form a liquid pool 5 (droplet trapping region) (FIG. 6). (B)).

  Examples of the first solution include 1M Tris-HCl buffer, TEMED, and pure water. These amounts may be appropriately set according to the concentration of the polyacrylamide gel, and the mixing ratio is not particularly limited. Examples of the first solution discharging means include a pipetter, a dispenser, and an ink jet head.

  The liquid discharged in the first discharge step may be any liquid that can form the droplet trapping region 5 in the gel adhesion region 2, but it is preferable that the liquid does not interfere with the formation of the gel. A solvent is more preferable, and a solution containing a reagent that promotes gelation of the second solution as in this embodiment is further preferable.

  The discharge amount of the first solution in the first discharge step may be appropriately set according to the film thickness of the gel to be formed. For example, when a thick gel is to be formed, the discharge is performed so that the liquid pool becomes thick, and the thin gel When it is desired to form the liquid, it is preferable to discharge the liquid so that the liquid pool becomes thin. For example, the liquid may be discharged so that a liquid pool of about 0.2 mm to 0.4 mm is formed.

  That is, when the gel solution is discharged onto the substrate 1, if the reaction system is in the gas phase, it is difficult for the discharged gel solution microdroplets to be sufficiently mixed on the substrate 1. Therefore, the liquid droplets are previously ejected to the gel adhesion region 2 to form a liquid pool, so that the coupling between the micro droplets is likely to occur. Therefore, the gel solution can be sufficiently mixed. At this time, if the surface modification treatment is performed so that the gel adhesion region 2 includes a hydrophilic region, the gel solution can be mixed more suitably.

  If the liquid is a solution containing a reagent related to gel formation, the reagent for forming the gel is discharged in multiple stages. In such a gel production method, it is possible to control the gelation time, and for example, it is possible to prevent the occurrence of a malfunction of the apparatus such as an unnecessary gelation reaction progressing and clogging of piping.

  After the first solution is discharged, the second solution is discharged to the gel adhesion region 2 in which the droplet trapping region 5 is formed (second discharge step). Examples of the second solution include a gel solution such as a 30% acrylamide mixed solution.

  Examples of the second solution discharging means include a pipetter, a dispenser, and an ink jet head. In particular, it is preferable to use an ink jet head that ejects fine liquid droplets from a fine nozzle to adhere to the substrate 1. As shown in FIG. 6C, if the ink droplets can be ejected from the inkjet head 11 with the second liquid droplets 6, the gel concentration and the formation region can be easily controlled.

  For example, when producing an IPG gel or a gradient gel, a high-definition gray scale (gradient) can be produced by discharging a 30% acrylamide mixed solution with a gradient using the inkjet head 11. Therefore, a high-performance IPG gel or SDS-PAGE gradient gel can be provided.

  The ejection method of the inkjet head 11 is mainly classified into a continuous ejection type (continuous inkjet) and an on-demand type (drop-on-demand inkjet). Further, as the continuous ink jet, for example, a charge control method for controlling charged micro droplets with an electric field can be mentioned, and as the drop-on-demand ink jet, for example, a thermal (bubble) method, an electrostatic actuator method or a piezo method can be mentioned. Etc.

  As in the present embodiment, when the droplets 6 of the second solution are ejected from the inkjet head 11, the droplet trapping region 5 (pool) is formed in the gel adhesion region 2. 5 can greatly promote the mixing of the fine droplets 6 discharged to the nozzle 5. Therefore, it is possible to prevent the deterioration of the electrophoretic characteristics that occurs when there is no droplet capturing region 5.

  Subsequently, the third solution is discharged to the mixed liquid 7 containing the droplet trapping region 5 and the second solution ((d) in FIG. 6) (third discharge step). The third solution contains, for example, APS and may be discharged using a pipetter, a dispenser, an inkjet head, or the like.

  Thereby, for example, when discharging the 1st-3rd solution of a total amount of 80 microliters with respect to the gel adhesion area | region 2 which has an area of 50 millimeters x 2.4 millimeters, it is 0.5-1.0 millimeters. A gel plate 10 on which the polyacrylamide gel 8 is formed is obtained.

  When the gel plate 10 is produced in the reactor, the inside of the reactor is made an inert gas such as argon or a nitrogen atmosphere during the production of the gel plate 10, and oxygen which becomes an inhibitory factor for the gelation reaction Is preferably discharged from the reactor.

  In the conventional typical gelation reaction, it is difficult to touch the atmosphere because the gel is cast on a gel preparation jig formed by a glass substrate or the like, but the gel plate 10 of this embodiment directly applies the gel solution to the substrate 1. Because of the drawing, most of the surface of the gel solution is exposed to the atmosphere and is susceptible to oxygen. Therefore, it is desirable to make the inside of the reactor an inert gas or nitrogen atmosphere.

  However, when the gel plate 10 is simply produced, the gelation reaction may be performed in the air without using a reactor or the like. In this case, it is preferable that the discharge amount of APS is, for example, about 5% to 20% with respect to the total amount of the first to third solutions discharged to the gel adhesion region 2 of the substrate 1.

  If APS is 5% or more, the gel solution can be sufficiently gelled. In addition, if APS is 20% or less, it is possible to suppress insufficient control due to a decrease in radicals necessary for the start of gelation, which is caused by the influence of APSs on each other to increase the gelation speed. Can do.

  By the way, it is preferable to discharge the APS as the third solution in the final stage as in this embodiment. This suppresses gelation of the gel solution in the preparation jig or gel preparation apparatus, and prevents problems such as clogging of pipes that are unnecessarily gelled in the gel preparation jig or gel preparation apparatus. Can do.

  Thus, according to the manufacturing method of the gel plate 10 of the present embodiment, each gel solution is discharged to the gel adhesion region 2 formed at an arbitrary position on the substrate 1, thereby having an arbitrary size. The gel 3 can be directly formed with a composition and concentration and high position reproducibility.

  Therefore, conventionally, since the gel plate is formed using a casting jig such as a glass substrate, the place where the gel is formed is limited. However, according to the manufacturing method of this embodiment, for example, the end face of the substrate 1 or the like. A gel can be formed at any place.

(Preparation of isoelectric focusing chip 20)
Next, a manufacturing method in the case of applying the isoelectric focusing chip 20 as the electrophoresis reaction tool according to the present invention will be described with reference to FIG. FIG. 7 is a cross-sectional view showing a flow when manufacturing the isoelectric focusing chip 20 according to the embodiment of the present invention. The IPG gel formed on the IEF chip 20 shown in FIG. 7 is a first medium (1D gel) disclosed in, for example, Japanese Patent Application Laid-Open No. 2007-64848 (published on March 15, 2007), that is, immobilization. It can be suitably applied to a pH gradient gel.

  In the present embodiment, a method for manufacturing the isoelectric focusing chip 20 (IEF chip) in the first dimension of two-dimensional electrophoresis, that is, an immobilized pH gradient gel (IPG gel) used for isoelectric focusing. Will be described.

  A generally known IEF chip used for isoelectric focusing is obtained by casting an IPG gel on a gel bond film and cutting it into a desired shape.

  For example, when the manufacturing method of the present embodiment is applied to such an IPG gel, the portions other than the portion where the gel adhesion region 2 is formed are masked with respect to PET which is a material of the gel bond film, and glow discharge or arc discharge is performed. By performing the oxygen plasma treatment, the gel adhesion region 2 can be formed.

  Thus, if the manufacturing method of this embodiment is used for the production of the IEF chip to which the IPG gel is fixed, the gel can be sufficiently formed even for shapes that have been difficult to produce.

  Examples of the reagent for forming the IPG gel include immobiline mixed solution, isoelectric focusing reagent, TEMED, APS, and pure water.

  The immobiline mixed solution is, for example, a solution in which two types of immobiline with different pH are mixed, and by mixing immobiline having various dissociation constants (pK) with an acrylamide derivative having a positive charge or a negative charge, An immobiline mixed solution having the desired pH is obtained. Moreover, the isoelectric focusing reagent (Ampholine) is an ampholyte mixture.

  First, as shown in FIG. 7A, the gel adhesion region 2 is formed on the upper end surface of the support base 12. As the support base 12, for example, a plastic substrate such as polymethyl methacrylate resin (PMMA) or a glass substrate can be used.

  Also in this embodiment, it is preferable to make the gel adhesion region 2 of the support base 12 hydrophilic and make the region other than the gel adhesion region 2 hydrophobic. For example, when the support base 12 is PMMA, the gel adhesion region 2 having a hydrophilic region can be formed by masking a region other than the gel adhesion region 2 and performing oxygen plasma treatment or sulfonation treatment.

  Next, the first solution is discharged to the gel adhesion region 2 of the support base 12. Examples of the first solution include an isoelectric focusing reagent, TEMED, and pure water. These mixing ratios are not particularly limited. Thereby, the droplet trapping region 13 is formed in the gel adhesion region 2.

  As the first solution discharge means, for example, a pipetter, a dispenser, an ink jet head, or the like can be used.

  After the first solution is discharged, the second solution is discharged to the gel adhesion region 2 where the droplet trapping region 13 is formed to form a gradient. As the second solution, for example, an immobiline mixed solution or the like can be used.

  As the second solution discharge means, for example, a pipetter, a dispenser, an ink jet head, or the like can be used. For example, as shown in FIG. 7B, the inkjet head 11 is supported on the support base 12 so that a concentration gradient of two types of immobiline mixed solution can be formed in the gel adhesion region 2 where the droplet capturing region 13 is formed. In the longitudinal direction (in the direction of the arrow indicated by “A” in FIG. 7B).

  For example, one immobiline mixed solution is adjusted to pH 3 and the other immobiline mixed solution adjusted to pH 10 is ejected from the inkjet head 11 as fine droplets. In addition, about the adjustment method of an immobiline mixed solution, since a general method should just be used, description is abbreviate | omitted.

  Note that the immobiline-containing gel solution 14 (FIG. 7C) formed by discharging the immobiline mixed solution from the inkjet head 11 to the droplet capturing region 13 does not undergo a gelation reaction until the next step. Can remain in solution.

  Subsequently, the third solution is discharged to the immobiline-containing gel solution 14. The third solution contains, for example, APS and may be discharged using a pipetter, a dispenser, an ink jet head, or the like, but an ink jet head is preferably used.

  For example, when APS is ejected using a pipetter or the like, the gelation of the region where APS is not ejected is suppressed with respect to the region where APS is dripped, so that the uniformity of the IPG gel deteriorates. Therefore, the discharge of APS using the inkjet head can be made uniform not only in the plane of the gel adhesion region 2 but also in the depth direction of the immobiline-line containing gel solution 14.

  Thereby, for example, the pH is 3 to 10, and the size of the IPG gel is 50 nm (isoelectric point gradient direction) × 2.4 mm × 0.5 m, and the support substrate 12 is fixed with high positional accuracy. Thus, the IEF chip 20 composed of the IPG gel formed can be obtained.

  In addition, when producing IPG gel other than inert gas atmosphere, such as nitrogen or argon, it is preferable that the volume ratio (APS discharge volume / total discharge volume) of APS is 5 to 20%, for example. However, when the IPG gel is produced in an inert gas atmosphere such as nitrogen or argon, that is, a deoxygenated atmosphere, the volume ratio of APS may be 1% or less.

(Preparation of SDS-PAGE chip 30)
Next, a manufacturing method when the SDS-PAGE chip 30 is applied as the electrophoresis reaction instrument according to the present invention will be described with reference to FIG. FIG. 8 is a cross-sectional view showing a flow when manufacturing the SDS-PAGE chip 30 according to the embodiment of the present invention.

  In the present embodiment, a manufacturing method of the SDS-PAGE chip 30 in which the second dimension of two-dimensional electrophoresis, that is, a gradient gel used for SDS-PAGE electrophoresis, is formed will be described.

  The SDS-PAGE chip 30 used for the generally known SDS-PAGE has a polyacrylamide gel cast on an instrument made of a plastic resin such as PMMA.

  However, if the manufacturing method of the SDS-PAGE chip 30 of this embodiment is used, it is not necessary to provide a casting structure like the IEF chip 20, and a structure such as a plastic flat plate or a glass flat plate may be used. Note that the gradient gel formed on the SDS-PAGE chip 30 is, for example, the second medium (2D gel) and the second separation unit disclosed in Japanese Patent Application Laid-Open No. 2007-64848 (published on March 15, 2007). (Sample instrument) Suitable for gradient gel.

  As a reagent for forming a gradient gel, for example, a solution similar to the polyacrylamide gel described above can be used.

  First, as shown in FIG. 8A, the gel adhesion region 2 is formed in a desired region of the support base 15 on which the gradient gel is provided. As the support base 15, for example, a plastic substrate such as PMMA or a glass substrate can be used.

  Also in this embodiment, it is preferable to make the gel adhesion region 2 of the support base 15 hydrophilic and make the region other than the gel adhesion region 2 hydrophobic. For example, the gel adhesion region 2 having a hydrophilic region can be formed by masking a region other than the gel adhesion region 2 of the support base 15 and performing oxygen plasma treatment, sulfonation treatment, nitration treatment, or the like.

  Next, the first solution is discharged to the gel adhesion region 2 of the support base 15. Examples of the first solution include 1M Tris-HCl buffer, TEMED, and pure water. These mixing ratios are not particularly limited. Thereby, as shown in FIG. 8B, the droplet trapping region 16 is formed in the gel adhesion region 2.

  As the first solution discharge means, for example, a pipetter, a dispenser, an ink jet head, or the like can be used.

  After the first solution is discharged, the second solution is discharged to the gel adhesion region 2 where the droplet trapping region 16 is formed to form a gradient. Examples of the second solution include an acrylamide mixed solution (acrylamide + N, N′-methylenebisacrylamide). The concentration of the acrylamide mixed solution can be, for example, a relatively high concentration of 30 to 50% (acrylamide: N, N′-methylenebisacrylamide = 37.5: 1).

  As the second solution discharging means, for example, a pipetter, a dispenser, an ink jet head, or the like can be used. In particular, the ink jet head 11 is preferably used. For example, the gradient can be suitably formed by scanning along the direction of the arrow indicated by “B” in FIG. 8B using the inkjet head 11.

  Note that the acrylamide mixture-containing gel solution 17 (FIG. 8C) formed by discharging the second solution from the inkjet head 11 to the droplet trapping region 16 does not undergo a gelation reaction until the next step. Can remain in solution.

  Subsequently, the third solution is discharged to the acrylamide mixture-containing gel solution 17. As the third solution, for example, APS may be used, and it may be discharged using a pipetter, a dispenser, an inkjet head, or the like, but an inkjet head is preferably used.

  Similar to the IEF chip 20, since it is desirable that the discharge of APS is uniform not only in the plane of the gel adhesion region 2 but also in the depth direction of the acrylamide mixture-containing gel solution 17, it is preferable to use an inkjet head. . Thus, a gradient gel is formed on the gel adhesion region 2 formed in a desired region of the support base 15 by discharging a desired amount of APS to the acrylamide mixture-containing gel solution 17 having a gradient. be able to.

  For example, the support base 15 is 4% on the low concentration side and 15% on the high concentration side, and the size of the gradient gel is 50 nm (concentration gradient direction) × 2.4 mm × 0.5 m, and the positional accuracy with respect to the support base 15 An SDS-PAGE chip 30 composed of a gradient gel that is well immobilized is obtained.

  In addition, when producing a gradient gel other than inert gas atmosphere, such as nitrogen or argon, it is preferable that the volume ratio (APS discharge volume / total discharge volume) of APS is 5% or more, for example. However, when an IPG gel is produced in an inert gas atmosphere such as nitrogen or argon, that is, a deoxygenated atmosphere, it is not limited to this.

(Substance to be separated)
The substance to be electrophoresed using each of the above-described electrophoresis reaction instruments may be any substance to be separated or analyzed by electrophoresis and transcription. For example, an individual organism, body fluid, cell line, tissue culture Preparations collected from biological materials such as products or tissue fragments can be suitably used. In particular, polypeptides or polynucleotides are more preferred.

(Electrophoresis kit)
Furthermore, this invention includes the kit for electrophoresis containing the base material for gel fixation which concerns on this invention.

  In addition to the gel fixing substrate according to the present invention, the kit may include, for example, a reagent for gel formation, a buffer solution for electrophoresis, an instrument for electrophoresis, and the like.

  The present invention is not limited to the above-described embodiments, and various modifications can be made within the scope of the claims, and embodiments obtained by appropriately combining the respective technical means disclosed are also included in the present invention. Included in the technical scope. Moreover, all the literatures described in this specification are used as reference.

  The present invention can be used for polyacrylamide gel electrophoresis or agarose gel electrophoresis for separating biopolymers such as protein, DNA or RNA, and particularly includes isoelectric focusing and SDS-PAGE electrophoresis. It can be suitably used for two-dimensional electrophoresis.

1 Substrate (Gel fixing base material)
2 Gel adhesion area 3 Gel 10 Gel plate (reaction equipment for electrophoresis)
20 IEF chip (reaction equipment for electrophoresis)
30 SDS-PAGE chip (Reaction equipment for electrophoresis)

Claims (7)

A gel fixing substrate for fixing a gel for electrophoresis,
At least a part of the surface for fixing the gel has a gel adhesion region subjected to a treatment for attaching the gel,
The gel fixing base material, wherein the gel adhesion region is formed in a convex shape.   The base material for gel fixation according to claim 1, wherein a plurality of uneven structures are formed in the gel adhesion region.   The at least part of the gel adhesion region has hydrophilicity, and the surface and at least a part of the region other than the gel adhesion region have hydrophobicity. Gel fixing substrate.   The gel fixing base material according to claim 3, wherein the hydrophilic region of the gel adhesion region has a composition containing an oxygen-containing functional group.   An electrophoresis reaction instrument, wherein an electrophoresis gel is fixed to the gel fixing base material according to any one of claims 1 to 4.   6. The electrophoresis reaction apparatus according to claim 5, wherein the gel is formed to have a gradient of gel concentration or pH.   An electrophoresis kit comprising the gel-fixing base material according to claim 1.

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