WO2015093282A1 - 生体分子分析装置 - Google Patents
生体分子分析装置 Download PDFInfo
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- WO2015093282A1 WO2015093282A1 PCT/JP2014/081856 JP2014081856W WO2015093282A1 WO 2015093282 A1 WO2015093282 A1 WO 2015093282A1 JP 2014081856 W JP2014081856 W JP 2014081856W WO 2015093282 A1 WO2015093282 A1 WO 2015093282A1
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- biomolecule
- sample
- opening
- separation
- electrode
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
- G01N27/44704—Details; Accessories
- G01N27/44717—Arrangements for investigating the separated zones, e.g. localising zones
- G01N27/44739—Collecting the separated zones, e.g. blotting to a membrane or punching of gel spots
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
- G01N27/44704—Details; Accessories
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
- G01N27/44756—Apparatus specially adapted therefor
- G01N27/44773—Multi-stage electrophoresis, e.g. two-dimensional electrophoresis
- G01N27/44778—Multi-stage electrophoresis, e.g. two-dimensional electrophoresis on a common gel carrier, i.e. 2D gel electrophoresis
Definitions
- the present invention relates to a biomolecule analyzer that separates a biomolecule sample in a separation medium, and continuously adsorbs the separated biomolecule sample to a sample adsorbing member.
- 2DE two-dimensional electrophoresis
- Western blotting is an indispensable step when comprehensively examining multiple biological characteristics of proteins such as increase / decrease in protein expression and presence / absence of post-translational modification using separation results by 2DE. I can say that.
- Patent Document 1 proposes a technique for performing electrophoresis and transfer with a single device in capillary electrophoresis (CE) using a capillary tube. Specifically, the sample discharged through the capillary (inside the gel or solution) is adsorbed to the transfer film as it is, and from separation to recovery (immobilization to the transfer film) is performed with one apparatus. It can be done. According to the present technology, electrophoresis and transfer can be performed continuously.
- CE capillary electrophoresis
- JP-A-4-264253 Japanese Patent Publication “JP-A-4-264253” (published on September 21, 1992)
- the present invention has been made in view of the above-mentioned problems, and the purpose thereof is to enable continuous execution from separation by electrophoresis to transfer to a sample adsorbing member, and a high-resolution sample.
- An object of the present invention is to provide a biomolecule analyzer that realizes adsorption.
- a biomolecule analyzer includes: A biomolecule analyzer that separates a sample in the separation medium by passing an electric current through the buffer solution through the buffer and adsorbs the separated biomolecule sample from the separation medium to the adsorption member, A first electrode; A second electrode; A separation portion that is provided with a first opening that opens on the first electrode side and a second opening that opens on the second electrode side, and stores the separation medium; A pressing tool located between the separation part and the second electrode, the first structure disposed on the separation part side, and the second structure disposed and fixed on the second electrode side; And a pressing tool sandwiching an elastic member having an insulating property by,
- the first structure body is provided with a first through hole penetrating from the separating portion side to the second electrode side at a position opposite to the second opening
- the second structure body is provided with a second through hole penetrating from the separation portion side to the second electrode side at a position opposite to the second opening,
- the adsorption member is
- biomolecule analyzer that can continuously perform separation from electrophoresis to transfer to a sample adsorbing member and realize high-resolution sample adsorption. Play.
- FIG. 1 It is a top view which shows the structure of one Embodiment of the biomolecule analyzer which concerns on this invention. It is sectional drawing of the biomolecule analyzer shown in FIG. It is the figure which showed typically the breadth of the electric line of force in the one part area
- Embodiment 1 Hereinafter, an embodiment of a biomolecule analyzer according to the present invention will be described in detail with reference to FIGS.
- FIG. 1 is a top view schematically showing the biomolecule analyzer 1
- FIG. 2 is a cross-sectional view taken along line AA ′ shown in FIG.
- the biomolecule analyzer 1 of the first embodiment is an instrument for separating biomolecule sample components according to molecular weight, and subsequently transferring (adsorbing) the separated biomolecule sample to a transfer film. Therefore, the biomolecule analyzer 1 according to the present embodiment includes a housing 2, a first buffer tank 3 in which a cathode 31 (first electrode) is disposed, and a first electrode in which an anode 41 (second electrode) is disposed. 2 buffer tank 4, sample separation unit 5 (separation unit, second-dimensional electrophoresis unit), presser 6, transfer film 7 (adsorption member), and transfer film moving arm 70 (adsorption member moving means) ( 2) and a sample introduction arm 82 (medium moving means) (FIG. 2). The first buffer tank 3, the second buffer tank 4, the sample separation unit 5, the presser 6 and the transfer film 7 are disposed in the housing 2.
- the sample separation unit 5 has a first opening 57 that opens toward the first buffer solution tank 3 and a second opening 58 that opens toward the second buffer solution tank 4.
- the first opening 57 opens to the cathode 31 side
- the second opening 58 opens to the anode 41 side.
- the anode 41 includes a buffer solution in two tanks (first buffer tank 3 and second buffer tank 4), a separation gel 53 (separation medium) (FIG. 2) stored in the sample separation unit 5, and a transfer. Electrical connection is made via the membrane 7.
- the biomolecule analyzer 1 separates the biomolecule sample introduced from the first opening 57 by the separation gel 53 (FIG. 2) of the sample separation unit 5 by applying a voltage to the cathode 31 and the anode 41.
- the separated components can be discharged from the second opening 58 and adsorbed onto the transfer film 7.
- the separation direction of the biomolecule sample defined by the cathode 31 and the anode 41 is the x-axis direction (first direction), and the movement direction of the transfer film 7 is the y-axis direction. (Second direction), and a direction perpendicular to both the x-axis and the y-axis is defined as a z-axis direction.
- the cathode 31 is arranged in the first buffer solution tank 3, and the anode 41 is arranged in the second buffer solution tank 4.
- the cathode 31 and the anode 41 are formed from a conductive material such as metal.
- a material for forming the cathode 31 and the anode 41 for example, platinum is preferable from the viewpoint of suppressing ionization of the electrode.
- the cathode 31, the second opening 58, and the anode 41 are arranged substantially in a straight line. Furthermore, it is preferable that a slit 61a (first through hole) and a through hole 62a (second through hole) of the pressing tool 6 described later are also arranged in the same straight line as these. In such an arrangement, if the transfer film 7 is arranged as shown in FIGS. 1 and 2, the lines of electric force passing through the second opening 58 are substantially perpendicular to the transfer film 7. The accuracy of adsorption of the biomolecule sample can be improved.
- the anode 41 is disposed away from the transfer film 7. Thereby, it is possible to suppress the bubbles generated from the anode 41 from adversely affecting the adsorption of the separation component to the transfer film 7.
- the cathode 31 and the anode 41 may be disposed in the first buffer solution tank 3 and the second buffer solution tank 4 in advance, but when a cover that covers the housing 2 is provided, the lower surface of the cover When the cathode 31 and the anode 41 are suspended from and covered with a cover, the cathode 31 and the anode 41 may be disposed in the first buffer solution tank 3 and the second buffer solution tank 4.
- the first buffer tank 3 and the second buffer tank 4 are formed by attaching the sample separation unit 5 in the housing 2 and dividing the housing 2 into two tanks.
- the buffer solution put in the first buffer solution tank 3 and the second buffer solution tank 4 is a conductive material used as a well-known electrophoretic buffer solution as long as there is no possibility of adversely affecting the separation gel 53 and the transfer membrane 7. Any buffer solution having the following can be adopted.
- a buffer solution containing 3-morpholinopropanesulfonic acid (MOPS) or trishydroxymethylaminomethane (Tris) is used as a cathode buffer solution (first buffer solution) to be placed in the first buffer solution tank 3, and ethanol is used.
- the buffer solution containing pH 6.5 to 8.8 is used as the anode buffer solution (second buffer solution) to be put in the second buffer solution tank 4.
- the separation gel 53 accommodated in the sample separation unit 5 shown in FIG. 2 is prepared using a buffer solution containing Bis-Tris (Tris) or Tris-hydroxymethylaminomethane (Tris). It is preferable to employ the gel prepared.
- a cathode buffer solution and an anode buffer solution having the following composition and a separation gel are used.
- Anode buffer solution 100 mM MOPS (pH 7.3) 50 mM Trishydroxymethylaminomethane (Tris) 50 mM Bis-Tris 20% ethanol
- Cathode buffer solution 100 mM MOPS (pH 7.2) 50 mM Tris 50 mM Bis-Tris 0.25% sodium dodecyl sulfate (SDS)
- the sample separation unit 5 includes a first opening 57 that is a sample supply medium connection portion that opens toward the first buffer solution tank 3 and a sample component discharge port that opens toward the second buffer solution tank 4.
- a second opening 58 is provided.
- the sample separation unit 5 includes two plates (a lower plate 51 and an upper plate 52) formed of an insulator such as glass or acrylic and having an xz plane in the y direction.
- the separation gel 53 (separation medium) is stored in the gap with a predetermined gap.
- the separation gel 53 faces the first buffer solution tank 3 through the first opening 57 and faces the second buffer solution tank 4 through the second opening 58.
- the end on the first opening 57 side of the lower plate 51 arranged on the lower side protrudes from the end of the upper plate 52.
- the first opening 57 side of the separation gel 53 is exposed on the upper surface, and the biomolecule sample can be introduced from this exposed portion.
- the separation gel 53 is a gel for separating the biomolecule sample component introduced from the first opening 57 according to the molecular weight.
- Examples of the separation gel 53 include an acrylamide gel and an agarose gel, and it is preferable to use a gel that is suitable for the above-described composition and matched to a buffer solution.
- the separation gel 53 can be formed by filling the sample separation part 5 before or after attaching the sample separation part 5 to the housing 2.
- the configuration in which the sample separation unit 5 is filled with the separation gel 53 is adopted.
- a large number of ultrafines called nanopillars are provided between two opposing plates constituting the sample separation unit 5.
- a configuration in which a column is provided may also be employed.
- the second opening 58 of the sample separation unit 5 may be covered with a coating part (conductive medium: not shown) formed of a porous material including the periphery thereof. Accordingly, it is possible to reduce the frictional resistance and damage that the transfer film 7 receives from the sample separation unit 5 and the separation gel 53 when the transfer film 7 that is in contact with or pressed against the second opening 58 is conveyed.
- a coating part conductive medium: not shown
- the porous material forming the covering portion is preferably a material having through-holes and a material having hydrophilicity, low sample adsorption ability, and high strength.
- separated component passes can pass the isolate
- the separation gel 53 is sufficiently filled into the second opening 58 when the separation gel 53 is filled, and the pores Is filled with the separation gel 53. Thereby, the transfer film 7 and the separation gel 53 can be brought into close contact with each other. Therefore, the separated component can be reliably prevented from diffusing into the buffer solution, and a stable energized state can be maintained.
- Examples of the material for forming the covering portion include film-like materials such as a hydrophilic PVDF (Polyvinylidene difluoride) film and a hydrophilic PTFE (Polytetrafluorethylene) film.
- Examples of the method for attaching the covering portion to the sample separation portion 5 include a method using an adhesive tape or an adhesive, and a method of fixing the sample separation portion 5 and the covering portion with a clip or the like.
- Examples of the method of including the separation gel 53 in the coating portion include a method of filling the sample separation portion 5 with the separation gel 53 after attaching the coating portion around the second opening 58 and the periphery thereof.
- the separation gel 53 when a polyacrylamide gel is used as the separation gel 53, the acrylamide solution before gel polymerization may be poured from the side of the first opening 57 of the sample separation portion 5 to which the covering portion is attached, and then gel polymerization may be performed.
- the sample component discharge port is covered with the coating part, the electric lines of force spread excessively while passing through the coating part, and as a result, the biomolecule sample further spreads before reaching the transfer film. It is not preferable.
- the biomolecule analyzer 1 of the first embodiment there is no problem because the second opening 58 is covered by the covering portion and is converged by the slit 61a as described later.
- the transfer film 7 is preferably a biomolecule sample adsorbing / holding body that enables the biomolecule sample separated by the separation gel 53 to be stably stored for a long period of time and further facilitates subsequent analysis.
- the material of the transfer film 7 is preferably a material having high strength and high sample binding ability (weight that can be adsorbed per unit area).
- a PVDF film or the like is suitable as the transfer film 7, when the sample is protein.
- the PVDF membrane is preferably hydrophilized in advance using methanol or the like.
- the biomolecule sample that can be separated and adsorbed by the biomolecule analyzer 1 can include, but is not limited to, protein, DNA, and RNA.
- a preparation from a biological material for example, an individual organism, a body fluid, a cell line, a tissue culture, or a tissue fragment
- a commercially available reagent can be used.
- a polypeptide or polynucleotide is mentioned.
- the transfer film 7 extends in the yz plane between the second opening 58 and the slit 61a of the slit structure 61 (first structure).
- the transfer film 7 is configured in advance so as to be pulled up in the y direction as will be described later. Therefore, one end of the long transfer film 7 enters under the bottom of the sample separation unit 5 and is at the bottom in the first buffer solution tank 3, and the other end is held by the transfer film moving arm 70.
- the transfer film 7 is transported in the direction of the arrow (+ y direction) in FIG. 2 when the biomolecule sample is separated and adsorbed.
- one end of the transfer film 7 is at the bottom of the first buffer solution tank 3, and the length of the transfer film from the one end to the other end is a predetermined length.
- the present invention is not limited to this, and the one end may constitute a transfer film transfer film roll around which the transfer film is wound and may be pulled out from the transfer film roll. .
- a transfer film having a desired length can be passed between the second opening 58 and the slit 61 a of the slit structure 61.
- the transfer film roll is rotatably attached to the inner wall of the main body of the biomolecule analyzer 1.
- the transfer membrane roll is preferably arranged at such a height as in the buffer solution. This is to prevent the transfer film 7 from being dried during the separation and adsorption of the biomolecule sample. Further, the transfer film roll is preferably disposed at a position separated from each electrode. This is to prevent bubbles generated from each electrode from adhering to the transfer film 7.
- a guide composed of a rotary shaft may be provided as appropriate.
- the biomolecule analyzer 1 may be provided in a state in which the transfer film 7 is attached or in a state in which the transfer film 7 is attached later by a user. In either case, the transfer film 7 is immersed in a buffer solution. It will be in the state.
- the transfer film moving arm 70 has a configuration in which the transfer film 7 is pulled up in the + y direction.
- the transfer film moving arm 70 is not limited to a pull-up type, and may be configured as a transfer film recovery member that winds up the transfer film 7 by a rotating operation. If the transfer film collecting member is used, it is not necessary to secure a wide driving range as in the transfer film moving arm 70 that pulls up the transfer film 7 in the + y direction, and the biomolecule analyzer 1 can be downsized.
- sample introduction arm 82 As shown in FIG. 2, the sample introduction arm 82 is used to introduce a biomolecule sample into the first opening 57 of the sample separation unit 5, and holds the gel strip 80 supported by the support plate 81. Since the gel strip 80 is generally thin and soft, it is not directly held by the sample introduction arm 82, but is fixed to the sample introduction arm 82 by fixing the gel strip 80 to a support plate 81 made of an acrylic plate, a resin film or the like. Retained.
- two arms ie, the transfer film moving arm 70 and the sample introduction arm 82 are used.
- the present invention is not limited to this, and one of them is omitted and only one moving arm is provided. There may be.
- one arm 70 or 82 may hold and transfer the transfer film 7 when the biomolecule sample is separated and transferred after the gel strip 80 is introduced into the first opening 57.
- the pressing tool 6 presses the transfer film 7 against the second opening 58 of the sample separation unit 5 with a predetermined pressure, and between the sample separation unit 5 and the anode 41, the slit 61a and the fixing of the slit 61a. It is a member for inhibiting current from passing through the tool 62 (second structure) other than the through hole 62a. That is, the pusher 6 regulates the path (current flow) of the lines of electric force generated from the charges induced in the cathode 31 and the anode 41 to which the voltage is applied between the sample separation unit 5 and the anode 41. It is a member for.
- the buffer solution that is the path through which the current flows in the second buffer solution tank 4 is pushed by the pusher 6 except for the slit 61a of the slit structure 61 and the through-hole 62a of the fixture 62 (second structure).
- the structure does not pass through the transfer film 7.
- the pusher 6 includes an elastic body 620 (elastic member) sandwiched between a slit structure 61 in which a slit 61a extends in the y direction and a fixture 62, and more specifically, the elastic body 620 is fixed. It arrange
- the slit structure 61 is located closest to the transfer film 7 in the pressing tool 6 and has a contact surface that contacts the transfer film 7.
- the slit structure 61 is provided with a slit 61 a having a width in the y direction of 50 to 300 ⁇ m that passes between the contact surface and the back surface, and the slit 61 a is located at a position facing the second opening 58. . By being in this position, the lines of electric force generated from the cathode 31 to the anode 41 are converged with the central position of the slit 61a as the convergence point.
- FIG. 3 is a diagram schematically showing electric lines of force in the transfer film 7 and the vicinity thereof, and is a diagram schematically showing electric lines of force in the partially enlarged sectional view of FIG.
- the slit 61a is located on the back surface side (the anode 41 side) of the transfer film 7, electric lines of force can be narrowed from the separation gel 53 to the slit 61a.
- the lines of electric force that have been subjected to the effect of the diaphragm pass over the transfer film 7 located on the front surface side (second opening 58 side) of the slit 61a. Since the biomolecule sample flows along the lines of electric force, it is adsorbed and held in a concentrated state on the transfer film 7 due to the influence of the convergence. That is, the biomolecule sample can be transferred to the transfer film 7 with high resolution.
- the width in the y direction of the slit structure 61 is configured to be narrower than the width in the y direction (1.0 to 1.2 mm) of the second opening 58 of the sample separation unit 5.
- the slit structure 61 is preferably made of a material having a low conductivity, and more preferably made of an insulating material.
- the slit structure 61 can be made of glass, ceramic, resin, or the like.
- a convex portion 61 b that protrudes toward the fixture 62 is provided on the anode 41 side of the slit structure 61.
- the fixture 62 is located closer to the anode 41 than the slit structure 61, and is adhesively fixed to the housing 2 at a contact portion with the housing 2. Similar to the slit structure 61, the fixture 62 can be made of glass, ceramic, resin, or the like.
- the through-hole 62a is provided in the fixture 62, and a recess 62b is provided on the surface of the fixture 62 on the slit structure 61 side so as to surround the through-hole 62a.
- the width of the through hole 62a is desirably equal to or greater than the width of the slit 61a, and more desirably larger than the width of the slit 61a.
- the recess 62b is open on the surface of the fixture 62 on the slit structure 61 side, and is formed as a groove formed in a square shape as shown in FIG. 4 which is an exploded perspective view of the pusher 6. Yes.
- the convex portion 61 b provided in the slit structure 61 described above can be inserted into a concave portion 62 b formed in a square shape on the fixture 62. It has a square shape so that it can.
- the elastic body 620 is disposed in the recess 62b so as to completely fill the recess 62b. Therefore, the convex portion 61 b applies pressure to the elastic body 620 but is pushed back by the restoring force of the elastic body 620.
- the slit structure 61 having the convex portion 61b is pushed back in the ⁇ x direction.
- the slit structure 61 that has received this push-back keeps the transfer film 7 between the second opening 58 and the slit structure 61 constant toward the second opening 58 from the second buffer solution tank 4 side of the transfer film 7. Press with pressure of.
- the slit structure 61 is pressed in the ⁇ x direction (toward the second opening 58) by the fixture 62 shown in FIG. 2 and the elastic body 620 disposed in the recess 62b of the fixture 62.
- the state in which the slit forming surface (contact surface) of the slit structure 61 is in contact with the transfer film 7 can be maintained, and the state in which the transfer film 7 is in contact with the second opening 58 can be maintained.
- the transfer film 7 can be arranged at a position near the convergence point of the electric force lines in the slit 61a, high-resolution sample adsorption can be realized at the time of biomolecule sample separation adsorption.
- the transfer film 7 When the transfer film 7 is pressed from the second buffer solution tank 4 side of the transfer film 7 toward the second opening 58, the transfer film 7 may be brought into contact with the second opening 58 if the pressing force is weak. On the other hand, if the pressing force is too strong, the transfer film moving arm 70 may pull up the transfer film 7 in the + y direction as shown in FIG. is there.
- the biomolecule analyzer 1 is provided with a structure in which the sample separation unit 5 can be positioned and fixed detachably in the housing 2, and the fixture 62 is positioned and fixed.
- the structure which can do is provided.
- the relative position between the sample separation unit 5 and the slit structure 61 can be accurately determined, and accordingly, the pressing force in the ⁇ x direction by the slit structure 61 can be set to a suitable magnitude.
- the pressure applied to the transfer film 7 by the slit structure 61 is preferably 0.1N to 10N, and more preferably 1N to 5N.
- the sample separation unit 5 is then placed in the housing 2 as shown in FIG. 8.
- the sample separation unit 5 can be smoothly set on the biomolecule analyzer 1 by the pusher 6 having cushioning properties.
- the slit structure 61 is pushed toward the fixture 62 in the direction of the arrow shown in FIG. 7 by the sample separator 5, and the slit structure 61 is pushed back toward the sample separator 5 by the restoring force of the elastic body 620. .
- the elastic force of the elastic body 620 is related to the contact of the transfer film 7 to the second opening 58 and the frictional resistance.
- the elastic body 620 can be formed using a stretchable material such as a polymer, rubber, gel, or sol and having a low electrical permeability, or an insulating material.
- a stretchable material such as a polymer, rubber, gel, or sol and having a low electrical permeability, or an insulating material.
- the rubber include nitrile rubber, fluorine rubber, silicone rubber, ethylene propylene rubber, chloroprene rubber, acrylic rubber, butyl rubber, urethane rubber, natural rubber, chlorosulfonated polyethylene rubber, and epichlorohydrin rubber.
- the elastic body 620 is realized by filling the concave portion 62b formed in a square shape of the fixture 62 with a polymer, rubber, gel, sol, or the like.
- a spring may be used as the elastic body 620.
- the elastic body 620 is more preferably made of a material having chemical resistance, heat resistance, and oxidation resistance, since it is easy to adjust the pressing force (pressure) of the slit structure 61 against the transfer film. However, it is not limited to this. Note that the pressing force (pressure) of the slit structure 61 against the transfer film is not limited to this, but may be, for example, 3.45 N ( ⁇ 15%).
- the region between the slit structure 61 and the fixture 62 has a ring shape (b-shaped) surrounding the opening on the anode 41 side in the slit 61 a.
- the body 620 and the convex portion 61b of the slit structure 61 form a cylindrical (restricted) space having one end opened by the slit 61a and the other end opened by the through hole 62a. Since this space is not only divided, but also electrically restricted (closed), the electric lines of force generated by applying a voltage to the cathode 31 and the anode 41 are generated by the slit structure 61. After passing through the slit 61a, it passes through the through hole 62a of the fixture 62.
- the path of the electric lines of force can be limited (limited) to the slit 61 a of the slit structure 61 by the elastic body 620 and the convex portion 61 b of the slit structure 61.
- the elastic body 620 also surrounds the opening of the through hole 62a on the fixing device 62 side.
- the fixture 62 is bonded and fixed to the housing 2, and the bottom of the fixture 62 and both side portions are in contact with the inner wall of the housing 2 and sealed.
- the fixture 62 is provided at a place other than the through hole 62 a provided at the center of the fixture 62.
- the slit structure 61 side and the second buffer solution tank 4 do not communicate with each other. With this structure, the path of the lines of electric force can be limited (limited) to the through hole 62a of the fixture 62.
- the electric line of force is x.
- the accuracy of the adsorption of the biomolecule sample to the transfer film 7 is lowered without converging in the axial direction and the lines of electric force are not substantially perpendicular to the transfer film 7.
- the accuracy of adsorption of the biomolecule sample to the transfer film 7 is improved by restricting the path and converging the electric lines of force on the slit 61a of the slit structure 61 as in the first embodiment. Can do.
- the pressing portion is provided so as to surround the portion where the electric lines of force converge, the entire surface of the transfer film contact surface in the slit structure 61 is made uniform with respect to the transfer film 7. Can be pressed. For example, if the slit structure 61 is pushed back from the second buffer solution tank 4 side using a spring instead of the fixture 62 and the elastic body 620, depending on the number and location of the spring, the slit In some cases, it is difficult for the structure 61 to uniformly press the transfer film 7, and as a result, the sample may not be favorably adsorbed to the transfer film 7.
- the transfer film 7 can be pressed against the second opening 58 with a uniform pressure, and the transfer film 7 can be brought into good contact with the second opening 58 to biomolecules.
- the sample can be adsorbed on the transfer film 7 with high resolution.
- the elastic body 620 is gelled by using a sol-like elastic precursor (for example, a resin monomer) into the concave portion 62b formed in a square shape of the fixture 62 (using an organic chemical or optical technique). 4), a rectangular-shaped elastic body 620 is formed in advance and formed into a rectangular shape of the fixture 62 as shown in FIG. It is also possible to form it by fitting it into the recessed portion 62b. For example, by adopting a method of gelling by pouring resin monomer, it has the merit that it can be easily manufactured, it can be used repeatedly, does not adversely affect electrophoresis, and is continuously reproducible There is also an advantage that it can be analyzed well.
- a sol-like elastic precursor for example, a resin monomer
- the sample introduction arm 82 holds the gel strip 80 supported by the support plate 81, and is attached to the sample introduction arm 82 of FIG. 2 until the gel strip 80 is inserted into or contacts the first opening 57. Move in the direction of the arrow.
- the gel strip 80 contains components obtained by separating a biomolecule sample one-dimensionally by isoelectric focusing.
- a voltage is applied between the cathode 31 and the anode 41 in a state where the gel strip 80 is inserted or brought into contact with the first opening 57. Thereby, each component contained in the gel strip 80 is further separated in the separation gel 53 according to their molecular weight.
- the first-dimensional electrophoresis unit may be incorporated in the biomolecule analyzer 1 according to the first embodiment. Accordingly, it is possible to automate from the first-dimensional isoelectric focusing separation to the second-dimensional electrophoresis separation and transfer to the transfer film.
- a well (dent) for filling the separation gel 53 with a biomolecule sample may be formed.
- the biomolecule sample is fixed using an agarose gel or the like to prevent the biomolecule sample from flowing out to the first buffer solution tank 3.
- the biomolecule sample may be introduced by mixing with an agarose gel and coagulated in the well.
- the above-mentioned well is formed by the same method as normal SDS-PAGE. That is, after pouring the gel monomer solution (the solution before being polymerized and gelled) into the first opening 57 and before the gel monomer is polymerized, a plurality of combs (usually having a height (depth) of about 5 mm). A comb-like plate having irregularities is inserted into the first opening 57 and then gelled. After gelling, the well is formed by removing the comb.
- the current is passed between the cathode 31 and the anode 41 to separate the biomolecule sample by electrophoresis.
- the value of current flowing between the electrodes is preferably 50 mA or less, and more preferably 20 mA or more and 30 mA or less. If it is the said range, heat_generation
- the transfer film 7 is gradually conveyed in the direction of the arrow in FIG. 2 by driving the transfer film moving arm 70 in accordance with the progress of electrophoresis in the sample separation unit 5.
- Whether or not the separated component has reached the second opening 58 is determined by mixing a marker stained with a biomolecule sample in advance and confirming the electrophoretic state according to the position of the marker, or measuring the voltage value with a monitor. Judgment can be made.
- the stained marker BPB (Bromophenol Blue) which is usually used for confirming the beginning of electrophoresis is preferable.
- the voltage monitor (voltage detection means: not shown) which monitors the voltage between the cathode 31 and the anode 41 is mentioned, for example.
- the biomolecule analyzer 1 automatically detects the discharge of the component from the separation gel 53 by incorporating a program for monitoring the voltage value, and starts to pull up the transfer film 7 by the transfer film moving arm 70. be able to.
- the pulling speed of the transfer film 7 after the start of component adsorption can also be controlled by the voltage value or the current value.
- the pulling speed of the transfer film 7 may be a speed at which the biomolecule sample can be adsorbed to the transfer film 7 with sufficient resolution, and such a speed can be appropriately set by those skilled in the art. According to these controls, it is possible to make the transfer result reproducible, avoid useless use of the transfer film 7 (occurrence of a portion not adsorbing components), and reduce the size of the biomolecule analyzer. Is possible.
- the first to second dimensional electrophoresis to the transfer can be performed continuously in one apparatus.
- the transfer film 7 is collected by the transfer film moving arm 70 and used for staining or immune reaction. Thereafter, a separation pattern of components adsorbed on the transfer film 7 is detected by a fluorescence detector or the like.
- a fluorescence detector may be incorporated in the biomolecule analyzer 1, thereby automating all the steps of electrophoresis, transcription, and detection.
- the biomolecule analyzer 1 is configured as a two-dimensional electrophoresis device in which the sample separation unit 5 is configured as a second-dimensional electrophoresis unit and the first-dimensional electrophoresis unit is incorporated. Can be configured.
- the gel strip 80 is used as a separation medium for the first-dimensional electrophoresis, and the gel strip 80 after the first-dimensional electrophoresis (isoelectric focusing) is performed is the sample introduction arm. 82 is inserted into the first opening 57 of the sample separator 5.
- the elastic body 620 presses the slit structure 61 toward the sample separation unit 5.
- the transfer film 7 disposed between the slit 61 a and the second opening 58 is close to the second opening 58.
- the separated biomolecule sample discharged from the second opening 58 can be adsorbed to the transfer film 7, and the separation from electrophoresis to transfer to the transfer film 7 can be continuously performed.
- the transfer film 7 is close to the second opening 58, the separated biomolecule sample discharged from the second opening 58 can be effectively adsorbed to the transfer film 7, and high-resolution sample adsorption can be achieved. Can be realized.
- the elastic body 620 restricts the area where the buffer solution exists between the second opening 58 side and the fixture 62 side of the slit structure 61 to the slit 61a. Thereby, the electric current which passes through the slit structure 61 other than the slit 61a and the through-hole 62a of the fixing tool 62 and flows from the sample separation part 5 side of the pressing tool 6 to the anode 41 side is inhibited. As a result, if there is no such configuration and a current flows in a portion other than the slit 61a of the slit structure 61 and further in a portion other than the through hole 62a of the fixture 62, electric lines of force are formed there. .
- the biomolecule sample may flow to an unspecified location other than the slit 61a of the slit structure 61 and the through hole 62a of the fixture 62.
- the biomolecule sample is not concentrated and adsorbed on a specific portion of the transfer film 7 and the resolution is lowered.
- the pusher 6 causes the buffer solution, which is a path through which current flows in the second buffer solution tank 4, to pass through other than the slit 61 a of the slit structure 61 and the through hole 62 a of the fixture 62.
- the transfer film 7 is not contacted. Therefore, the biomolecule sample is concentrated and adsorbed on a specific portion of the transfer film 7. Thereby, high-resolution sample adsorption can be realized.
- the biomolecule analyzer 1 is a horizontal device that performs sample separation in a substantially horizontal direction, but the present invention is not limited to this and may be a vertical device.
- the path of the electric lines of force is regulated by the square-shaped elastic body 620 and the square-shaped convex portion 61 b and the fixing fixing of the fixture 62 to the housing 2.
- the present invention is not limited to this, and the path of the electric lines of force may be restricted only by the square-shaped elastic body 620 and the square-shaped convex portion 61b.
- the above-described path restriction of the electric lines of force is performed only by the square-shaped elastic body 620 and the square-shaped convex portion 61b. According to this, it is not necessary to hermetically fix the fixing tool 62, and it is sufficient that the fixing tool 62 is fixed to the housing 2 to such an extent that the slit structure 61 can be supported.
- region in which the square-shaped elastic body 620 in 62 is formed may be provided.
- the water level of the buffer solution for the anode is lower than the upper end of the fixture 62, but the water level is higher than the upper end of the fixture 62. Also good.
- FIG. 9 is a cross-sectional view of the pusher provided in the biomolecule analyzer of the second embodiment, and is a view seen from the same direction as FIG.
- the difference between the pressing tool 6 ′ of the second embodiment and the pressing tool 6 of the first embodiment is the unevenness and the location where the elastic body is disposed.
- the pressing tool 6 ′ of the second embodiment is provided with a recess 61 c on the fixing tool 62 side of the slit structure 61, and on the contrary, the slit structure of the fixing tool 62.
- a convex portion 62 c that protrudes toward the slit structure 61 is provided on the body 61 side.
- the elastic body 610 (elastic member) is held in the recess 61c of the slit structure 61, and the elastic body 610 held in the recess 61c of the slit structure 61 is installed when the sample separation unit 5 is installed.
- the protrusion 62c of the fixture 62 is inserted from the side of the fixture 62 of the elastic body 610, and the slit structure 61 is pushed back by the restoring force of the elastic body 610, so that the slit structure 61 is transferred as in the first embodiment.
- a mechanism for pressing the membrane toward the second opening is realized.
- FIG. 10 is a cross-sectional view of the pusher provided in the biomolecule analyzer of the third embodiment, and is a view seen from the same direction as FIG.
- the difference between the pressing tool 6 ′′ of the third embodiment and the pressing tool 6 of the first embodiment is that the pressing tool 6 ′′ of the third embodiment has no unevenness.
- the pressing tool 6 ′′ of the third embodiment includes a fixing structure 62 ′ side of the slit structure 61 ′ and a slit structure structure 61 ′ side of the fixing tool 62 ′. Both are flat and have no irregularities.
- the slit structure 61 ′ is provided with a slit 61 a
- the fixture 62 is provided with a through hole 62 a.
- a square-shaped elastic body 620 that forms a cylindrical (restricted) space having one end opened by a slit 61a and the other end opened by a through hole 62a. ing.
- the elastic body 620 is pressed so that the convex portion 61b is recessed, but in the third embodiment, the surface of the slit structure 61 ′ on the fixture 62 ′ side is pressed against the elastic body 620.
- a restoring force of the elastic body 620 is generated to realize a mechanism in which the slit structure 61 ′ presses the transfer film toward the second opening as in the first embodiment.
- the elastic body 620 is simply sandwiched between the slit structure 61 ′ and the fixture 62 ′, so that the elastic body 620 can be easily replaced, and the elasticity of any thickness and hardness can be obtained. You can choose your body. In addition, when it deteriorates with time, there is also an advantage that only the elastic body can be easily replaced.
- the flat bottom and side portions of the fixture 6 are bonded and fixed to the flat surface inside the housing 2.
- the present invention is not limited to this, and in the biomolecule analyzer 1 ′ shown in FIG. 11, the concave groove 2 a is formed at a predetermined position inside the housing 2, and the fixing tool 62 side is formed.
- a convex groove 622 that fits into the concave groove 2 a is formed in the part, and the fixture 62 ′′ can be fixed at a predetermined position inside the housing 2 by fitting these. Thereby, positioning of the fixture 62 ′′ in the housing 2 is facilitated, it is not necessary to use an adhesive, and the fixing of the fixture 62 ′′ to the housing 2 can be easily realized.
- the fitting 62 ′′ is fixed to the housing 2 with high reliability by fitting the concave groove 2a and the convex groove 622, so that the slit structure 61 is pressed (toward the transfer film). Can be realized reliably.
- the biomolecule analyzer according to aspect 1 of the present invention is An electric current is passed through the separation medium (separation gel 53) through the buffer solution to separate the biomolecule sample in the separation medium (separation gel 53), and the separated biomolecule sample is separated from the separation medium (separation gel 53).
- a second through hole (through hole 62a of fixtures 62, 62 ′, 62 ′′) is provided,
- the adsorption member (transfer film 7) is disposed between the second opening 58 and the first through hole (slit 61a),
- the elastic members (elastic bodies 620, 610) press the first structure (slit structure 61) toward the separation part (sample separation part 5),
- the elastic member (elastic bodies 620, 610) surrounds the opening on the second electrode (anode 41) side in the first through hole (slit 61a) and also in the second through hole (through hole 62a). It has an annular shape surrounding the opening on the separation part side, and the flow path of the buffer solution (anode buffer solution) between the openings is limited by the annular elastic member (elastic body 620, 610). It is characterized by that.
- an elastic member elastic body 620,610
- a 1st structure (slit) An adsorbing member (transfer film 7) disposed between the first through hole (slit 61 a) of the structure 61) and the second opening 58 is close to the second opening 58.
- the separated biomolecule sample discharged from the second opening 58 can be adsorbed to the adsorbing member (transfer film 7), and the process from separation by electrophoresis to transfer to the adsorbing member (transfer film 7) is continuously performed. Can be done automatically.
- the adsorption member (transfer film 7) is close to the second opening 58, the separated biomolecule sample discharged from the second opening 58 can be effectively adsorbed to the adsorption member (transfer film 7). And high-resolution sample adsorption can be realized.
- an insulating elastic member (elastic body 620, 610) surrounds the opening on the second electrode (anode 41) side in the first through hole (slit 61a), and the second through hole (through hole).
- 62a) has an annular shape surrounding the opening on the separation portion side, and the flow path of the buffer solution between the openings is limited by the annular elastic member (elastic body 620, 610). Yes. Accordingly, the buffer solution, which is a path through which current flows in the second buffer solution tank 4, is restricted so as not to contact the transfer film 7 through other than the slit 61 a of the slit structure 61 and the through hole 62 a of the fixture 62. can do.
- the buffer solution that is the path through which the current flows in the second buffer solution tank 4 is the first structure (slit structure 61).
- the biomolecule sample is not concentrated and adsorbed on a specific portion of the adsorption member (transfer film 7), and the resolution is lowered.
- the flow path of the buffer solution is limited as described above, so that the biomolecule sample is concentrated and adsorbed on a specific portion of the adsorption member (transfer film 7). Thereby, high-resolution sample adsorption can be realized.
- the first direction is defined by the first electrode (cathode 31) and the second electrode (anode 41).
- the sample separation pattern can be obtained by moving the adsorption member (transfer film 7) in the second direction perpendicular to the vertical direction.
- the medium on which the first-dimensional electrophoresis has been performed is set as a biomolecule sample in the separation unit (sample separation unit 5), whereby the second-dimensional electrophoresis and transfer are continuously performed. Can be done.
- the biomolecule analyzer according to aspect 2 of the present invention is the above aspect 1, On the first structure (slit structure 61) side of the second structure (fixing tool 62) or on the second structure (fixing tool 62) side of the first structure (slit structure 61), Recesses (62b, 61c) for holding the elastic members (elastic bodies 620, 610) are provided.
- the elastic members (elastic bodies 620, 610) can be held, the elastic members (elastic bodies 620, 610) are not unnecessarily detached, and are continuously and reproducible.
- a biomolecule analyzer that can be analyzed well can be provided.
- the biomolecule analyzer according to aspect 3 of the present invention is the above aspect 1 or 2, wherein Either on the first structure (slit structure 61) side of the second structure (fixing tool 62) or on the second structure (fixing tool 62) side of the first structure (slit structure 61). Are provided with concave portions (62b, 61c) for holding the elastic members (elastic bodies 620, 610), and on the other side, convex portions (61b, 61c) inserted into the elastic members (elastic bodies 620, 610). 62c).
- region where a buffer solution exists is restrict
- the biomolecule analyzer according to aspect 4 of the present invention is the above aspect 1 to 3,
- the elastic members (elastic bodies 620 and 610) can be made of a polymer, rubber, gel, or sol.
- the biomolecule analyzer according to aspect 5 of the present invention is the above aspect 4,
- the rubber nitrile rubber, fluorine rubber, silicone rubber, ethylene propylene rubber, chloroprene rubber, acrylic rubber, butyl rubber, urethane rubber, natural rubber, chlorosulfonated polyethylene rubber or epichlorohydrin rubber can be used.
- the biomolecule analyzer according to aspect 6 of the present invention is the above aspect 1 to 5, In the second direction perpendicular to the first direction defined by the first electrode (cathode 31) and the second electrode (anode 41), the width of the first through hole (slit 61a) is the second width. It is narrower than the opening 58.
- the first through hole (slit 61a) can converge the lines of electric force to a width narrower than that of the second opening 58.
- the biomolecule analyzer according to aspect 7 of the present invention is the above aspect 1 to 6,
- the first electrode (cathode 31), the second opening 58, the first through hole (slit 61a), the second through hole (through hole 62a), and the second electrode (anode 41) are arranged in a straight line. Has been.
- the flow of electric lines of force in the vicinity of the second opening 58 of the separation part is perpendicular to the adsorption member (transfer film 7), and thus is discharged from the second opening 58.
- the biomolecule sample is adsorbed from a direction perpendicular to the adsorbing member (transfer film 7).
- the biomolecule analyzer according to aspect 8 of the present invention is the above aspect 1-7, At the position facing the second opening 58, the adsorption member (transfer film 7) is perpendicular to the first direction defined by the first electrode (cathode 31) and the second electrode (anode 41). Further, an adsorption member moving means (transfer film moving arm 70) for moving in the second direction is further provided.
- the biomolecule analyzer according to aspect 9 of the present invention is the above aspect 1 to 8, A first buffer solution tank 3 in which the first electrode (cathode 31) is disposed; A second buffer solution tank 4 in which the second electrode (anode 41) is disposed;
- the first buffer solution (cathode buffer solution) put into the first buffer solution tank 3 is a pH 6.5-8.8 buffer solution containing ethanol,
- the second buffer solution (anode buffer solution) placed in the second buffer solution tank 4 is a buffer solution containing MOPS or Tris.
- the biomolecule analyzer according to aspect 10 of the present invention is the above aspects 1 to 9, A first-dimensional electrophoresis unit that performs first-dimensional electrophoresis;
- the separation unit (sample separation unit 5) is configured as a second-dimensional electrophoresis unit that performs second-dimensional electrophoresis.
- the biomolecule analyzer according to aspect 11 of the present invention is the above aspect 10, Medium moving means (sample introduction arm) for moving the separation medium (gel strip 80) for the first dimension electrophoresis from which the biomolecule sample has been separated in the first dimension electrophoresis section to the separation section (sample separation section 5) 82).
- the two-dimensional electrophoresis apparatus capable of automatically and continuously performing the first-dimensional electrophoresis, the second-dimensional electrophoresis in the separation unit, and the sample adsorption by the adsorption member. Can be provided.
- the biomolecule analyzer according to aspect 12 of the present invention is the above aspect 1 to 11, As the biomolecule sample, protein, DNA or RNA can be used.
- width means a dimension in the Y direction
- length means a dimension in the X direction
- thickness means a dimension in the Z direction.
- this example is based on the configuration of the first embodiment.
- sample separation part As an example of the sample separation unit 5 shown in FIG. 2, a glass plate having a size of 60 mm in width ⁇ 30 mm in length ⁇ 5 mm in thickness is used, and a separation medium with a thickness of 1 mm is filled therebetween. Was used.
- the second opening of the sample separation part was covered with a porous membrane (coating part) using a commercially available 0.65 ⁇ m Durapore (registered trademark) membrane filter (Millipore).
- the separation medium is a protein separation gel (10 mm wide x 25 mm long x 1 mm thick) using a 10% polyacrylamide gel using Bis-Tris buffer at pH 6.4 on the second opening side of the sample separation section.
- a separation medium in which a protein concentration gel (width 60 mm ⁇ length 5 mm ⁇ thickness 1 mm) using a 3% polyacrylamide gel is located on the first opening side of the sample separation portion was used.
- the slit structure 61 shown in FIG. 2 As an example of the slit structure 61 shown in FIG. 2, an acrylic material is used, the width in the z direction is 75 mm, the width in the y direction is 24 mm, and the width in the x direction (from the contact surface to the convex portion 61b (FIG. 2)).
- a slit structure with a width of 8 mm at the tip was prepared.
- the protruding length of the convex portion 61b shown in FIG. 1 was 4 mm, and the width of the slit in the y direction was 300 ⁇ m. Further, the width of the square-shaped convex portion 61b (FIG. 2) in the y direction was set to 9 mm.
- the fixture 62 shown in FIG. 2 was made of an acrylic material, with a z-direction width of 75 mm, a y-direction width of 24 mm, and an x-direction width of 8 mm.
- the through hole 62a (FIG. 2) had a width in the z direction of 65 mm and a width in the y direction of 10 mm ⁇ m.
- the recess 62b (FIG. 2) had a width in the z direction of 65 mm, a width in the y direction of 5 mm, and a depth (x direction) of 8 mm.
- the elastic body 620 shown in FIG. 2 was formed by first pouring silicone rubber into the concave portion of the fixture, completely filling the concave portion, and then polymerizing.
- the sample separation unit to which the porous membrane (transfer assistant) is attached is set in the biomolecule analyzer, and the first buffer tank in which the cathode is arranged is a pH 7.3 MOPS buffer (manufactured by Invitrogen). Satisfied.
- positions an anode was filled with 100 mM MOPS (pH 7.3) and 20% ethanol.
- the cathode produced with the platinum wire was arrange
- the transfer auxiliary body is a hydrophilic film having a pore size (pore size) of 100 ⁇ m or less, and can be adhered to the second opening of the sample separation unit using an adhesive, thermocompression bonding, double-sided tape, or the like. .
- Immobilon FL manufactured by Millipore
- ethanol a commercially available PVDF membrane
- the upper part of the transfer film was fixed to the transfer film moving arm 70 shown in FIG.
- FIG. 12 shows a photograph of the transfer film after sample adsorption. From FIG. 12, it was shown that the biomolecule sample can be separated and developed in the two-dimensional direction on the transfer film by this example.
- the biomolecule analyzer according to the present invention can be used for, for example, drug discovery research and development, and can also be applied to medical equipment for diagnosis.
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Abstract
Description
緩衝液を介して分離媒体に電流を流して、該分離媒体中のサンプルを分離し、且つ分離された生体分子サンプルを該分離媒体から吸着部材へ吸着させる生体分子分析装置であって、
第1電極と、
第2電極と、
上記第1電極側に開口する第1開口および上記第2電極側に開口する第2開口が設けられ、且つ上記分離媒体を格納する分離部と、
上記分離部と上記第2電極との間に在る押し具であって、上記分離部側に配置された第1構造体と上記第2電極側に配置され位置固定された第2構造体とによって絶縁性を有した弾性部材を挟持した押し具と、を備え、
上記第1構造体には、上記第2開口の対向位置に、上記分離部側から上記第2電極側に貫通する第1貫通孔が設けられ、
上記第2構造体には、上記第2開口の対向位置に、上記分離部側から上記第2電極側に貫通する第2貫通孔が設けられ、
上記吸着部材は、上記第2開口と上記第1貫通孔との間に配置され、
上記弾性部材は、上記第1構造体を上記分離部に向かって押圧し、
上記弾性部材は、上記第1貫通孔における上記第2電極側の開口部を取り囲むとともに、上記第2貫通孔における上記分離部側の開口部を取り囲む環形状を有しており、該開口部同士の間の緩衝液の流路を該環形状の弾性部材によって制限していることを特徴としている。
以下、本発明に係る生体分子分析装置の一実施形態について、図1から図8を用いて詳細に説明する。
本実施形態1に係る生体分子分析装置の概略的な構成について、図1および図2を参照して説明する。図1は、生体分子分析装置1を概略的に示す上面図であり、図2は、図1に示す切断線A-A´における矢視断面図である。
陰極31は第1緩衝液槽3内に配置されており、陽極41は第2緩衝液槽4内に配置されている。
第1緩衝液槽3および第2緩衝液槽4は、筐体2内にサンプル分離部5を取り付けて、筐体2内を2つの槽に分けることによって形成されている。
○陽極用緩衝液
100mM MOPS(pH7.3)
50mM トリスヒドロキシメチルアミノメタン(Tris)
50mM Bis-Tris
20% エタノール
○陰極用緩衝液
100mM MOPS(pH7.2)
50mM Tris
50mM Bis-Tris
0.25% ドデシル硫酸ナトリウム(SDS)
○分離ゲル
pH6.8のTris-HClバッファーを用いた10%ポリアクリルアミドゲル
(サンプル分離部5)
サンプル分離部5は、上述したように第1緩衝液槽3に向かって開口するサンプル供給媒体接続部である第1開口57、および第2緩衝液槽4に向かって開口するサンプル成分排出口である第2開口58を有している。
転写膜7は、分離ゲル53によって分離された生体分子サンプルを長期間にわたって安定に保存可能にし、さらに、その後の分析を容易にする生体分子サンプルの吸着・保持体であることが好ましい。転写膜7の材質としては、高い強度を有し、且つサンプル結合能(単位面積当たりに吸着可能な重量)が高いものが好ましい。転写膜7としては、サンプルがタンパク質である場合にはPVDF膜などが適している。なお、PVDF膜は予めメタノールなどを用いて親水化処理を行っておくことが好ましい。これ以外には、ニトロセルロース膜またはナイロン膜など、従来からタンパク質、DNAおよび核酸の吸着に利用されている膜も使用可能である。
転写膜移動アーム70は、図2に示すように、転写膜7を+y方向に引き上げる構成を有する。なお、転写膜移動アーム70は、引き上げ式に限らず、回転動作によって転写膜7を巻き取る転写膜回収部材として構成されてもよい。転写膜回収部材を用いれば、転写膜7を+y方向に引き上げる転写膜移動アーム70のように広い駆動範囲を確保する必要がなく、生体分子分析装置1を小型化し得る。
サンプル導入アーム82は、図2に示すように、サンプル分離部5の第1開口57に対して生体分子サンプルを導入するために用いられ、支持板81に支持されたゲルストリップ80を保持する。ゲルストリップ80は、一般的に薄く且つ軟らかいため、サンプル導入アーム82によって直接に保持するのではなく、アクリル板、樹脂フィルムなどからなる支持板81にゲルストリップ80を固定してサンプル導入アーム82に保持される。
押し具6は、サンプル分離部5の第2開口58に対して転写膜7を所定の圧力で押圧するとともに、サンプル分離部5から陽極41までの間において、スリット構造体61のスリット61aおよび固定具62(第2構造体)の貫通孔62a以外を電流が通過することを阻害するための部材である。すなわち、押し具6は、サンプル分離部5から陽極41までの間において、電圧が印加された陰極31および陽極41に誘起された電荷から発生する電気力線の経路(電流の流れ)を規制するための部材である。具体的には、押し具6により、第2緩衝液槽4において電流が流れる経路である緩衝液が、スリット構造体61のスリット61aおよび固定具62(第2構造体)の貫通孔62a以外を通って転写膜7に接することの無い構成となっている。
スリット構造体61は、押し具6における最も転写膜7側に位置し、転写膜7に接触する接触面を有している。
固定具62は、スリット構造体61よりも陽極41側に在って、筐体2との接触部分において筐体2に接着固定されている。固定具62は、スリット構造体61と同様に、ガラス、セラミック、樹脂などから構成することができる。
次に、生体分子分析装置1における生体分子サンプルの分離および吸着の流れについて、図2を参照して説明する。
先述したように、生体分子分析装置1は、サンプル分離部5を二次元目電気泳動部として構成し、一次元目電気泳動部を組み込んだ二次元電気泳動装置として構成することができる。一次元目電気泳動部では一次元目電気泳動用の分離媒体としてゲルストリップ80が用いられ、一次元目の電気泳動(等電点電気泳動)が行われた後のゲルストリップ80がサンプル導入アーム82によってサンプル分離部5の第1開口57に挿入される。
本実施形態1の生体分子分析装置1によれば、弾性体620がスリット構造体61をサンプル分離部5に向かって押圧することから、スリット構造体61のスリット61aと第2開口58との間に配置される転写膜7が第2開口58に近接する。これにより、第2開口58から排出された分離後の生体分子サンプルを転写膜7に吸着することができ、電気泳動による分離から転写膜7への転写までを連続的に行うことが可能である。また、第2開口58に転写膜7が近接することから、第2開口58から排出された分離後の生体分子サンプルを効果的に転写膜7に吸着することができ、高分解能なサンプル吸着を実現することができる。
上記実施形態1では、電気力線の経路の規制を、ロの字型の弾性体620およびロの字型の凸部61bと、筐体2への固定具62の密閉固定とによっておこなっている。しかしながら、本発明はこれに限定されるものではなく、ロの字型の弾性体620およびロの字型の凸部61bのみによって、電気力線の経路の規制をおこなってもよい。
スリット構造体、弾性体、固定具の形状は、実施形態1に示すものに限られず、図9に示す形状も適用可能である。そこで、本発明の他の実施形態について、図9に基づいて説明すれば、以下のとおりである。なお、説明の便宜上、上記実施形態1にて説明した部材と同じ機能を有する部材については、同じ符号を付記し、その説明を省略する。
スリット構造体、弾性体、固定具の形状は、実施形態1に示すものに限られず、図10に示す形状も適用可能である。そこで、本発明の他の実施形態について、図10に基づいて説明すれば、以下のとおりである。なお、説明の便宜上、上記実施形態1にて説明した部材と同じ機能を有する部材については、同じ符号を付記し、その説明を省略する。
筐体への固定具の固定方法は、実施形態1に示すものに限られず、図11に示す形状も適用可能である。そこで、本発明の他の実施形態について、図11に基づいて説明すれば、以下のとおりである。なお、説明の便宜上、上記実施形態1にて説明した部材と同じ機能を有する部材については、同じ符号を付記し、その説明を省略する。
以上のように、本発明の態様1に係る生体分子分析装置は、
緩衝液を介して分離媒体(分離ゲル53)に電流を流して、該分離媒体(分離ゲル53)中の生体分子サンプルを分離し、且つ分離された生体分子サンプルを該分離媒体(分離ゲル53)から吸着部材(転写膜7)へ吸着させる生体分子分析装置であって、
第1電極(陰極31)と、
第2電極(陽極41)と、
上記第1電極(陰極31)側に開口する第1開口57および第2電極(陽極41)側に開口する第2開口58が設けられ、且つ上記分離媒体(分離ゲル53)を格納する分離部(サンプル分離部5)と、
上記分離部(サンプル分離部5)と上記第2電極(陽極41)との間に在る押し具(6、6´、6´´)であって、上記分離部(サンプル分離部5)側に配置された第1構造体(スリット構造体61)と上記第2電極(陽極41)側に配置され位置固定された第2構造体(固定具62、62´、62´´)とによって絶縁性を有した弾性部材(弾性体620、610)を挟持した押し具(6、6´、6´´)と、を備え、
上記第1構造体(スリット構造体61)には、上記第2開口58の対向位置に、上記分離部(サンプル分離部5)側から上記第2電極(陽極41)側に貫通する第1貫通孔(スリット61a)が設けられ、
上記第2構造体(固定具62、62´、62´´)には、上記第2開口58の対向位置に、上記分離部(サンプル分離部5)側から上記第2電極(陽極41)側に貫通する第2貫通孔(固定具62、62´、62´´の貫通孔62a)が設けられ、
上記吸着部材(転写膜7)は、上記第2開口58と上記第1貫通孔(スリット61a)との間に配置され、
上記弾性部材(弾性体620、610)は、上記第1構造体(スリット構造体61)を分離部(サンプル分離部5)に向かって押圧し、
上記弾性部材(弾性体620、610)は、上記第1貫通孔(スリット61a)における上記第2電極(陽極41)側の開口部を取り囲むとともに、上記第2貫通孔(貫通孔62a)における上記分離部側の開口部を取り囲む環形状を有しており、該開口部同士の間の緩衝液(陽極用緩衝液)の流路を該環形状の弾性部材(弾性体620、610)によって制限していることを特徴としている。
上記第2構造体(固定具62)の上記第1構造体(スリット構造体61)側または上記第1構造体(スリット構造体61)の上記第2構造体(固定具62)側には、上記弾性部材(弾性体620、610)を保持する凹部(62b、61c)が設けられている。
上記第2構造体(固定具62)の上記第1構造体(スリット構造体61)側または上記第1構造体(スリット構造体61)の上記第2構造体(固定具62)側の一方には、上記弾性部材(弾性体620、610)を保持する凹部(62b、61c)が設けられており、他方には、上記弾性部材(弾性体620、610)に挿入される凸部(61b、62c)が設けられている。
上記弾性部材(弾性体620、610)は、高分子、ゴム、ゲルまたはゾルから構成することができる。
上記ゴムとして、ニトリルゴム、フッ素ゴム、シリコーンゴム、エチレンプロピレンゴム、クロロプレンゴム、アクリルゴム、ブチルゴム、ウレタンゴム、天然ゴム、クロロスルフォン化ポリエチレンゴムまたはエピクロルヒドリンゴムを用いることができる。
上記第1電極(陰極31)と上記第2電極(陽極41)とにより規定される第1方向に対して垂直な第2方向において、上記第1貫通孔(スリット61a)の幅は上記第2開口58よりも狭い。
上記第1電極(陰極31)、上記第2開口58、上記第1貫通孔(スリット61a)、上記第2貫通孔(貫通孔62a)、および上記第2電極(陽極41)が一直線上に配置されている。
上記第2開口58の対向位置において、上記吸着部材(転写膜7)を、上記第1電極(陰極31)と上記第2電極(陽極41)とにより規定される第1方向に対して垂直な第2方向に移動させる吸着部材移動手段(転写膜移動アーム70)を更に備えている。
上記第1電極(陰極31)を内部に配置する第1緩衝液槽3と、
上記第2電極(陽極41)を内部に配置する第2緩衝液槽4とを更に備え、
上記第1緩衝液槽3に入れる第1緩衝液(陰極用緩衝液)は、エタノールを含むpH6.5~8.8の緩衝液であり、
上記第2緩衝液槽4に入れる第2緩衝液(陽極用緩衝液)は、MOPSまたはトリスを含む緩衝液である。
一次元目の電気泳動をおこなう一次元目電気泳動部を更に備え、
上記分離部(サンプル分離部5)が、二次元目の電気泳動をおこなう二次元目電気泳動部として構成される。
上記一次元目電気泳動部において生体分子サンプルが分離された一次元目電気泳動用の分離媒体(ゲルストリップ80)を、上記分離部(サンプル分離部5)に移動する媒体移動手段(サンプル導入アーム82)を更に備えている。
上記生体分子サンプルとして、タンパク質、DNAまたはRNAを用いることができる。
図2に示したサンプル分離部5の実施例として、幅60mm×長さ30mm×厚さ5mmの寸法のガラス板2枚を用いて、その間に厚さ1mmの分離媒体を充填して形成したものを用いた。
図2に示したスリット構造体61の実施例として、アクリル材を用いて、z方向の幅を75mm、y方向の幅を24mm、x方向の幅(接触面から凸部61b(図2)の先端までの幅)を8mmとしたスリット構造体を作製した。図1に示した凸部61bの突出長を4mmとし、スリットのy方向の幅を300μmとした。また、ロの字型の凸部61b(図2)のy方向の幅を9mmとした。
次に、多孔質膜(転写補助体)を取り付けたサンプル分離部を、生体分子分析装置にセットし、陰極を配置する第1緩衝液槽を、pH7.3のMOPSバッファー(Invitrogen社製)で満たした。また、陽極を配置する第2緩衝液槽を、100mM MOPS(pH7.3)、20%エタノールで満たした。なお、第1緩衝液槽には白金線で作製した陰極を配置し、第2緩衝液槽にも白金線で作製した陽極を配置した。なお、上記転写補助体とは、親水性で孔径(ポアサイズ)が100μm以下である膜であり、接着剤、熱圧着、両面テープなど利用してサンプル分離部の第2開口に接着することができる。
2 筐体
2a 凹溝
3 第1緩衝液槽
4 第2緩衝液槽
5 サンプル分離部
6、6´、6´´ 押し具
7 転写膜
31 陰極(第1電極)
41 陽極(第2電極)
51 下板
52 上板
53 分離ゲル
57 第1開口
58 第2開口
61、61´ スリット構造体(第1構造体)
61a スリット(第1貫通孔)
61b 凸部
61c 凹部
62、62´、62´´ 固定具(第2構造体)
62a 貫通孔(第2貫通孔)
62b 凹部
62c 凸部
70 転写膜移動アーム(吸着部材移動手段)
80 ゲルストリップ(一次元目電気泳動用の分離媒体)
81 支持板
82 サンプル導入アーム(媒体移動手段)
610、620 弾性体(弾性部材)
622 凸溝
Claims (12)
- 緩衝液を介して分離媒体に電流を流して、該分離媒体中の生体分子サンプルを分離し、且つ分離された生体分子サンプルを該分離媒体から吸着部材へ吸着させる生体分子分析装置であって、
第1電極と、
第2電極と、
上記第1電極側に開口する第1開口および上記第2電極側に開口する第2開口が設けられ、且つ上記分離媒体を格納する分離部と、
上記分離部と上記第2電極との間に在る押し具であって、上記分離部側に配置された第1構造体と上記第2電極側に配置され位置固定された第2構造体とによって絶縁性を有した弾性部材を挟持した押し具と、を備え、
上記第1構造体には、上記第2開口の対向位置に、上記分離部側から上記第2電極側に貫通する第1貫通孔が設けられ、
上記第2構造体には、上記第2開口の対向位置に、上記分離部側から上記第2電極側に貫通する第2貫通孔が設けられ、
上記吸着部材は、上記第2開口と上記第1貫通孔との間に配置され、
上記弾性部材は、上記第1構造体を上記分離部に向かって押圧し、
上記弾性部材は、上記第1貫通孔における上記第2電極側の開口部を取り囲むとともに、上記第2貫通孔における上記分離部側の開口部を取り囲む環形状を有しており、該開口部同士の間の緩衝液の流路を該環形状の弾性部材によって制限していることを特徴とする生体分子分析装置。 - 上記第2構造体の上記第1構造体側または上記第1構造体の上記第2構造体側には、上記弾性部材を保持する凹部が設けられていることを特徴とする請求項1に記載の生体分子分析装置。
- 上記第2構造体の上記第1構造体側または上記第1構造体の上記第2構造体側の一方には、上記弾性部材を保持する凹部が設けられており、他方には、該弾性部材に挿入される凸部が設けられていることを特徴とする請求項1または2に記載の生体分子分析装置。
- 上記弾性部材は、高分子、ゴム、ゲルまたはゾルから構成されていることを特徴とする請求項1から3までの何れか1項に記載の生体分子分析装置。
- 上記ゴムは、ニトリルゴム、フッ素ゴム、シリコーンゴム、エチレンプロピレンゴム、クロロプレンゴム、アクリルゴム、ブチルゴム、ウレタンゴム、天然ゴム、クロロスルフォン化ポリエチレンゴムまたはエピクロルヒドリンゴムであることを特徴とする請求項4に記載の生体分子分析装置。
- 上記第1電極と上記第2電極とにより規定される第1方向に対して垂直な第2方向において、上記第1貫通孔の幅は上記第2開口よりも狭いことを特徴とする請求項1から5までの何れか1項に記載の生体分子分析装置。
- 上記第1電極、上記第2開口、上記第1貫通孔、上記第2貫通孔、および上記第2電極が一直線上に配置されていることを特徴とする請求項1から6までの何れか1項に記載の生体分子分析装置。
- 上記第2開口の対向位置において、上記吸着部材を、上記第1電極と上記第2電極とにより規定される第1方向に対して垂直な第2方向に移動させる吸着部材移動手段を更に備えることを特徴とする請求項1から7までの何れか1項に記載の生体分子分析装置。
- 上記第1電極を内部に配置する第1緩衝液槽と、
上記第2電極を内部に配置する第2緩衝液槽とを更に備え、
上記第1緩衝液槽に入れる第1緩衝液は、エタノールを含むpH6.5~8.8の緩衝液であり、
上記第2緩衝液槽に入れる第2緩衝液は、MOPSまたはトリスを含む緩衝液であることを特徴とする請求項1から8までの何れか1項に記載の生体分子分析装置。 - 一次元目の電気泳動をおこなう一次元目電気泳動部を更に備え、
上記分離部が、二次元目の電気泳動をおこなう二次元目電気泳動部として構成されることを特徴とする請求項1から9までの何れか1項に記載の生体分子分析装置。 - 上記一次元目電気泳動部において生体分子サンプルが分離された一次元目電気泳動用の分離媒体を、上記分離部に移動する媒体移動手段を更に備えている請求項10に記載の生体分子分析装置。
- 上記生体分子サンプルは、タンパク質、DNAまたはRNAであることを特徴とする請求項1から11までの何れか1項に記載の生体分子分析装置。
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