JP2011145137A - Sample separation and suction instrument - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a sample separation and suction instrument, providing a sample suction pattern of high resolving power. <P>SOLUTION: In the sample separation and suction instrument 100, a line of electric force is produced in the direction going toward the second opening 36 of a sample separation part 31 from the first opening 35. The sample in the separation medium 33 housed in the sample separation part 31 is migrated toward the second opening 36 along the line of electric force to be separated and discharged from the second opening 36 to be suctioned by an suction member 6. This sample separation and suction instrument 100 is equipped with a line-of-electric force converging structure composed of a first slit structure 46 and a second slit structure 30. Since the line of electric force is converged between the first and second slit structures 46 and 30, the suction expansion of the sample to the suction member 6 is suppressed. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a sample separation and adsorption device for separating a biological sample and sequentially adsorbing the separated sample onto an adsorption member.

  After the completion of the Human Genome Project, proteome research has been actively conducted. By “proteome” is intended a whole cell that is translated and produced in a specific cell, organ, and organ, and its research includes protein profiling.

  One of the most widely used methods for profiling proteins is protein electrophoresis, particularly two-dimensional electrophoresis. Because proteins have unique properties of charge and molecular weight, they combine a proteome, which is a mixture of many proteins, rather than separating individual proteins into their components based on charge alone or molecular weight alone. As a result, more proteins can be separated with high resolution.

  Although proteins are separated by electrophoresis and / or molecular weight by electrophoresis, it is difficult to specify biological properties from the separation position of proteins separated only by such physical properties. In addition, it is known that the function of a protein is controlled by being subjected to chemical modification (post-translational modification) such as phosphorylation after being synthesized. It is difficult to obtain information on such post-translational modifications by electrophoresis alone.

  The Western blotting method is a method of transferring a protein in a slab gel separated by electrophoresis to a membrane (see, for example, Patent Document 1). If a specific antibody is overlaid on the membrane onto which the protein has been transferred by Western blotting, the protein can be specified to some extent based on the antigen-antibody reaction. In addition, regarding phosphorylation, which is one of the post-translational modifications, it is possible to detect the presence or absence of phosphorylation and the difference in phosphorylation sites by overlaying an anti-phosphorylated protein antibody on the membrane on which the protein is transcribed. Become.

  Thus, the combination of the electrophoresis method and the Western blotting method is a very useful method for specifying the biochemical properties of the protein (see, for example, Non-Patent Document 1).

  Conventionally, electrophoresis and Western blotting are performed manually by researchers using independent devices. For example, after performing isoelectric focusing and SDS-PAGE with an electrophoresis apparatus, the gel is removed from the apparatus and transferred to a transfer apparatus, and a transfer film is set and transferred (blotting). It is common to manually overlay the probe. Since the gel used for this operation is a very soft material and difficult to handle, the operation becomes complicated, and the operation requires skill.

  Therefore, a technology that automates these operations has been developed. For example, Patent Document 2 discloses a sample separation and adsorption device that automates a series of operations from electrophoresis to blotting.

In the sample separation adsorption device described in Patent Document 2, when a voltage is applied between the first electrode and the second electrode, the sample is separated by a separation medium (gel) in the sample separation unit. The separated sample is discharged from the opening of the sample separation section, moves along the electric lines of force generated from the first electrode toward the second electrode, and is adsorbed (transferred) to the sample adsorption member (transfer film).

Japanese Patent Laid-Open No. 7-63763 (published on March 10, 1995) JP 2007-292616 A (published on November 8, 2007)

Protein experiment note (bottom): From separation identification to functional analysis (Yodosha, 2005, paragraphs 38-47)

  When performing electrophoresis and transfer continuously, it is possible to use a blotting system in which separated molecules are transferred from the end face of the electrophoresis medium to the transfer film using the electrode pair used for electrophoresis. However, in such a blotting method, the electric lines of force due to the electrode pair have a spread from the end face of the electrophoresis medium toward the second electrode. For this reason, the separated molecules discharged from the end face of the electrophoretic medium are transferred to the transfer film while spreading according to the lines of electric force. This causes a problem that the transfer pattern in the transfer film becomes unclear.

  The present invention has been made in view of the above problems, and an object of the present invention is to provide a sample separation / adsorption instrument capable of obtaining a high-resolution sample adsorption pattern.

  In order to solve the above-described problems, the sample separation / adsorption device according to the present invention separates a sample in the separation medium by passing an electric current through the buffer to the separation medium, and removes the separated sample. A sample separation / adsorption device for adsorbing to a sample adsorption member from the separation medium, wherein the first electrode, the second electrode, the first opening that opens on the side facing the first electrode, and the second electrode are opposed to each other A sample separation part for storing the separation medium, and a portion facing the second opening in the sample adsorption member between the front and rear sides in the sample separation direction. The electric force line converging structure composed of the first and second insulating members forming the slit at the sandwiched position, and the relative position of the sample adsorbing member with respect to the slit, the separation direction Is characterized by comprising moving means for moving in a direction substantially perpendicular.

  In the above configuration, when a voltage is applied to the first electrode and the second electrode, a current flows through the separation medium via the buffer solution. At this time, the sample in the separation medium moves from the first opening toward the second opening, and is separated according to the mobility of each component. The separated sample is discharged from the second opening of the sample separation unit. The sample discharged from the second opening sequentially reaches the sample adsorbing member along electric lines of force generated from the first electrode toward the second electrode. At this time, the moving means moves the sample adsorption member in a direction substantially perpendicular to the sample separation direction. For this reason, the sample discharged | emitted from 2nd opening is continuously adsorbed by the member for sample adsorption.

Here, the sample separating and adsorbing device according to the present invention is configured to converge the electric lines of force composed of the first and second slit structures having a slit at a position sandwiching a portion facing the second opening in the sample adsorbing member. It has a structure. The slits in the first and second slit structures are made of an insulating material. Therefore, the electric lines of force generated between the first electrode and the second electrode converge when passing through the slits of the first and second slit structures. For this reason, in the area | region where the member for sample adsorption | suction is arrange | positioned between the 1st slit structure and the 2nd slit structure, the spreading | diffusion of the said electric-force line is suppressed.

  The moving means may move the sample separation unit and the two slit structures in a state where the sample adsorption member is fixed.

  According to the above configuration, the spread of the sample adsorption with respect to the sample adsorption member can be suppressed, and a sample adsorption pattern with high resolution can be obtained.

  Further, in the sample separation / adsorption device according to the present invention, the first insulating member is disposed in the second opening of the sample separation portion, or the second opening is formed as the slit. It is configured as a part of the sample separation part, and the second insulating member is preferably disposed between the sample adsorbing member and the second electrode.

  According to the above configuration, the sample that has passed through the first structure in the second opening reaches the adsorbing member along the lines of electric force whose spread is suppressed. Therefore, a sample adsorption pattern with higher resolution can be obtained.

  In the sample separation / adsorption device according to the present invention, the second insulating member is provided with an elastic material that allows the buffer solution to pass therethrough, which presses the sample adsorption member toward the second opening. It is preferable.

  According to the above-described configuration, the sample discharged from the second opening can be prevented from diffusing into the buffer solution by bringing the sample adsorbing member into close contact with the second opening. Therefore, a sample adsorption pattern with higher resolution can be obtained.

  Further, in the sample separation and adsorption device according to the present invention, the surface of the first insulating member facing the sample adsorption member, and the sample adsorption member in the second insulating member or the elastic material It is preferable that a hydrophilic film is provided on the opposite surface.

  According to the above configuration, the sample adsorbing member sandwiched between the first and second insulating members is sandwiched between the front and back surfaces of the same material called a hydrophilic film. For this reason, when the sample suction member is pulled up in the vertical direction, the front surface and the back surface receive the same frictional force, and the sample suction pattern is prevented from being disturbed due to the difference in the frictional force generated during the lifting. be able to.

  In addition, by providing a hydrophilic film on the sample adsorbing member side of the first insulating member (or second opening), the sample adsorbing member is securely adhered to the first insulating member (or second opening). You can also.

  In the sample separation / adsorption device according to the present invention, the first insulating member may be formed by applying or attaching an insulating material to the surface of the hydrophilic film, leaving an area to be the slit. Preferably formed.

  Moreover, in the sample separation and adsorption device according to the present invention, the second insulating member is formed by applying or affixing an insulating material on the surface of the elastic material or the hydrophilic film, leaving the region to be the slit. It is preferable to form the slit.

  According to the said structure, the slit in each of the said 1st insulating member and the said 2nd insulating member can be formed suitably.

  The sample separation / adsorption device according to the present invention further includes a slit block having a through hole at a position facing the slit formed by the second insulating member and supporting the second insulating member. Is preferred.

  According to the said structure, the member for sample adsorption | suction can be pressed more uniformly in the case of pressing by an elastic material. Therefore, a sample adsorption pattern with higher resolution can be obtained.

  The sample separation / adsorption device according to the present invention further includes a first buffer solution tank in which the first electrode is disposed, and a second buffer solution tank in which the second electrode is disposed, and the penetrating through the slit block. It is preferable that the hole is filled with a buffer solution filled in the second buffer solution tank.

  According to the above configuration, since the electric charge is always supplied into the through hole of the slit block, the voltage value in the sample separation adsorption process can be kept stable. Therefore, even long-time sample separation and adsorption can be performed stably.

  In the sample separation / adsorption device according to the present invention, the elastic material may be made of an elastic material having a through hole or a material that expands when water is absorbed.

  The sample separation / adsorption device according to the present invention separates a sample in the separation medium by passing an electric current through the buffer to the separation medium, and separates the separated sample from the separation medium to the sample adsorption member. A sample separation / adsorption device to be adsorbed, comprising: a first electrode; a second electrode; a first opening that opens on a side facing the first electrode; and a second opening that opens on a side facing the second electrode. And a sample separation portion for storing the separation medium, and a portion facing the second opening in the sample adsorption member at a position sandwiched between front and rear in the sample separation direction by an insulating material. The electric force line converging structure composed of the first and second slit structures having the slits, and the relative position of the sample adsorbing member with respect to the slits, the separation direction Because and a moving means for moving in a substantially vertical direction, it is possible to obtain a high-resolution sample pickup patterns.

It is sectional drawing which shows the principal part structure of the sample separation adsorption instrument which concerns on one Embodiment of this invention. It is sectional drawing which shows the said sample separation adsorption instrument roughly. It is sectional drawing which shows the sample separation part and 1st slit structure in the said sample separation adsorption instrument. (A) (b) is sectional drawing which shows the example of the slit structure in the said sample separation adsorption instrument. (A) (b) is sectional drawing which shows the other example of the slit structure in the said sample separation adsorption instrument. It is sectional drawing which shows another example of the principal part structure of the said sample separation adsorption instrument. (A) is a side view which shows the other example of the said sample separation part, (b) is the sectional drawing. It is sectional drawing which shows another example of the principal part structure of the said sample separation adsorption instrument. (A) is a perspective view which shows the slit block in the said sample separation adsorption | suction instrument, (b) is the sectional drawing. (A) (b) It is sectional drawing which shows the said slit structure installed in the said slit block. It is sectional drawing which shows another example of the principal part structure of the said sample separation adsorption instrument. It is a perspective view which shows the other example of the said slit block. It is a figure which shows the other example of the said slit structure. It is a perspective view which shows the principal part structure of the said sample separation adsorption instrument. It is a perspective view which shows the principal part structure of the said sample separation adsorption instrument. It is sectional drawing which shows the principal part structure of the said sample separation adsorption instrument. (A) is sectional drawing which shows the sample separation part in Example 1 and 2, (b) is sectional drawing which shows a sample separation part toward the 2nd opening side from the 1st opening side. It is a figure which shows the simulation model of the electric force line in the said sample separation adsorption | suction instrument. It is a figure which shows an example of the 1st simulation result of the electric force line in the said sample separation adsorption | suction instrument. It is a graph which shows the 1st simulation result of the electric line of force in the said sample separation adsorption instrument. It is a figure which shows the 2nd simulation model of the electric force line in the said sample separation adsorption | suction instrument. It is a figure which shows an example of the 1st simulation result of the electric force line in the said sample separation adsorption | suction instrument. It is a graph which shows the 2nd simulation result of the electric force line in the above-mentioned sample separation adsorption instrument. (A) is a figure which shows the migration locus | trajectory of the sample in a comparative example, (b) is the enlarged view. (A) is a figure which shows the migration locus | trajectory of the sample in an Example, (b) is the enlarged view.

  An embodiment of the present invention will be described below with reference to the drawings.

(Configuration of sample separation / adsorption device 100)
First, a schematic configuration of the sample separation / adsorption device 100 according to the present embodiment will be described with reference to FIGS. 1 and 2. FIG. 1 is an enlarged view showing a sample adsorption field in the sample separation / adsorption instrument 100. FIG. 2 is a cross-sectional view showing the configuration of the sample separation / adsorption instrument 100.

  In the following description, in the sample separation / adsorption instrument 100, the vertical direction (vertical direction) shown in FIG. 2 is the Z-axis direction, and the SDS-PAGE direction of the sample defined by the first electrode 41 and the second electrode 42 is the same. The direction is the Y-axis direction, and the direction perpendicular to both the Y-axis and the Z-axis is the X-axis direction.

  As shown in FIGS. 1 and 2, the sample separation / adsorption device 100 includes a first buffer tank 1 in which a first electrode 41 is disposed, a second buffer tank 2 in which a second electrode 42 is disposed, and a first slit. The structure 46, the second slit structure 30, and the sample separation unit 31 for storing the separation medium 33 are provided. The sample separation / adsorption device 100 includes a moving mechanism (for example, a moving arm 61) for moving the adsorbing member (sample adsorbing member) 6.

  Below, each main member is demonstrated in detail.

The first buffer solution tank 1 and the second buffer solution tank 2 are tanks filled with the first buffer solution and the second buffer solution, respectively, and the first electrode 41 and the second electrode 42 are arranged inside thereof. The
It does not specifically limit as a 1st buffer solution and a 2nd buffer solution, According to a use or the objective, it can select suitably from the buffer solution of the composition generally used for electrophoresis. In the present embodiment, the first electrode 41 is connected to the negative electrode of a power source (not shown), and the second electrode 42 is connected to the positive electrode.

  The first electrode 41 is disposed to face the first opening 35 in the first buffer solution tank 1, while the second electrode 42 is disposed to face the second opening 36 in the second buffer solution tank 2. Is done. In order to ensure the arrangement of these electrodes, the first electrode 41 and the second electrode 42 were installed in the first electrode fixing part 411 and the second buffer solution tank 2 installed in the first buffer solution tank 1. It is preferable that the second electrode fixing portions 412 are fixed respectively.

  Moreover, although the 1st electrode 41 and the 2nd electrode 42 should just be formed from an electroconductive raw material, in order to prevent ionization of an electrode, it is preferable to use platinum as a raw material. The shape of the second electrode 42 is preferably the same as the second opening 36 or smaller than the second opening 36, and may be, for example, linear.

  The sample separation unit 31 is disposed between the first buffer tank 1 and the second buffer tank 2. For example, the sample separation unit 31 may be placed in the separation unit place 8 as shown in FIG. The placed sample separation unit 31 may be fixed by fitting or fastening with the second buffer solution tank 2 or the separation unit placement site 8 in order to fix the movement in the Y-axis direction. In addition, the sample separation unit 31 may be restrained by a rise prevention claw 80 provided in the second buffer solution tank 2 or the separation unit storage 8 in order to fix the movement in the Z-axis direction.

  Further, the sample separation unit 31 has openings at both ends in the Y-axis direction. Specifically, the sample separation unit 31 has a first opening 35 that opens to face the first electrode 41, and a second opening 36 that opens to face the second electrode 42. The shapes of the first opening 35 and the second opening 36 may be rectangular, for example.

  The sample separation unit 31 stores a separation medium 33 therein. In the present embodiment, the separation medium 33 stored in the sample separation unit 31 faces the first buffer solution tank 1 through the first opening 35 and the second buffer solution tank 2 through the second opening 36. Facing. Moreover, the sample separation part 31 can be comprised from the insulating plate 34 of 2 sheets, and the spacer (not shown) installed in order to ensure a space between them. On the first opening 35 side of the sample separation unit 31, it is desirable that the upper insulating plate 34 is missing so that the sample can be brought into contact with the exposed portion of the separation medium 33.

  The separation medium 33 is a medium for separating a sample by electrophoresis, and a medium generally used for electrophoresis, for example, polyacrylamide gel or agarose gel can be used.

As used herein, “sample” is intended to mean “biological sample” or an equivalent thereof. A “biological sample” is intended to be any preparation obtained from biological material as a source (eg, an individual, body fluid, cell line, tissue culture or tissue section). Biological samples include body fluids (eg, blood, saliva, plaque, serum, plasma, urine, synovial fluid and cerebrospinal fluid) and tissue sources. A preferred biological sample is a subject sample. Preferred subject samples are skin lesions, sputum, pharyngeal mucus, nasal mucus, pus or secretions obtained from the subject. As used herein, the term “tissue sample” intends a biological sample obtained from a tissue source. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art. As used herein, the term “sample” includes, in addition to the biological sample and the tissue sample, a protein sample extracted from the biological sample and the tissue sample, a genomic DNA sample, and / or A total RNA sample is also included. The “sample component” is intended to mean various factors (components) constituting the “sample”.

  The first slit structure 46 is provided in the second opening 36 of the sample separation unit 31. FIG. 3 is a diagram illustrating the sample separation unit 31 provided with the first slit structure 46. As shown in FIGS. 1 and 3, the first slit structure 46 includes an insulating portion (first insulating member) 44, and the insulating portion 44 forms a slit 45 at a position facing the second opening 36. is doing. The slit 45 formed by the insulating portion 44 has a slit width in the Z-axis direction. It is preferable that the slit 45 is filled with an elastic material (for example, gel) that can transmit a buffer solution.

  The second slit structure 30 is disposed between the adsorption member 6 and the second electrode 42 in the second buffer solution tank 2. The second slit structure 30 includes an insulating part (second insulating member) 32, and the insulating part 32 forms a slit 29 at a position corresponding to the second opening 36. The slit 29 formed by the insulating portion 32 has a slit width in the Z-axis direction.

In the present embodiment, the slit 45 formed by the insulating portion 44 installed in the second opening 36 is referred to as a first slit, and the slit 29 formed by the insulating portion 32 is referred to as a second slit. The electric field line focusing structure according to this embodiment includes an insulating portion 44 that forms a first slit and an insulating portion 32 that forms a second slit.
As will be described in detail later, in such an electric force line converging structure, the spread of the electric force lines can be converged by arranging the first slit and the second slit as a set.

  The adsorbing member 6 is a member for adsorbing a sample. The adsorption member 6 in the present embodiment is disposed between the first slit structure 46 and the second slit structure 30 in the second buffer solution tank 2 as shown in FIG. The adsorption member 6 is fixed to the moving arm 61 and is moved in a direction substantially perpendicular to the sample separation direction by a moving mechanism (moving means) described later.

  The adsorbing member 6 is preferably made of a material that can ensure strength. For example, when the sample is a protein, a PVDF (Polyvinylidene fluoride) film or the like can be used. Note that the PVDF membrane is preferably hydrophilized in advance using methanol or the like. In addition, a conventionally used membrane such as nylon or nitrocellulose, which is easily bound to nucleic acid or protein, can be used.

(Sample separation and adsorption)
Next, the flow of sample separation and adsorption in the sample separation / adsorption device 100 will be described with reference to FIGS. 1 and 2.

  First, the sample separation unit 31 is installed in the sample separation unit place 8, and the first buffer solution tank 1 is filled with the first buffer solution, and the second buffer solution tank 2 is filled with the second buffer solution. After the sample adsorbing member 6 fixed to the moving arm is immersed in the second buffer solution, the first electrode 41 and the second electrode 42 are arranged.

  When performing two-dimensional electrophoresis, the sample medium 50 that has been subjected to the first-dimensional isoelectric focusing in advance is used. When introducing the sample, the sample medium 50 is fixed to the support 51, moved by the moving arm 52, and placed on the separation medium 33 above the first opening 35. In addition, when the first-dimensional electrophoresis is not performed, such as SDS-PAGE, the sample may be introduced into the well formed in the separation medium 33.

  When a voltage is applied between the first electrode 41 and the second electrode 42 after introducing the sample, a current flows through the separation medium 33 via the buffer solution. As a result, the sample in the separation medium 33 is electrophoresed in the Y-axis direction from the first opening 35 toward the second opening 36, and is separated based on the difference in mobility generated between the sample components. The separated sample is further electrophoresed in the Y-axis direction, and sequentially passes through a slit 45 (first slit) provided in the second opening 36 and is discharged. Further, after the start of electrophoresis, the moving arm 61 starts to pull up the adsorption member 6. For this reason, the sample discharged through the first slit provided in the second opening 36 is continuously adsorbed to the adsorbing member 6.

  Here, electricity generated between the first electrode 41 and the second electrode 42 between the first slit installed in the second opening 36 and the slit 29 (second slit) of the second slit structure 30. The force lines are converged in the Z-axis direction. For this reason, the spread of the electric lines of force in the sample adsorbing member 6 is suppressed.

  The sample discharged through the first slit provided in the second opening 36 reaches the adsorbing member 6 along the lines of electric force whose spread is suppressed by the first and second slits. Therefore, the sample discharged through the first slit provided in the second opening 36 is adsorbed by the adsorbing member 6 with high accuracy while being prevented from being diffused.

(First slit structure 46 and second slit structure 30)
Next, the configuration of the first slit structure 46 and the second slit structure 30 will be described in detail.

  As described above, the first slit structure 46 includes the insulating portion 44, and the insulating portion 44 forms a slit 45 (first slit). The second slit structure 30 includes an insulating portion 32, and the insulating portion 32 forms a slit 29 (second slit).

  The second slit structure 30 can include an elastic material 38. The elastic member 38 is made of an elastic material that is permeable to a buffer solution, such as a material that expands when absorbing water or an elastic material having a through hole. As a material that expands upon water absorption or an elastic material having a through hole, filter paper having different thicknesses or compression degrees, polyvinyl alcohol, vinylon, polyvinyl acetate, polyurethane, or the like can be used. For example, as shown in FIGS. 4A and 4B, the elastic member 38 may be provided on any of the surfaces having the slit openings in the insulating portion 32. In addition, the 2nd slit structure 30 shown in FIG. 1 uses the 2nd slit structure 30 shown to Fig.4 (a).

  When the second slit structure 30 includes the elastic member 38, the sample adsorbing member 6 is pressed against the second opening 36 side of the sample separation unit 31 due to the elasticity of the elastic member 38, thereby bringing the members into close contact with each other. Can do. As a result, the sample discharged from the second opening 36 can be prevented from diffusing into the second buffer solution, and a more accurate transfer pattern can be obtained.

  The 2nd slit structure 30 provided with the elastic material 38 can be produced with the method shown below.

  For example, the insulating material may be applied to the surface of one side of the elastic material constituting the elastic material 38, leaving a region to be a slit. The insulating material applied at this time constitutes the insulating portion 32. As the insulating material, phenol resin, epoxy resin, or the like can be used.

  Alternatively, an insulating film may be attached to the surface of one side of the elastic material constituting the elastic material 38, leaving a region to be a slit. The insulating film pasted at this time constitutes the insulating portion 32. As the insulating film, polyethylene, polypropylene, vinyl chloride, polymethyl methacrylate, polyvinyl alcohol, or the like can be used.

  Alternatively, a porous material may be used as the elastic material constituting the elastic material 38, and the insulating material may be introduced on the surface or inside thereof, leaving a region to be a slit.

  In addition, each of the first slit structure 46 and the second slit structure 30 preferably includes a hydrophilic film. As the hydrophilic film, for example, a hydrophilic PVDF film can be used.

  The hydrophilic film is preferably provided on the side facing the sample adsorption member 6 in each of the first slit structure 46 and the second slit structure 30. For example, the hydrophilic film 37 is provided on the second slit structure 30 including the elastic member 38 as shown in FIGS. Thereby, when the sample suction member 6 is pulled up, the frictional force applied to the front surface and the back surface of the sample suction member 6 is the same, so that the sample suction pattern can be prevented from being disturbed. Further, since the hydrophilic film is in close contact with the adsorbing member 6, each member from the adsorbing member 6 to the second opening 36 of the sample separation unit 31 can be more intimately adhered.

  The 1st slit structure 46 provided with a hydrophilic film | membrane can be produced by leaving the area | region used as a slit with respect to a hydrophilic film | membrane, and apply | coating an insulating material. Alternatively, it can be produced by attaching an insulating film while leaving a region to be a slit with respect to the hydrophilic film. At this time, the applied insulating material or the pasted insulating film constitutes the insulating portion 44.

  Moreover, the 2nd slit structure 30 provided with the elastic material 38 and the hydrophilic film | membrane 37 is producible with the method shown below.

  For example, as described above, an insulating material is applied or an insulating film is applied to the surface of one side of the elastic material constituting the elastic material 38, leaving a region to be a slit. Alternatively, an insulating material is introduced leaving a region to be a slit on the surface or inside of the porous material constituting the elastic material 38. At this time, the affixed insulating film or the applied insulating material constitutes the insulating portion 32. Thereafter, the hydrophilic film 37 is affixed to the configured insulating portion 32 or elastic material 38. Thus, the second slit structure 30 including the elastic material 38 and the hydrophilic film 37 is produced.

  Alternatively, first, an insulating material is applied or an insulating film is applied to the surface on one side of the hydrophilic film 37, leaving a region to be a slit. At this time, the affixed insulating film or the applied insulating material constitutes the insulating portion 32. Then, the elastic material which comprises the elastic material 38 is affixed on the comprised insulation part 32. FIG. Thus, the second slit structure 30 including the elastic material 38 and the hydrophilic film 37 is produced.

  For the insulating material and the insulating film, the same materials as those described above can be used.

(Other examples of the first slit)
A modification of the first slit in the electric force line focusing structure will be described below with reference to FIGS.

  In the above description, the first slit in the electric field line focusing structure is configured as the slit 45 formed by the insulating portion 44 of the first slit structure 46, but the present invention is not limited to this.

  For example, as shown in FIG. 6, instead of arranging the first slit structure 46, the sample separation unit 31 may form the second opening 36 as the first slit. At this time, a part of the sample separation part 31 constitutes a first insulating member. FIG. 6 is a diagram showing a sample adsorption field according to this modification.

  In the present modification, the electric field line focusing structure includes a sample separation part 31 that forms the first slit and an insulating part 32 that forms the second slit.

  FIG. 7A is a side view showing the sample separation portion 31 that forms the first slit, and FIG. 7B is a cross-sectional view thereof. As shown in FIG. 7, the second opening 36 is preferably formed as a first slit, for example, in a tapered shape (tapered shape).

  FIG. 8 is a diagram showing a sample adsorption field when a hydrophilic film is arranged in the configuration shown in FIG. Also in this modified example, as shown in FIG. 8, the hydrophilic film 43 is provided in the second opening 36 (first slit) of the sample separation portion 31, and the slit 29 (second slit) formed by the insulating portion 32. Is preferably provided with a hydrophilic film 37.

  According to the electric force line converging structure configured as described above, electric force is generated between the second opening 36 (first slit) and the slit 29 (second slit), that is, in the region where the adsorption member 6 is disposed. Line spread can be converged. Thereby, the diffusion of the sample adsorbed on the adsorbing member 6 can be suppressed.

  In addition, the 1st slit structure 46 may be provided with respect to the taper-shaped 2nd opening 36 in this modification (for example, refer FIG. 13).

(Slit block 40)
Next, the slit block 40 will be described.

  It is preferable that the 2nd slit structure 30 is hold | maintained at the slit block 40 as shown to Fig.9 (a) (b). That is, the slit block 40 has a configuration for holding the second slit structure 30. For example, the slit block 40 is fixed to the second buffer solution tank 2 by fitting or fastening. Moreover, in order to prevent the movement of the slit block 40 in the Z-axis direction, a rise prevention claw 80 set in the second buffer solution tank 2 may press the slit block 40 (see, for example, FIG. 2).

  FIGS. 10A and 10B are sectional views showing the slit block 40 provided with the second slit structure 30. As shown in FIGS. 10A and 10B, the slit block 40 is provided with a slit 39 penetrating in the Y-axis direction. The slit 39 is preferably filled with the second buffer solution. As a result, since electric charge is always supplied into the slit 39, the voltage value in the sample separation and adsorption process can be kept stable.

  FIG. 11 is an enlarged view showing a sample adsorption field in the sample separation / adsorption instrument 100. Since the slit block 40 holds the second slit structure 30, the elastic member 38 can suitably press the suction member 6 toward the second opening 36. In addition, FIG. 11 has shown about the case where the sample separation part 31 which forms a 1st slit is used.

  Moreover, the slit block 40 is not restricted to the structure shown to Fig.9 (a) (b), For example, as shown in FIG. 12, you may be provided with the mandrel 62 for hold | maintaining the member 6 for adsorption | suction. When the film-like sample adsorbing member 6 is used, the experimenter's operation can be simplified by winding the adsorbing member 6 around the mandrel 62 and storing it in a roll shape.

(Adsorption member 6 or sample separation unit 31 and the movement mechanism of the two slit structures)
Next, an example of a moving mechanism that moves the adsorption member 6 will be described.

  14 and 15 are perspective views showing a moving mechanism provided in the sample separation / adsorption device 100. FIG. In FIG. 14 and FIG. 15, the first electrode fixing portion 411 and the second electrode fixing portion 412 are omitted to simplify the drawing. Further, in FIG. 15, the moving arm 52 and the support body 51 are omitted in order to simplify the drawing.

  As shown in FIGS. 14 and 15, the entire sample separation / adsorption device 100 including the driving means 7 is fixed to the base 9. Here, the adsorbing member 6 is fixed to the moving arm 61. For example, the moving arm 61 may include a sample suction member fixing jig, and the suction member 6 may be fixed by the sample suction member fixing jig.

  The moving arm 61 is driven by the driving means 7. Specifically, the moving arm 61 is connected to the first driving means 71, and the first driving means 71 is connected to the second driving means 72 fixed on the base 9. The moving arm 61 is driven in the Y-axis direction and the Z-axis direction by the first driving means 71 and the second driving means 72. Thereby, the movement of the adsorption member 6 can be controlled.

  Note that the present invention is not limited to moving the adsorption member 6, and the adsorption member 6 may be fixed and the driving means may be used to move the sample separation unit and the two slits.

  FIG. 16 is a cross-sectional view showing the sample separation / adsorption device 100. As shown in FIG. 16, the first electrode 41 and the second electrode 42 are connected to the negative electrode and the positive electrode of the power supply 111 via the wiring 112, respectively.

  An ammeter 113 is disposed between the power source 111 and the second electrode 42. The data processing device 114 controls the power supply 111 so that a constant current flows between the first electrode 41 and the second electrode 42. The ammeter 113 may be disposed between the first electrode 41 and the power source 111.

  Further, as shown in FIG. 16, the driving means 7 (first driving means and second driving means) is controlled by a driving means control unit 70. The drive means controller 70 may change the pulling speed of the sample adsorbing member 6 by the drive means 7 stepwise over time. Further, the data processing device 114 may control the driving means control unit 70 based on the current value measured by the ammeter 113.

  Although not shown, the moving arm 52 can also be driven using the same means as the moving mechanism described above, whereby the introduction of the sample can be controlled.

(Other configuration examples)
The electric force line convergence structure can also be expressed as follows.

The first slit is disposed between the sample adsorption member and the first electrode, and at any position from the surface facing the second opening to the second opening in the sample adsorption member. A first insulating member to be formed; and a second insulating member disposed between the sample adsorbing member and the second electrode and forming a second slit at a position facing the first slit. Electric field line convergence structure composed of

  EXAMPLES Hereinafter, although this invention is demonstrated in detail based on an Example, this invention is not limited to a following example.

Example 1
In Example 1, the second separation electrophoresis and sample adsorption of two-dimensional electrophoresis were performed using the sample separation / adsorption instrument 100 according to the present embodiment.

[First-dimensional electrophoresis (sample medium 50)]
An immobilized gel with a pH gradient isoelectric focusing by immobiline was cut into 1 mm × 60 mm and used. A sample medium 50 was obtained by performing sample introduction and gel swelling and performing electrophoresis at 6000 V for 1 hour.

  As a sample, a mouse liver sample stained with a fluorescent dye was used.

[Sample separation unit 31]
As the two insulating plates 34, quartz glass having a thickness of 60 mm × 30 mm × 2 mm and a thickness of 60 mm × 28 mm × 2 mm was used. As shown in FIG. 16, the two quartz glasses were arranged such that the first opening 35 had a width of 1000 μm and the second opening 36 had a taper shape of 100 μm.
A hydrophilic PVDF film was attached to the outside of the two quartz glasses with Kapton tape. At this time, care was taken so that there was no gap between the hydrophilic PVDF film and the glass surface, and the Kapton tape was made not to inhibit the second opening 36.

  Both ends of this quartz glass protrude 0.5 mm from the center, and the thickness of the separation medium is defined by these protrusions.

  A sample separation unit 31 prepared as described above and filled with polyacrylamide gel was placed in the sample separation unit place 8 and fixed with a rise prevention claw 80.

[First slit structure 46]
A slit structure 46 prepared by applying an insulating material leaving a region (60 mm × 1 mm) to be a slit on the surface of the hydrophilic film is adhered and adhered so as to coincide with the second opening 36 of the sample separation portion 31. I attached.

[Second slit structure 30]
As the elastic material 38, a filter paper having a thickness of 2.5 mm when dried or polyurethane having fine pores was used, and a hydrophilic PVDF membrane was used as the hydrophilic membrane 37. An elastic material 38 with a hydrophilic film 37 attached thereto was provided on the slit surface of the insulating portion 32 to produce the second slit structure 30. The size of the second slit structure 30 was 60 mm × 10 mm × 10 mm, and the slit width was 1 mm.

The second slit structure 30 produced as described above was installed by being fitted into the second buffer solution tank 2, and fixed by the rise prevention claw 80. At this time, the surface of the hydrophilic film 37 is on the sample adsorbing member 6 side, and the elastic material 38 connects the sample adsorbing member 6 to the second part of the sample separating unit 31.
The second slit structure 30 was disposed so as to be in close contact with the opening 36.

[First buffer tank 1]
A first buffer solution tank 70 of 70 mm × 70 mm × depth 15 mm (depth 8 mm in the sample separation part storage 8) was provided and filled with the first buffer solution. A commercially available MOPS / SDS buffer was used as the first buffer solution. A platinum wire was used as the first electrode 41 disposed in the first buffer tank 1. The first electrode 41 was connected to the negative electrode of the power source 111.

[Second buffer tank 2]
A second buffer solution tank 2 of 70 mm × 70 mm × depth 15 mm was provided and filled with the second buffer solution. A commercially available MOPS buffer was used as the second buffer solution. A platinum wire was used as the second electrode 42 disposed in the second buffer solution tank 2. At this time, the second electrode 42 was connected to the positive electrode of the power source 111.

[Adsorption member 6]
As the adsorbing member 6, a hydrophobic PVDF hydrophilized with methanol was used. With the adsorbing member 6 immersed in the second buffer solution in the second buffer solution tank 2, the upper end side thereof was fixed to the moving arm 61.

[Introduction of the sample medium 50 into the separation medium 33]
The sample medium 50 containing the first-dimensional electrophoresis sample is brought into close contact with the portion where the separation medium 33 is exposed in the first opening 35 of the sample separation section 31 by the moving arm 52.

[Second-dimensional electrophoresis]
With the sample medium 50 and the separation medium 33 in close contact, a constant current was passed between the first electrode 41 and the second electrode 42 for 25 minutes so that the value of the ammeter 113 was 25 mA.

[Sample adsorption]
A constant current was passed between the first electrode 41 and the second electrode 42 for 60 minutes so that the value of the ammeter 113 was 40 mA. At this time, the first driving means 71 lifted the suction member 6 upward. The pulling speed was gradually reduced from 32 μm / sec to 2 μm / sec by the drive means controller 70.

〔result〕
After adsorbing the sample, the adsorbing member 6 was collected and the sample was detected. As a result, according to Example 1, a sample adsorption pattern with high resolution could be obtained while performing sample separation and sample adsorption continuously with the same instrument. The reproducibility was also confirmed.

(Example 2)
In Example 2, SDS-PAGE and sample adsorption were performed using the sample separation / adsorption device 100 according to the present embodiment. In the second embodiment, the description of the same as the first embodiment is omitted, and only the differences will be described below.

[Sample separation unit 31]
The sample separation unit 31 was filled with polyacrylamide gel as the separation medium 33. Before the polyacrylamide gel was polymerized, a comb was inserted into the upper portion of the first opening 35 to prepare a well for introducing a one-dimensional electrophoresis sample. 10 μL of a molecular weight visible marker sample was poured into the prepared well, and the well was covered with an agarose gel. A hydrophilic film 43 was wound around the outside of the second opening 36.

[SDS-PAGE]
A constant current was passed between the first electrode 41 and the second electrode 42 for 25 minutes so that the value of the ammeter 113 was 25 mA.

[Sample adsorption]
A constant current was passed between the first electrode 41 and the second electrode 42 for 60 minutes so that the value of the ammeter 113 was 40 mA. At this time, the first driving means 71 lifted the suction member 6 upward. The pulling speed was gradually reduced from 32 μm / sec to 2 μm / sec by the drive means controller 70.

〔result〕
After adsorbing the sample, the adsorbing member 6 was collected and the sample was detected. As a result, according to Example 2, it was possible to obtain a sample adsorption pattern with high resolution while continuously performing sample separation and sample adsorption with the same instrument. The reproducibility was also confirmed.

(Simulation of electric field lines)
Below, the result of having performed the simulation about the behavior of the electric line of force produced toward the 2nd electrode 42 from the 1st electrode 41 is explained.

  First, a first simulation using the sample separation / adsorption instrument 100 having the configuration shown in FIG. 18 will be described. FIG. 18 is a cross-sectional view showing the main configuration of the sample separation / adsorption instrument 100. In FIG. 18, each of the sample separation unit 31 and the insulating unit 32 is provided with a hydrophilic film 43 or 37, and the adsorption member 6 is in close contact with the second opening 36.

  In the first simulation, the width of the slit 29 in the Z-axis direction was changed stepwise from 2000 μm to 100 μm, and the behavior of the lines of electric force in each was simulated. The width of the second opening 36 in the Z-axis direction was 100 μm, and the applied voltage was 200 V, which is close to the actual applied voltage.

  An example of the result of the first simulation is shown in FIG. FIG. 19 shows the result of a simulation performed with the slit 29 having a width of 300 μm.

  Further, based on the result of the first simulation, the lines of electric force reach from the surface of the suction member 6 facing the second opening 36 to the back surface of the suction member 6 facing the slit 29 side. The extent of the spread was calculated. The result is shown in FIG. FIG. 20 is a graph in which the extent of electric field lines (%) is on the vertical axis and the width (μm) of the slit 29 is on the horizontal axis.

  Referring to FIG. 20, it can be seen that as the width of the slit 29 is narrowed, the electric lines of force in the attracting member 6 are narrowed. It can be seen that the slit width is minimized around 200 μm.

  Next, a second simulation using the sample separation / adsorption instrument 100 having the configuration shown in FIG. 21 will be described. FIG. 21 is a cross-sectional view showing the main configuration of the sample separation / adsorption instrument 100. In FIG. 21, the sample separation unit 31 is provided with a hydrophilic film 43, and the adsorption member 6 is in close contact with the second opening 36.

In the second simulation, the width of the slit 29 in the Z-axis direction is changed from 2000 μm to 200 μm.
The behavior was changed stepwise over μm, and the behavior of the electric lines of force in each was simulated. The width of the second opening 36 in the Z-axis direction was 100 μm, and the applied voltage was 200 V, which is close to the actual applied voltage.

  An example of the result of the second simulation is shown in FIG. FIG. 22 shows the result of a simulation performed with the slit 29 having a width of 200 μm.

  Further, based on the result of the second simulation, the extent of the spread of the electric lines of force from the front surface to the back surface of the adsorption member 6 was calculated. The result is shown in FIG. FIG. 23 is a graph in which the extent of electric field lines (%) is on the vertical axis and the width (μm) of the slit 29 is on the horizontal axis.

  Referring to FIG. 23, as in the first simulation, in the second simulation, it can be seen that the electric lines of force in the suction member 6 are narrowed as the width of the slit 29 is reduced.

  According to the above first and second simulation results, when the width of the second opening 36 in the Z-axis direction is 100 μm and the width of the slit 29 in the Z-axis direction is between 100 μm and 1000 μm, the lines of electric force Can be effectively converged. Therefore, when the width of the second opening in the Z-axis direction is 1, the width of the slit 29 in the Z-axis direction is preferably in the range of 1-10.

  In the first and second simulation results, when the width of the slit 29 in the Z-axis direction is larger than 1000 μm, the lines of electric force are not effectively converged, and the width of the slit 29 in the Z-axis direction is When it is smaller than 100 μm, the electric lines of force are easily diffused around the slit.

(Sample migration trajectory)
Below, the result of having performed the simulation about the migration locus of a sample is explained. In this simulation, a configuration in which only the first slit structure 46 is arranged is used as a comparative example, and a configuration in which the first slit structure 46 and the second slit structure 30 are arranged is used as an example.

  In this simulation, the sample medium 50 obtained by subjecting five proteins to the first dimension electrophoresis was introduced into the separation medium 33, and the second dimension electrophoresis and the sample adsorption were performed. The results are shown in FIGS.

  FIG. 24A is a diagram showing the migration trajectory of the sample in the comparative example, and FIG. 24B is an enlarged view thereof. On the other hand, FIG. 25 (a) is a diagram showing the migration trajectory of the sample in the example, and FIG. 25 (b) is an enlarged view thereof.

  Note that the five lines shown in FIGS. 24 and 25 represent the migration loci of the five proteins.

  Comparing FIG. 24 and FIG. 25, it can be seen that the convergence of the sample is realized by providing two slit structures. That is, according to the electric force line convergence structure including two slit structures, it can be seen that the sample is adsorbed to the adsorbing member 6 with high accuracy.

The present invention can further develop proteome research that is being actively conducted. Moreover, a market can be activated by producing and selling the sample separation adsorption instrument and the various members used in the instrument according to the present invention separately.

DESCRIPTION OF SYMBOLS 1 1st buffer solution tank 2 2nd buffer solution tank 6 Adsorption member 29 Slit 30 2nd slit structure 31 Sample separation part 32 Insulation part 33 Separation medium 35 1st opening 36 2nd opening 37 Hydrophilic film | membrane 38 Elastic material 39 Slit 40 Slit block 41 1st electrode 42 2nd electrode 44 Insulation part 45 Slit 46 1st slit structure 61 Moving arm 100 Sample separation adsorption instrument

Claims (9)

A sample separation / adsorption instrument for separating a sample in the separation medium by passing an electric current through the buffer to the separation medium and adsorbing the separated sample from the separation medium to a sample adsorption member,
A first electrode;
A second electrode;
A sample separation unit that has a first opening that opens on the side facing the first electrode and a second opening that opens on the side facing the second electrode, and stores the separation medium;
In the sample adsorbing member, an electric force line converging structure composed of first and second insulating members forming a slit at a position sandwiching a portion facing the second opening from the front and rear in the sample separation direction;
A sample separation / adsorption device comprising: a moving means for moving a relative position of the sample adsorption member with respect to the slit in a direction substantially perpendicular to the separation direction. The first insulating member is arranged in the second opening of the sample separation part, or is configured as a part of the sample separation part so as to form the second opening as the slit,
The sample separation and adsorption device according to claim 1, wherein the second insulating member is disposed between the sample adsorption member and the second electrode.   3. The sample according to claim 2, wherein the second insulating member is provided with an elastic material capable of transmitting a buffer solution that presses the sample adsorbing member toward the second opening. 4. Separation adsorption device.   A hydrophilic film is formed on a surface of the first insulating member facing the sample adsorbing member and a surface of the second insulating member or the elastic material facing the sample adsorbing member. The sample separation / adsorption device according to claim 3, wherein the sample separation / adsorption device is provided.   The said 1st insulating member leaves the area | region used as the said slit on the surface of a hydrophilic film | membrane, forms the said slit by apply | coating or sticking an insulating material, The said slit is formed. Sample separation adsorption equipment.   The said 2nd insulating member leaves the area | region used as the said slit on the surface of an elastic material or a hydrophilic film | membrane, and forms the said slit by apply | coating or sticking an insulating material, It is characterized by the above-mentioned. The sample separation / adsorption device according to any one of 3 to 5.   The slit block which has a through-hole in the position which opposes the said slit which the said 2nd insulating member forms, and supports the said 2nd insulating member is further provided. 2. The sample separation / adsorption device according to item 1. A first buffer solution tank in which the first electrode is disposed;
A second buffer tank in which the second electrode is disposed;
The sample separation / adsorption device according to claim 7, wherein the through hole in the slit block is filled with a buffer solution filled in the second buffer solution tank.   The sample separating and adsorbing device according to any one of claims 3 to 6, wherein the elastic material is made of an elastic material having a through hole or a material that expands when water is absorbed.

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