JP2013040792A - Electrophoresis gel chip, and method and kit for manufacturing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an electrophoresis gel chip having high preservability or transportability, and a technique for suitably manufacturing the electrophoresis gel chip.SOLUTION: An electrophoresis gel chip 50 includes: a support medium 51 made of an insulator; a belt-like electrophoresis gel 53 fixed to the support medium 51; and a protective film 54 which covers an opposite side of the electrophoresis gel 53 from the support medium 51.

Description

  The present invention relates to an electrophoresis gel chip used for electrophoresis and a technique for producing the electrophoresis gel chip.

  In recent years, proteome analysis has attracted attention as a research subject in the post-genome. Proteome analysis is a large-scale study of protein structure and function. In proteome analysis, usually, a protein contained in a sample is first separated into individual proteins. At this time, two-dimensional electrophoresis is widely used as one method for separating proteins.

  Two-dimensional electrophoresis is a technique for two-dimensionally separating proteins by two-stage electrophoresis. In two-dimensional electrophoresis, for example, after isoelectric focusing (IEF), proteins are separated in the first dimension based on the isoelectric point, and then sodium dodecyl sulfate / polyacrylamide gel electrophoresis (SDS) is used. -PAGE: isolates proteins in the second dimension based on molecular weight or the like by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Such two-dimensional electrophoresis has a very high resolution and can separate several thousand types of proteins into spots.

  In general, a strip-shaped strip gel is used as the first-dimensional electrophoresis gel. Such a strip gel is used by being peeled off from a package for packing goods. In many cases, the SDS-PAGE gel used as the second-dimensional electrophoresis gel is composed of a concentrated gel for concentrating the sample and adjusting the start point, and a separation gel for separating the sample by the difference in molecular weight. Composed.

  Here, as a method for suitably performing the two-dimensional electrophoresis method, there is a technique described in Patent Document 1. A sample separation device described in Patent Document 1 is a first direction separation device for separating a sample in a first direction in a first medium fixed to a side end surface of a first medium support that is a plate-like insulator. A sample separation device having an insulator for housing a second medium for further separating the separated sample separated in the first direction in the first medium in a second direction different from the first direction; Means for holding the medium support so that the first medium is on the lower side and automatically bringing the first medium after sample separation in the first direction into close contact with the second medium, The insulator has a first opening and a second opening that define a second direction for energizing the second medium, and the second medium contacts the outside of the insulator in the first opening and the second opening. The second medium is accommodated as possible, and the second opening includes the first medium containing the separated sample. It has a shape for adhering the second medium along the first direction.

JP 2007-64848 A (published on March 15, 2007) Japanese Patent Laid-Open No. 5-223780 (released on August 31, 1993)

  The electrophoresis gel chip in which the electrophoresis gel is fixed to the support can be suitably used in an electrophoresis apparatus that automatically conveys the electrophoresis gel as described in Patent Document 1. Therefore, a technique for suitably providing such an electrophoresis gel chip to the user is required. In particular, an electrophoresis gel chip having high storage stability or transportability, and a technique for suitably producing such an electrophoresis gel chip are useful.

  The present invention has been made in view of the above problems, and has as its main object to provide an electrophoresis gel chip having high storage stability or transportability, and a technique for suitably producing such an electrophoresis gel chip. To do.

  In order to solve the above problems, a method for producing an electrophoresis gel chip according to the present invention includes a support made of an insulator, a strip-shaped electrophoresis gel fixed to the support, and the strip-shaped electrophoresis gel. A method for producing an electrophoresis gel chip comprising a protective film covering a surface opposite to the support, the sheet-like electrophoresis gel having one surface covered with a protective film, and cutting the sheet gel A cutting step for separating the electrophoresis gel, and a fixing step for fixing the strip-shaped electrophoresis gel separated in the cutting step to the support.

  According to said structure, an electrophoretic gel chip with high preservability or transportability can be manufactured. That is, since the electrophoresis gel chip manufactured by the above method includes a protective film, physical damage to the surface of the electrophoresis gel, proteins, and salts during processing, storage, and delivery (transport) Etc. can be prevented, and the preservability or transportability of the electrophoresis gel can be improved.

  Moreover, according to said structure, the electrophoresis gel chip mentioned above can be manufactured suitably. That is, by manufacturing an electrophoresis gel chip by cutting a sheet-like electrophoresis gel whose one surface is covered with a protective film and fixing it to a support, for example, a very long electrophoretic gel having a width of several millimeters. The difficult task of covering the electrophoresis gel with a protective film can be easily performed, and can also contribute to mass production.

  In the method for producing an electrophoresis gel chip according to the present invention, in the cutting step, the blade is moved relative to the sheet-like electrophoresis gel so that the blade crosses the sheet-like electrophoresis gel. Is preferred.

  According to said structure, an electrophoretic gel chip with high preservability can be manufactured suitably. That is, in order to prevent deterioration of the electrophoresis gel due to hydrolysis, the amount of water contained in the electrophoresis gel may be reduced. When such a dried electrophoresis gel is pushed all at once as in Patent Document 2, for example, the electrophoresis gel may be damaged. Here, according to the above configuration, since the blade is moved relative to the sheet-like electrophoresis gel so that the blade crosses the sheet-like electrophoresis gel, the dried electrophoresis gel is preferably used. Can be cut.

  In the method for producing an electrophoresis gel chip, in the cutting step, after separating the one band-shaped electrophoresis gel from the sheet-like electrophoresis gel, the second direction perpendicular to the first direction traversed by the blade Adjusting the relative position between the blade and the sheet-like electrophoresis gel, and again with respect to the sheet-like electrophoresis gel, the blade so that the blade crosses the sheet-like electrophoresis gel. Is relatively moved.

  According to said structure, since it can cut | disconnect while feeding sheet-like electrophoresis gel in order, many strip | belt-shaped electrophoresis gel can be isolate | separated successfully.

  In the method for manufacturing an electrophoretic gel chip, it is preferable that the automatic feeding device adjusts the relative position in the second direction in the cutting step.

  According to said structure, as shown in the Example mentioned later by adjusting the relative position of the blade and sheet-like electrophoresis gel in a 2nd direction with automatic feeders, such as a movable stage, for example, it is obtained. Therefore, it is possible to manufacture a high-quality electrophoresis gel chip by suppressing the variation in the width of the strip-shaped electrophoresis gel.

  In the method for producing an electrophoresis gel chip, it is preferable that the relative position in the second direction is adjusted in the cutting step in a state where the blade and the sheet-like electrophoresis gel are separated from each other.

  According to the above configuration, in order to adjust the relative position between the blade and the sheet-like electrophoresis gel in the second direction in a state where the blade and the sheet-like electrophoresis gel are separated, when adjusting the relative position, It is possible to prevent the blade from touching the sheet-like electrophoresis gel and damaging the sheet-like electrophoresis gel, and a high-quality electrophoresis gel chip can be manufactured.

  In the method for producing an electrophoresis gel chip according to the present invention, in the cutting step, it is preferable that the sheet-shaped electrophoresis gel is cut in a state where a fixing device is fixed with the sheet-shaped electrophoresis gel sandwiched therebetween.

  According to said structure, as shown in the Example mentioned later by cut | disconnecting a sheet-like electrophoresis gel in the state which the fixing tool has pinched | interposed and fixed the sheet-like electrophoresis gel, the obtained strip | belt-shaped electrophoresis gel is obtained. It is possible to manufacture a high-quality electrophoresis gel chip while suppressing variations in the width of the electrophoresis gel.

  In the method for producing an electrophoresis gel chip according to the present invention, in the cutting step, the sheet-like electrophoresis gel is cut together with the adhesive tape in a state where the adhesive tape is attached to the sheet-like electrophoresis gel. It may be.

  According to said structure, since the cut | disconnected adhesive tape remains in the isolate | separated strip-shaped electrophoresis gel, since the said adhesive tape can be used as a support body of the isolate | separated strip-shaped electrophoresis gel, after that This improves the handling of the work. In addition, the electrophoresis gel can be more suitably fixed with an adhesive tape.

  In the method for manufacturing an electrophoresis gel chip according to the present invention, in the fixing step, the band-shaped electrophoresis gel is fixed to the support in a state where the band-shaped electrophoresis gel is inserted into a groove provided in a fixing jig. It is preferable to do.

  According to the above configuration, by inserting the band-shaped electrophoresis gel into the groove of the fixing jig and further inserting the support, the groove serves as a guide, and the band-shaped electrophoresis gel is used as an appropriate part of the support. Can be fixed in position.

  In the method for producing an electrophoresis gel chip according to the present invention, the support is a plate-like insulator, and in the fixing step, the belt-shaped electrophoresis gel is fixed to a side end surface of the support. Also good.

  According to said structure, it can apply suitably for the electrophoresis apparatus of patent document 1, for example.

  In the method for producing an electrophoresis gel chip according to the present invention, the length of the band-shaped electrophoresis gel separated in the cutting step is longer than the length of the region where the band-shaped electrophoresis gel is fixed on the support. Further, after the fixing step, a removal step of removing a portion protruding from the support in the band-shaped electrophoresis gel may be further included.

  According to the above configuration, a band-shaped electrophoresis gel is prepared in advance with respect to the support, and the excess portion is removed after fixing to the support, thereby absorbing variations in the manufacturing process and providing high-quality electricity. An electrophoretic gel chip can be manufactured.

  An electrophoresis gel chip manufacturing kit according to the present invention includes a support made of an insulator, a strip-shaped electrophoresis gel fixed to the support, and the opposite side of the support in the strip-shaped electrophoresis gel. A electrophoresis gel chip manufacturing kit comprising a protective film covering the surface, and cutting the sheet-shaped electrophoresis gel whose one surface is covered with the protective film to separate the band-shaped electrophoresis gel It is characterized by having a device.

  Further, in the electrophoresis gel chip manufacturing kit according to the present invention, the cutting device includes a blade and a driving unit, and the driving unit is configured so that the blade traverses the sheet-like electrophoresis gel. It is preferable to move the blade relative to the sheet-like electrophoresis gel.

  In the electrophoresis gel chip manufacturing kit, the driving means separates the one band-shaped electrophoresis gel from the sheet-shaped electrophoresis gel, and then perpendicularly intersects the first direction in which the blade crosses. Adjust the relative position of the blade and the sheet-like electrophoresis gel in two directions, and again with respect to the sheet-like electrophoresis gel so that the blade crosses the sheet-like electrophoresis gel It is preferable to move the blade relatively.

  In the electrophoresis gel chip manufacturing kit, it is preferable that the driving unit automatically adjusts the relative position in the second direction.

  In the electrophoresis gel chip manufacturing kit, the driving unit preferably adjusts the relative position in the second direction in a state where the blade and the sheet-like electrophoresis gel are separated from each other.

  In the electrophoresis gel chip manufacturing kit according to the present invention, it is preferable that the cutting device further includes a fixing means for fixing the sheet-like electrophoresis gel.

  The electrophoresis gel chip manufacturing kit according to the present invention preferably further includes a fixing jig having a groove for inserting the band-shaped electrophoresis gel.

  According to said structure, it can use suitably for the manufacturing method of the electrophoresis gel chip concerning this invention, and there exists an effect equivalent to the manufacturing method of the electrophoresis gel chip concerning this invention.

  An electrophoresis gel chip according to the present invention covers a support made of an insulator, a strip-shaped electrophoresis gel fixed to the support, and a surface of the strip-shaped electrophoresis gel opposite to the support. It is characterized by having a protective film.

  According to said structure, the electrophoresis gel chip | tip with high preservability or transportability can be provided. In other words, according to the above configuration, since the protective film is provided, physical damage to the surface of the electrophoresis gel and generation of contamination such as proteins and salts during processing, storage, and delivery (transport) Can be prevented, and the preservability or transportability of the electrophoresis gel can be improved.

  According to the electrophoresis gel chip and the manufacturing method and manufacturing kit thereof according to the present invention, it is possible to provide an electrophoresis gel chip having high storage stability or transportability, and a technique for suitably manufacturing such an electrophoresis gel chip. it can.

It is sectional drawing which shows schematic structure of the electrophoresis gel chip which concerns on one Embodiment of this invention. It is a process perspective view explaining each process of a manufacturing method of an electrophoresis gel chip concerning one embodiment of the present invention. It is a figure explaining the cutting process of the manufacturing method of the electrophoresis gel chip concerning one embodiment of the present invention. It is a perspective view which shows the mode of the sheet-like electrophoresis gel with which the adhesive tape was stuck, and the strip | belt-shaped electrophoresis gel. It is a graph which shows the effect of fixing a sheet-like electrophoresis gel with a fixture in a cutting process, and (a) is a separation when cut in a state where a sheet-like electrophoresis gel is fixed with a fixture. (B) shows the variation in the width of the separated electrophoretic gel when the electrophoretic gel is cut without being fixed by a fixing device. Indicates. It is a graph which shows the effect of automatically feeding a sheet-like electrophoresis gel with an automatic feeder in a cutting process, and (a) is a separation when automatically feeding a sheet-like electrophoresis gel with an automatic feeder. The width variation of the strip | belt-shaped electrophoresis gel shown is shown, (b) shows the width variation of the separated strip | belt-shaped electrophoresis gel when the sheet-like electrophoresis gel is manually fed. It is a figure explaining the fixing process of the manufacturing method of the electrophoresis gel chip concerning one embodiment of the present invention. It is a schematic diagram which shows the principal part structure of an automated two-dimensional electrophoresis apparatus. It is sectional drawing which shows the principal part structure of an automated two-dimensional electrophoresis apparatus. It is sectional drawing which shows the principal part structure of an automated two-dimensional electrophoresis apparatus. It is a top view which shows the principal part structure of an automated two-dimensional electrophoresis apparatus. It is sectional drawing which shows the principal part structure of an automated two-dimensional electrophoresis apparatus. It is sectional drawing which shows the principal part structure of an automated two-dimensional electrophoresis apparatus. It is sectional drawing which shows the structure of the sample separation instrument which is a structural member of an automatic two-dimensional electrophoresis apparatus.

(Electrophoresis gel chip)
FIG. 1 is a cross-sectional view showing a schematic configuration of an electrophoresis gel chip 50 according to an embodiment of the present invention. As shown in FIG. 1, an electrophoresis gel chip 50 includes a support (chip) 51, an electrophoresis gel 53, an adhesive layer 52, and a protective film (protective film) 54 stacked in this order.

  The support 51 is a member for enabling the electrophoretic gel chip 50 to be automatically transported to an arbitrary place, and is a portion for the transport means to hold when the electrophoretic gel chip 50 is transported.

  The support 51 is preferably made of an insulating material. Examples of the insulating material include plastic materials such as polymethyl methacrylate resin (PMMA), polyethylene terephthalate (PET), and polycarbonate, quartz glass, Pyrex (registered trademark) glass, and alkali-free glass. Glass materials such as alumina, and ceramic materials such as alumina and low-temperature co-fired ceramics can be mentioned, but the material is not limited thereto.

  The shape of the support 51 can be appropriately designed according to the shape of the electrophoresis gel 53 and the conveying means of the electrophoresis apparatus using the electrophoresis gel chip 50. For example, by using a plate-like support, it can be suitably used in an automated two-dimensional electrophoresis apparatus 100 (described later) that holds the support by a vacuum suction means. For example, a PMMA plate having a size of 60 mm × 23 mm and a thickness of 1.2 mm can be used as the support 51. Such a support 51 can be manufactured by using a technique such as injection molding.

  The electrophoresis gel 2 is used as an electrophoresis medium, and polyacrylamide gel, agarose gel, and the like that are generally used as an electrophoresis medium can be used. In particular, the gel used in the first dimension of the two-dimensional electrophoresis is transported to a different location from the isoelectric focusing after completing the electrophoresis in the isoelectric focusing direction (first dimension electrophoresis), and the molecular weight direction Therefore, it can be suitably used as the electrophoresis gel 2 in the electrophoresis gel chip 51 according to the present embodiment provided with the support 51.

  The shape of the electrophoresis gel 53 can be appropriately designed according to the electrophoresis tank of the electrophoresis apparatus using the electrophoresis gel chip 50. For example, when it is used as a gel used in the first dimension of two-dimensional electrophoresis, it can be configured as a band-shaped electrophoresis gel having a width of 1 mm to 5 mm, preferably 1 mm to 2 mm. Although the length of a longitudinal direction is not specifically limited, For example, it can set suitably in the range of 50 mm or more and 240 mm or less. As an example, a belt-shaped electrophoresis gel having a longitudinal direction of 52 mm and a lateral direction (width) of 1.2 mm can be obtained.

  When the electrophoresis gel 53 is used as a gel used in the first dimension of two-dimensional electrophoresis, for example, an immobilized pH gradient (IPG) gel is suitable as the electrophoresis gel 53. The IPG gel is, for example, an acrylamide / bisacrylamide mixed solution that forms a gel skeleton (for example, a mixing ratio: 37.5 / 1), several acrylamide buffers that define the pH in the gel skeleton, and a polymerization initiator. Ammonium persulfate (APS), N, N, N ′, N′-tetramethylethylenediamine (TEMED; N, N, N ′, N′-tetramethylethylenediamine), glycerol (Glycerol), which are polymerization accelerators Can be used.

  The adhesive layer 52 adheres the support 51 and the electrophoresis gel 53. By using such an adhesive layer 52, it is possible to suppress the separation of the electrophoresis gel 53 from the support 51 and to improve the reliability of the electrophoresis gel chip 50.

  The adhesive layer 52 can be, for example, a surface coating agent attached to the support 51. As the surface coating agent, an acrylamide derivative-based coating agent, an agarose derivative-based coating agent, PVDC and the like are suitable, but are not limited thereto.

  Moreover, preferably, as shown in FIG. 1, the adhesive layer 52 can be composed of a chip adhesive layer (support adhesive layer) 52a and a gel adhesive layer 52b.

  The gel adhesive layer 52b has a hydrophilic surface, and adheres to the electrophoretic gel 53 via the hydrophilic surface. As the gel adhesive layer, for example, a commercially available gel bond film, a PET film coated with an oxygen plasma treatment or a hydrophilic coating agent, or the like can be used. The chip adhesive layer 52a adheres the support 51 and the gel adhesive layer 52b, and is preferably made of a material having insulation properties and excellent chemical resistance. For example, an adhesive, a double-sided tape, or the like is used. it can. As another embodiment, the gel adhesive layer 52b and the chip adhesive layer 52a may be the same member. In this case, a gel adhesive layer 52b may be provided on one surface of PET or the like, and a chip adhesive layer 52a may be provided on the other surface.

  Then, by constituting the adhesive layer 52 from the chip adhesive layer 52a and the gel adhesive layer 52b, the peeling of the electrophoretic gel 53 from the support 51 can be more suitably suppressed, and the reproducibility of the electrophoretic analysis is improved. Is possible.

  The protective film 54 is for protecting the electrophoresis gel 53. That is, in the gel chip according to the prior art, since the electrophoresis gel is exposed, physical damage and damage to the surface of the electrophoresis gel during processing, storage, and delivery (transport) Contamination such as proteins and salts may occur. Since the electrophoresis gel chip 51 according to the present embodiment includes the protective film 54 that covers the surface of the electrophoresis gel 53, it is possible to prevent the gel surface from being contaminated by physical damage and contamination to the electrophoresis gel 53. It becomes. Note that the protective film 54 is peeled off from the electrophoresis gel 53 when the electrophoresis gel chip 51 is used.

  As the protective film 54, for example, a resin film can be suitably used. Although not particularly limited, a commercially available PET film can be used as the resin film. In addition, it is preferable to provide an appropriate surface coat on the surface of the protective film 54 that is in contact with the electrophoretic gel 53, for example, a surface coat that can easily adhere to / separate from the electrophoretic gel 53. Moreover, as a protective film, it is preferable to provide a transparent film.

(Method for producing electrophoresis gel chip)
Hereinafter, the manufacturing method of the electrophoresis gel chip concerning one embodiment of the present invention and the manufacturing kit which can be used conveniently for the manufacturing method are explained.

  FIG. 2 is a process perspective view for explaining each process of the electrophoresis gel chip manufacturing method according to the embodiment of the present invention.

  First, as shown in FIG. 2A, a sheet-like electrophoresis gel 53 having a gel adhesive layer 52b on one side and a protective film 54 on the other side is formed (electrophoresis gel sheet forming step). Such a sheet-like electrophoresis gel 53 can be formed, for example, by forming the sheet-like electrophoresis gel 53 on the gel adhesive layer 52b and then attaching a protective film 54 thereto.

  In one example, a procedure in the case of using an IPG gel as the electrophoresis gel 53 will be described in detail. First, an acrylamide / bisacrylamide mixed solution (mixing ratio: 37.5 / 1), four acrylamide derivatives (acid dissociation constants: 3.6, 6.2, 7.0, 8.5), ammonium persulfate (APS; Preparation of acidic side monomer solution and basic side monomer solution adjusted at desired mixing ratio using ammonium persulfate), tetramethylethylenediamine (TEMED; N, N, N ′, N′-tetramethylethylenediamine), glycerol and pure water (See, for example, PG Righetti: Immobilized pH gradients: theory and methodology, Elsevier, Amsterdam, 1990).

  Then, the prepared acidic side monomer solution and basic side monomer solution are mixed using a mixer, and the resulting mixed solution is prepared as an IPG gel in which a sheet-like gel adhesive layer 52b (for example, a gel bond film) is arranged. Fill the instrument and gel. The length of the electrophoresis gel 53 can be arbitrarily designed from 50 mm to 240 mm, for example.

  Then, washing | cleaning and drying are performed and the protective film 54 is affixed on the surface on the opposite side to the gel contact bonding layer 52b in the electrophoresis gel 53. FIG. The protective film 54 is preferably attached carefully using a roller or the like so as to prevent air bubbles, dust and the like from being mixed.

  Next, as shown in FIG. 2B, a chip adhesive layer 52a is provided on the gel adhesive layer 52b (chip adhesive layer forming step). For example, an adhesive may be applied on the gel adhesive layer 52b, or a double-sided tape may be applied. When applying a double-sided tape, it is preferable to use a roller or the like carefully so that air bubbles and dust do not enter. Thus, a sheet-like electrophoresis gel 55 having a protective film 54 provided on one side is formed.

  When the adhesive layer 52 is composed of a single member, the chip adhesive layer forming step may be omitted. Further, when the adhesive layer 52 is a surface coating agent applied to the support 51, an adhesive layer forming step for applying a surface coat to the support 51 may be performed instead of the above-described chip adhesive layer forming step. Good. That is, the formation of the adhesive layer 52, the gel adhesive layer 52b, and the chip adhesive layer 52a may be appropriately performed before the fixing step described later, and is not limited to the above-described configuration.

(Cutting process)
Subsequently, as shown in FIG. 2C, the sheet-like electrophoresis gel 55 is cut to separate the belt-like electrophoresis gel (gel strip) 55a (cutting step). In this way, by cutting the sheet-like electrophoresis gel 55 whose one surface is covered with the protective film 54, for example, it is difficult to cover a very long electrophoresis gel 53 with a width of several millimeters with the protective film 54. Can be easily performed, and can contribute to mass production.

  In one embodiment, the cutting step can be performed using a cutting device 60 as shown in FIG. FIG. 3 is a diagram for explaining a cutting step of the method for producing an electrophoresis gel chip according to an embodiment of the present invention, (a) is a perspective view showing a schematic configuration of a cutting device 60 used in the cutting step, (B) is a side view of the cutting device 60, and (c) is a diagram showing the state of the cutting device 60 during cutting of the gel.

  As shown in FIG. 3, the cutting device 60 includes a movable stage (driving means, automatic feeding device, fixing tool, fixing means) 61, a blade driving unit (driving means) 62, a blade 62a, a frame and a stopper (fixing tool, fixing). Means) 63 and fasteners (fixing device, fixing means) 63a.

  The movable stage 61 is a stage on which the sheet-like electrophoresis gel 55 is placed, and the blade 62 a is relatively positioned with respect to the sheet-like electrophoresis gel 55 in the Y direction (first direction) and the X direction ( This is a driving means for driving in the second direction. The blade 62 a is a cutting blade for cutting the sheet-like electrophoresis gel 55. The blade driving unit 62 drives the blade 62a in the Z direction in FIG. The frame, the stopper 63, and the fastening device 63a are fixing devices that hold the sheet-like electrophoresis gel 55 together with the movable stage 61. The movable stage 61 does not drive the blade 62 a relative to the sheet-like electrophoresis gel 55, but the blade driving unit 62 makes the blade 62 a relative to the sheet-like electrophoresis gel 55. You may comprise so that it may drive.

  3C, the sheet-like electrophoresis gel 55 is fixed to the movable stage 61 by the frame, the stopper 63, and the fastening tool 63a, and the blade 62a is fixed by the movable stage 61 to the sheet-like electrophoresis. It moves so as to cross the gel 55, cuts the sheet-like electrophoresis gel 55 into strips, and separates the belt-like electrophoresis gel 55a. In this way, by cutting the blade 62a across the sheet-like electrophoresis gel 55, the amount of water contained in the electrophoresis gel 53 has been reduced in order to prevent degradation of the electrophoresis gel 53 due to hydrolysis. Even when it is (dried), it can cut suitably.

  As shown in FIG. 3B, the sheet-like electrophoresis gel 55 is sandwiched between the frame and the stopper 63 and the movable stage 61, and is firmly fixed by the fastening tool 63a. The gel-facing surface (stopper) of the frame and the stopper 63 is preferably made of a material having a high frictional force against the sheet-like electrophoresis gel 55 and the movable stage 61. For example, the material of the frame and the gel-facing surface (stopper) of the stopper 63 is preferably polydimethylsiloxane (PDMS; Polydimethylsiloxane), silicone resin, or the like.

  By fixing the sheet-like electrophoresis gel 55 in this way, it is possible to suppress variations in the width of the obtained belt-like electrophoresis gel 55a as shown in the examples described later.

  Next, the cutting method will be described in detail. First, the sheet-like electrophoresis gel 55 fixed to the movable stage 61 is driven by driving the movable stage 61 in the X and Y directions, and the sheet cutting line of the sheet-like electrophoresis gel 55 (dotted line in FIG. 2C). ) To the first cutting start position (first cutting step). Next, the blade drive unit 62 lowers the blade 62a in the Z direction to a position in contact with the surface of the sheet-like electrophoresis gel 55, and then further lowers the depth to be cut at once (second cutting step). Although the cutting depth of one time differs depending on whether the sheet-like electrophoresis gel 55 is hard or soft, it is preferably set to 10 μm or more and 50 μm or less, for example. In one example, the cutting depth for one time can be set to 20 μm.

  Then, the sheet-like electrophoresis gel 55 is cut along the sheet cutting line by driving the movable stage 61 in the Y direction (third cutting step). Finally, the blade drive unit 62 raises the blade 62a to a height that does not interfere with the sheet-like electrophoresis gel 55 and the frame and the stopper 63, moves to the first cutting start position, and then moves to the first cutting start position. It moves in the width X direction in the short direction of 55a (moves to the second cutting start location. Fourth movable step). And the 1st-4th cutting process is repeated and the sheet-like electrophoresis gel 55 is cut | disconnected.

  As described above, in the cutting step, after separating one band-shaped electrophoresis gel 55a from the sheet-like electrophoresis gel 55, the relative position between the blade 62a and the sheet-like electrophoresis gel 55 in the X direction is adjusted. The blade 62a is preferably moved relative to the sheet-like electrophoresis gel 55 so that the blade 62a crosses the sheet-like electrophoresis gel 55 again. As a result, the sheet-like electrophoresis gel 55 can be cut while being fed in order, so that a large number of belt-like electrophoresis gels 55a can be successfully separated.

  In the cutting process, it is preferable that the automatic feeder adjusts the relative position in the X direction. Thereby, as shown in the Example mentioned later, the variation in the width | variety of the strip | belt-shaped electrophoresis gel 55a obtained can be suppressed.

  In the cutting step, it is preferable to adjust the relative position in the X direction in a state where the blade 62a and the sheet-like electrophoresis gel 55 are separated from each other. Thereby, it is possible to prevent the blade 62a from touching the sheet-like electrophoresis gel 55 and damaging the sheet-like electrophoresis gel 55 when the relative position is adjusted.

  In one embodiment, as shown in FIG. 4, in the cutting step, the adhesive tape 64a (for example, Kapton tape) is applied to the sheet-like electrophoresis gel 55, and the adhesive tape 64a and the sheet-like electrophoresis are performed. The gel 55 may be cut.

  FIG. 4 is a perspective view showing a state of the sheet-like electrophoresis gel 55 and the belt-like electrophoresis gel 55a to which the adhesive tape 64a is attached. As shown in FIG. 4, the adhesive tape 64a is affixed to both ends of the sheet-like electrophoresis gel 55 in the direction (Y direction) that the blade 62a crosses. And when cut | disconnected, the adhesive tape 64a remains on the both ends of the longitudinal direction of the strip | belt-shaped electrophoresis gel 55a. At this time, since the adhesive tape 64a can be used as a support for transporting the separated strip-shaped electrophoresis gel 55a, handling properties in subsequent operations are improved. Further, the sheet-like electrophoresis gel 55 can be more suitably fixed in the cutting step by the adhesive tape 64a.

  Hereinafter, the effect of each condition in the cutting process was demonstrated.

(Example of fixing)
FIG. 5 is a graph showing the effect of fixing the sheet-like electrophoresis gel 55 with a fixing device (the frame and the stopper 63 and the fastening tool 63a). Fig. 5 shows the width variation of the separated band-shaped electrophoresis gel 55a when cut in a state of being fixed with a fixing device, and (b) was cut without fixing the sheet-like electrophoresis gel 55 with a fixing device. The variation in the width | variety of the separated strip | belt-shaped electrophoresis gel 55a at the time is shown.

  As is clear from comparison between FIGS. 5A and 5B, by fixing the sheet-shaped electrophoresis gel 55 using a fixing device, the variation in the width of the separated band-shaped electrophoresis gel 55a can be reduced. I was able to suppress it.

(Example of automatic feeding)
FIG. 6 is a graph showing the effect of automatically feeding (moving in the X direction) the sheet-like electrophoresis gel 55 by the automatic feeding device (movable stage 61). (A) is a sheet-like electrophoresis. The width of the separated band-shaped electrophoresis gel 55a when the gel 55 is automatically fed by the automatic feeding device is shown. (B) shows the separation when the sheet-like electrophoresis gel 55 is manually fed. The variation in the width of the strip-shaped electrophoresis gel 55a is shown.

  As is clear from comparison between FIGS. 5A and 5B, by fixing the sheet-shaped electrophoresis gel 55 using a fixing device, the variation in the width of the separated band-shaped electrophoresis gel 55a can be reduced. It could be suppressed to 5% or less (cutting variation within 1.2 mm ± 0.06 mm with respect to the strip-shaped electrophoresis gel 55a having a longitudinal direction of 52 mm × a lateral direction of 1.2 mm).

(Fixing process)
Subsequently, as shown in FIG. 2D, the electrophoresis gel chip 50 is manufactured by fixing the band-shaped electrophoresis gel 55a obtained in the cutting step to the support 51 (fixing step). As described above, since the belt-like electrophoresis gel 55a (or the support 51) is provided with an adhesive layer, the belt-like electrophoresis is performed by pressing the support 51 against the belt-like electrophoresis gel 55a. The gel 55a can be fixed to the support 51.

  However, when the belt-shaped electrophoresis gel 55a is fixed to the support body 51, the belt-shaped electrophoresis gel 55a may not be properly fixed because it is bent. Therefore, in the fixing step, it is preferable to fix the belt-shaped electrophoresis gel 55a to the support 51 using a fixing jig 70 as shown in FIG.

  FIG. 7 is a diagram for explaining a fixing process of the method for manufacturing an electrophoresis gel chip according to the embodiment of the present invention. FIG. 7A is a perspective view showing a schematic configuration of the fixing jig 70. As shown in FIG. 7A, the fixing jig 70 is provided with a groove 70a having a width D into which the belt-like electrophoresis gel 55a can be inserted. In one example, the groove 70a can have a width of 1.2 mm and a depth of 3 mm.

  First, as shown in FIG. 7B, a band-shaped electrophoresis gel 55 a is inserted into the groove 70 a of the fixing jig 70. Then, as shown in FIG. 7C, in a state where the belt-shaped electrophoresis gel 55a is inserted into the groove 70a of the fixing jig 70, the support body 51 is further inserted into the groove 70a and pushed into the belt 70a. The electrophoresis gel 55a can be fixed to the support 51. At this time, since the groove 70 a serves as a guide, the belt-shaped electrophoresis gel 55 a can be fixed at an appropriate position of the support 51.

  In addition, as shown in FIG.7 (d), when the strip | belt-shaped electrophoresis gel 55a protrudes from the support body 51, the protrusion part is cut | disconnected. As described above, the electrophoresis gel chip 50 can be manufactured as shown in FIG.

  In addition, as shown in FIG.7 (d), by making the belt-shaped electrophoresis gel 55a long with respect to the support body 51 previously, and fixing to the support body 51, it is made to remove an excess part, It is possible to manufacture a high-quality electrophoresis gel chip 50 by absorbing variations in the manufacturing process (for example, variations in the length of the belt-shaped electrophoresis gel 55a).

(Two-dimensional electrophoresis)
Next, a method for using the electrophoresis gel chip 50 according to the present invention will be described. In one embodiment, the electrophoresis gel chip 50 according to the present invention can be applied to an automated two-dimensional electrophoresis apparatus 100 as described below, for example. However, the use of the electrophoresis gel chip 50 is not limited to the case where it is applied to the automated two-dimensional electrophoresis apparatus 100, and can be suitably applied to the general implementation of the electrophoresis method involving the transportation of the gel.

  FIG. 8A is a perspective view showing a main configuration of the automated two-dimensional electrophoresis apparatus 100. FIG. FIG. 8B shows a configuration in which the electrophoresis gel chip 56 from which the protective film 54 has been peeled off from the electrophoresis gel chip 50 is coupled to the arm 31 of the holding means 3. 8C and 8D are a cross-sectional view and a top view showing the configuration of the second separation unit (sample separation instrument) 20 used in the automated two-dimensional electrophoresis apparatus 100. FIG.

  That is, the electrophoresis gel chip 50 is first set on the automated two-dimensional electrophoresis apparatus 100 (the arm 31) after the protective film 54 is peeled off.

  An automated two-dimensional electrophoresis apparatus 100 according to this embodiment includes a fixing unit 1 that fixes a second separation unit (sample separation instrument) 20 that performs second-dimensional separation of a sample, and an electrophoresis gel 53 that performs first-dimensional separation. Holding means 3 having an arm 31 for holding a support 51 to which the motor is fixed, and a driving means 4 (41 and 42) for moving the fixing means 1 and / or the holding means 3 to change the relative positions of both. ing.

  In the automated two-dimensional electrophoresis apparatus 100, the sample is separated in the first direction (Y direction in the figure) by the first separation unit (not shown), and then conveyed to the second opening 26 of the second separation unit 20. After being bonded, the second separation unit 20 separates the second direction (X direction in the figure).

  As for the shape of the second opening 26, as shown in FIGS. 8C and 8D, the width of the opening through the upper insulating plate 22 in the second buffer solution tank 28b is the groove width of the corresponding lower insulating plate 21. Wider. Due to this difference, the electrophoresis gel 53 and the second medium 24 can be brought into close contact with each other, and as a result, the second-dimensional separation of the sample in the electrophoresis gel 53 after the first-dimensional separation can be successfully performed.

  In order to adhere the electrophoretic gel 53 containing the separated sample to the second medium 24, the insulator 20a covering the second medium 24 must have a portion for bringing the electrophoretic gel 53 and the second medium 24 into close contact with each other. . Such a portion may be the second opening 26 or a further opening (third opening) 26 ′ may be provided between the first opening 25 and the second opening 26. The first medium supply port has an appropriate size and shape for adhering the electrophoresis gel 53 including the separated sample to the second medium 24 in a plane perpendicular to the first direction.

  When the second separation unit 20 includes the second medium 24, the second medium 24 is inserted from the second opening 26 in order to bring the electrophoresis gel 53 and the second medium 24 into close contact with each other in the first medium supply unit. It is preferable that the protrusion protrudes, and in order to increase the degree of adhesion, it is more preferable that the protruding second medium portion has no irregularities. When the second medium 24 does not protrude from the second opening 26, an adhesive member (not shown) for bringing the electrophoresis gel 53 and the second medium 24 into close contact with the second opening 26 is provided. It only has to be. Preferred adhesive members include, but are not limited to, agarose, gels such as acrylamide with low viscosity (about 1 to 3%), and high viscosity liquids such as glycerin, polyethylene glycol, and hydroxypropylcellulose.

  The parameters defining the separation in the first direction (Y direction in the figure) and the separation in the second direction (X direction in the figure) may be the same, but may be different for improving the separation performance. preferable. Parameters that define the separation in these two directions include isoelectric point of protein, molecular weight, surface charge per unit size (zone electrophoresis), distribution coefficient to micelle (micelle electrokinetic chromatography), stationary phase-transfer Examples include the partition coefficient to phases (electrochromatography) and affinity constants for interacting substances (affinity binding electrophoresis). In normal two-dimensional electrophoresis, separation in the first direction is based on the isoelectric point. Thus, the separation in the second direction is performed based on the molecular weight.

  As shown in FIG. 8 (a), in the automated two-dimensional electrophoresis apparatus 100, the driving means 4 includes a vertical direction driving means 41 and a parallel direction driving means 42. Specifically, the holding means 3 (supporting) The arm 31) is held so as to be movable in the Z-axis direction by the vertical driving means 41 via a recess (holding means connecting portion) 41 ′ of the vertical driving means 41. Further, the vertical direction driving means 41 is held so as to be movable in the X-axis direction by the parallel direction driving means 42 via a recess (Z-axis stage coupling portion) 42 ′ of the parallel direction driving means 42. Therefore, the fixing means 1 and / or the holding means 3 are moved in the vertical direction by the vertical direction driving means 41 and in the parallel direction by the parallel direction driving means 42 with respect to the plane composed of the first direction and the second direction. obtain.

  The term “sample” is used interchangeably with specimens, preparations in the art, and as used herein, a “biological sample” or equivalent thereof is intended. 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 fluids) 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, genomic DNA sample and / or extracted from the biological sample and the tissue sample. A total RNA sample is also included.

  The two-dimensional electrophoresis will be described below by taking as an example the case where the first dimension is performed by gel isoelectric focusing and the second dimension is performed by SDS-PAGE, but the present invention is not limited to this embodiment.

  In the automated two-dimensional electrophoresis apparatus 100, the first medium voltage applying unit 5 and the second medium voltage applying unit 6 are connected to the first separation unit 10 and the second separation unit 20 in order to automate the entire process. Wiring means 7 and 8 are provided. The wiring means 7 has a third electrode 14 (a pair of anode and cathode) provided in the first separation tank 11d at its tip, and the wiring means 8 at its tip. The first electrode 29a and the second electrode 29b are respectively provided in the first buffer solution tank 28a and the second buffer solution tank 28b (see FIG. 9).

  FIG. 9 shows a cross-sectional view of the fixing means 1 of the automated two-dimensional electrophoresis apparatus 100 according to this embodiment and members disposed in the vicinity thereof. In FIG. 9, two-dimensional electrophoresis is performed from left to right.

  On the fixing means 1 of the automated two-dimensional electrophoresis apparatus 100 according to the present embodiment, a first separation unit 10 that performs first-dimensional separation of a sample and a second separation unit that performs second-dimensional separation of a sample (sample separation instrument) 20) is fixed by vacuum suction. The first separation unit 10 is provided with a plurality of tanks, one of which is provided with an electrode (third electrode) 14. The second separation unit 20 is provided with two tanks (a first buffer solution tank 28a and a second buffer solution tank 28b), and electrodes (a first electrode 29a and a second electrode 29b) are provided respectively. .

  The support 51 is held by an arm 31 that is a part of the holding means 3 (not shown) of the automated two-dimensional electrophoresis apparatus 100 according to this embodiment. The arm 31 can be moved in the X direction and / or the Z direction as shown in the figure by the driving means 4 of the automated two-dimensional electrophoresis apparatus 100 according to the present embodiment.

  After the electrophoretic gel chip 56 is coupled by vacuum suction, the arm 31 is moved in the direction of arrow 2 in FIG. As the electrophoresis gel chip 56 is moved by the driving means 4, the electrophoresis gel 53 is subjected to desired processing in each tank provided in the first separation unit 10, and is subsequently conveyed to the second separation unit 20.

  In addition, the movement of the arm 31 by the drive means 4 is performed as follows. The parallel direction driving means 42 drives the arm 31 together with the vertical direction driving means 41 to the desired X position of the first medium placement tank 11a, and then the driving means 41 lowers the arm 31 to the desired Z position. Subsequently, by controlling the adsorption of the electrophoresis gel chip 56 arranged in the first medium arrangement tank 11 a to the arm 31 by the control means, the electrophoresis gel 53 can be moved by the driving means 4. The adsorption control to the arm 31 can be automatically controlled by using, for example, an electromagnetic valve.

  The electrophoresis gel 53 has a very thin shape compared to its width and length. Therefore, it is difficult not only to distinguish the front and back surfaces of the gel and the direction of the pH gradient, but also to cause warping and twisting, and it is difficult to keep the shape constant. This can be a factor in poor reproducibility of electrophoresis results. Furthermore, since the operation of the electrophoresis gel 53 in each step of electrophoresis is not easy, it is difficult to improve the positional accuracy when moving the electrophoresis gel 53. In order to overcome such problems and stably hold and operate the electrophoretic gel 53 with an automatic apparatus, the present inventors used the electrophoretic gel 53 fixed to the support 51.

  The configurations of the first separation unit 10 and the second separation unit 20 in FIG. 9 are shown in more detail in FIGS. 10 and 11. FIG. 10 shows a cross-sectional view of the fixing means 1 to which the first separation unit 10 that performs the first-dimensional separation of the sample and the second separation unit 20 that performs the second-dimensional separation of the sample are fixed. A top view is shown.

  As shown in FIG. 10, on the fixing means 1 of the automated two-dimensional electrophoresis apparatus 100 according to this embodiment, a first separation unit 10 that performs first-dimensional separation of a sample and a second separation that performs second-dimensional separation of a sample. The two separation parts 20 are fixed. The first separator 10 is provided with a plurality of reagent tanks 11 (11a to 11d) and 12 (12a to 12d), and the second separator 20 includes two tanks (a first buffer tank 28a and a second buffer tank 2). A buffer bath 28b) is provided. In FIG. 9, the first separation unit 10 uses a reagent tank 11 consisting of four tanks and a reagent tank 12 consisting of seven tanks. However, in FIGS. In order to do this, the first separation unit 10 has a configuration including a reagent tank 11 composed of four tanks and a reagent tank 12 composed of four tanks.

  In the first separation unit 10, the step of introducing the sample into the electrophoresis gel 53, the step of swelling the electrophoresis gel 53, the step of applying a voltage to the electrophoresis gel 53 and separating the sample in the first direction, the electrophoresis gel The process of dye | staining the isolation | separation sample in 53 and the process of equilibrating to the environment in the 2nd isolation | separation part 20 are performed. The first separation unit 10 has a preferable shape for treating the electrophoresis gel 53 in this way. By separately adding the sample to the electrophoresis gel 53 and swelling the first medium, the swelling speed can be improved.

  The staining step may be performed before the first direction separation or after the second direction separation of the sample as usual, but the staining operation is complicated and time-consuming, and it is very difficult to try automation. Therefore, it is preferable to bind the protein (or DNA) in the sample to be stained with the fluorescent substance after the sample separation in the first direction and before the sample separation in the second direction. Examples of the binding mode between the protein (or DNA) in the sample and the fluorescent substance include, but are not limited to, a covalent bond, an ionic bond, a coordinate bond, and an intercalation.

  The first separation unit 10 has a configuration in which grooves (reagent tanks) 11 and 12 are provided in a single insulator. The first reagent tank 11 is for storing reagents necessary for the process until the first-dimensional separation, and the second reagent tank 12 is necessary after the first-dimensional separation and before the second-dimensional separation. It is for storing the reagent. Specifically, the first reagent tank 11 includes a first medium placement tank 11 a, a sample tank 11 b, a swelling tank 11 c, and a first separation tank 11 d, and the sample tank 11 b has a sample introduction part 13. The second reagent tank 12 includes a first equilibration tank 12a, a staining tank 12b, a washing tank 12c, and a second equilibration tank 12d. The first separation tank 11d is a part where the first dimensional separation is performed in the electrophoresis gel 53, and is filled with a buffer solution necessary for the first dimensional separation. However, when the reagent stored in the swelling tank 11c contains a buffer solution necessary for the first-dimensional separation, the buffer solution necessary for the first-dimensional separation may not be filled in the first separation tank 11d. In the first separation tank 11d, the sample in the electrophoresis gel 53 is separated by applying a voltage from the first medium voltage applying means 5. The first equilibration tank 12a is preferably provided to store a buffer solution for replacing the buffer solution used for the first direction separation and increasing the efficiency of the staining performed after the first direction separation. The washing tank 12c is preferably provided for storing a buffer solution for washing excess fluorescent dye attached in the staining tank 12b in which the fluorescent dye is stored. The second equilibration tank 12d stores a reagent preferable for performing the second direction separation. For example, a reagent for reducing the protein in the electrophoresis gel 53 and a reagent for converting the protein into SDS are stored. . In addition, a buffer solution, a surfactant, an enzyme, an interacting substance, or the like may be stored depending on the second direction separation method.

  In the second medium 24, the separated sample separated in the first direction in the electrophoresis gel 53 is further separated in a second direction different from the first direction. In order to execute the sample separation in the second direction, the second separation unit 20 closely attaches the electrophoresis gel 53 including the sample separated in the first direction to the second medium 24; Is applied to separate the sample in the second direction. In the automated two-dimensional electrophoresis apparatus 100, a step of detecting a sample being separated in the second direction is also performed.

  The second separation unit 20 includes two grooves (first buffer solution tanks 28a) provided in the lower insulating part through the upper insulating part 22 in the insulating part 20a in which the lower insulating plate 21 and the upper insulating plate 22 are overlapped. And a second buffer tank 28b). The lower insulating plate 21 is provided with a groove (second medium storage portion) 24 ′ for covering and storing the second medium 24 between the lower insulating plate 21 and the upper insulating plate 22. The second medium 24 (not shown) stored in the second medium storage unit 24 ′ is covered with an insulating unit 20 a including the lower insulating plate 21 and the upper insulating plate 22, and the first opening 25 and the second opening 26 are covered. It can contact with the outside of the insulating part 20a.

  The first opening 25 and the second opening 26 face the first buffer solution tank 28a and the second buffer solution tank 28b provided in the second separation unit 20, respectively. In order to perform the sample separation in the second direction, the first buffer tank 28a and the second buffer tank 28b have the second medium 24 and the first opening 25 stored in the second medium storage section 24 ′. The first buffer solution and the second buffer solution that are in contact with each other via the second opening 26 are filled. The first buffer tank 28a and the second buffer tank 28b are provided with a first electrode 29a and a second electrode 29b, and the second medium voltage application means 6 passes through the first electrode 29a and the second electrode 29b. When a voltage is applied to the second medium 24, a current flows from the first opening 25 toward the second opening 26 and the sample is expanded / separated from the second opening 26 toward the first opening 25. The

  The automated two-dimensional electrophoresis apparatus 100 sets the sample, reagent, and separation medium necessary for two-dimensional electrophoresis at predetermined positions, and then appropriately controls each of the above-described means and automatically executes all processes. Control means. Information from a position sensor or the like for detecting the relative position of the fixing means 1 or the holding means 3 is input to the control means, and the control means performs the automation successfully by the fixing means 1 and the holding means 3 (arms). 31), the control signal of the drive means 4, the 1st medium voltage application means 5, the 2nd medium voltage application means 6, the 1st wiring means 7, and the 2nd wiring means 8 is output. The control unit included in the automated two-dimensional electrophoresis apparatus 100 according to the present embodiment is preferably a CPU that executes program codes that are manufactured using various languages and stored in a recording medium such as a ROM and / or a RAM. It may be a computing means.

  By starting the control, the driving unit 4 moves (conveys) the holding unit 3. Since the electrophoresis gel chip 56 is held by the arm 31 which is a part of the holding means 3, the electrophoresis gel chip 56 on which the electrophoresis gel 53 is immobilized is indirectly by the driving means 4 driven by the control means. The desired processing is performed as follows after being moved (conveyed).

  The electrophoresis gel chip 56 arranged in the first medium arrangement tank 11a is conveyed to the sample tank 11b. The electrophoresis gel chip 56 is maintained in the sample tank 11b until the sample is absorbed by the electrophoresis gel 53. The time required for sample absorption is recorded in the storage of the control means. Next, the electrophoresis gel chip 56 is conveyed to the swelling tank 11c, maintained in the swelling tank 11c until the electrophoresis gel 53 swells, and is shaken minutely as necessary. Information relating to the time required for the electrophoresis gel 53 to swell and the micro-shaking operation are also recorded in the storage section of the control means. The electrophoretic gel 53 swollen on the electrophoretic gel chip 56 is transported to the first separation tank 11d and disposed between the third electrodes 14 in the first separation tank 11d. Here, a voltage is applied to the electrophoresis gel 53 by the first medium voltage application means 5, and the sample is separated in the first direction in the electrophoresis gel 53. Information about the time required for sample separation and the required voltage is also recorded in the storage of the control means. Each information described above is appropriately selected and executed according to the type of electrophoresis gel 53 to be used, the type of sample, and the type of each reagent by a program recorded in the storage unit of the control means.

  After the separation in the first direction in the electrophoresis gel 53 is completed, the electrophoresis gel 53 is transported to the first equilibration tank 12a, and if necessary, the subsequent staining is successfully performed. Equilibrated to do. Next, the equilibrated electrophoresis gel 53 is transported to the staining tank 12b, and is micro-shaken as necessary to stain the sample in the electrophoresis gel 53. The dyed electrophoresis gel 53 is conveyed to the washing tank 12c, and excessive dye is washed appropriately by being shaken slightly. Next, the electrophoretic gel 53 after decolorization is transported to the second equilibration tank 12d and finely shaken as necessary in order to successfully perform separation in the second direction in the second medium. , And so that subsequent separation in the second direction in the second medium is carried out successfully. The equilibrated electrophoretic gel 53 is conveyed to the first medium supply unit 26 of the second medium 24 and is in close contact with the second medium 24.

  After the electrophoresis gel 53 is brought into close contact with the second medium 24, a voltage is applied to the second medium 24 by the second medium voltage applying means 6, whereby the sample is separated in the second direction in the second medium 24. The When real-time monitoring is performed, it may be performed via a detection means (not shown) during the separation in the second direction. Information such as the time required for separation in the reagent vessels 12a to 12d and the second medium 24 is also recorded in the storage unit of the control means. Each piece of information described above is appropriately selected and executed according to the type of electrophoresis gel 53 and second medium 24 to be used, the type of sample, and the type of each reagent by a program recorded in the storage unit of the control means.

  As described above, in the automated two-dimensional electrophoresis apparatus 100, the above-described control by the control unit is executed, whereby the two-dimensional electrophoresis process can be performed fully automatically. In addition, since the automated two-dimensional electrophoresis apparatus 100 includes a control unit that performs the control as described above, it is possible to easily select and / or introduce various protocols and pursue sample separation performance. . In addition, a two-dimensional high voltage application control system for feedback control of a voltage application program for two-dimensional electrophoresis can be introduced and controlled in cooperation with an automatic stage.

  Note that the second medium 24 may be manufactured in the second medium storage unit 24 ′, or the separately manufactured second medium 24 may be moved and fixed to the second medium storage unit 24 ′. When the second medium 24 is not manufactured by the second separation unit 20, the second medium storage unit 24 ′ does not need to be a groove, and in that case, a spacer (not shown) equal to the thickness of the second medium 24 is provided below. What is necessary is just to arrange | position so that the site | part which fixes the 2nd medium 24 of the insulating plate 21 may be enclosed, and to adhere | attach the lower insulating plate 21 and the upper insulating plate 22 via a spacer.

  Since the second medium 24 is preferably in contact with the buffer solution only in the first opening 25 and the second opening 26, the insulator 20a covering the second medium 24 is preferably made of a highly waterproof material. In addition, in order to detect a sample without removing the second medium 24 from the insulator 20a, such as in real-time monitoring, the insulator 20a is preferably made of a material having high light transmittance. Examples of the substance having such characteristics include glass and resin, and examples of the resin material include PMMA, PDMS, COP, polycarbonate, polystyrene, PET, vinyl chloride, and the like from the viewpoint of weight, operability, and productivity. An acrylic resin (for example, polymethyl methacrylate (PMMA) etc.) is preferable.

  In the automated two-dimensional electrophoresis apparatus 100, various processes are executed while the electrophoresis gel chip 56 is sequentially moved. Therefore, the three-dimensional positional accuracy of the electrophoresis gel chip 56 is important. In this case, not only the electrophoresis gel chip 56 is firmly fixed, but also the first separation unit 10 and the second separation unit 20 that move relatively need to be firmly fixed.

  Considering that the first separation unit 10, the second separation unit 20, and the electrophoresis gel chip 56 are exchanged for each sample, it is preferable that these immobilizations are detachable. As a mechanism for fixing the first separation unit 10, the second separation unit 20, and the electrophoresis gel chip 56 to the fixing unit 1 and the holding unit 3, in addition to the vacuum adsorption mechanism as described above, a pinching and fixing mechanism, magnetic force fixing Examples include, but are not limited to, mechanisms and electrostatic attraction mechanisms. Moreover, when employ | adopting a vacuum suction mechanism, it is preferable to fix through a vacuum suction plate.

  In the case where the first separation unit 10 and the second separation unit 20 are made of transparent PMMA, the excitation light is transmitted to the stage (fixing means) below the first separation unit 10 and the second separation unit 20 when detecting fluorescence of the sample. ) Irradiates to 1. If the stage (fixing means) 1 has irregularities, the excitation light and / or fluorescence wavelength is reflected non-uniformly, increasing the background at the time of detection and preventing successful detection. Therefore, the vacuum suction plate has a color tone with little reflection, is processed into a flat surface without unevenness, and a suction hole provided for vacuum suction is provided in a portion other than directly below the detection unit of the second separation unit 20 Is preferred.

  The first electrode 29a and the second electrode 29b provided in the first buffer tank 28a and the second buffer tank 28b, respectively, and the third electrode 14 provided in the first separation tank 11d may or may not be fixed. May be. When fixed, the electrodes 28a, 28b and 14 may be conductors patterned in the respective tanks 28a, 28b and 14. Further, when the electrode is transported / removed by automation, as shown in FIG. 12, the arm 31 transports the electrodes 29a, 29b and 14 to the tanks 28a, 28b and 11d, respectively, and the electrodes provided in each tank It can be attached to and detached from a fixing part (not shown). Alternatively, the electrodes 29a, 29b, and 14 may be immersed in the filled buffer solution without being fixed to the first buffer solution tank 28a, the second buffer solution tank 28b, and the first separation tank 11d, respectively. When the electrodes 29a, 29b and 14 are used in a transportable form, each electrode can be successfully cleaned by providing an electrode cleaning tank 40 as shown in FIG. In addition, the installation site | part of the electrode washing tank 40 is not restricted to the site | part shown in FIG. 12, It can be provided in the arbitrary site | parts of the 1st separation part 10. FIG.

  As described above, the first separation unit 10 includes a plurality of tanks (reagent tanks) 11 and 12. The plurality of reagent tanks are preferably filled with a reagent necessary for sample separation in the electrophoresis gel 53 and a reagent necessary for staining the electrophoresis gel 53 after separation (or the sample in the electrophoresis gel 53). The reagent to be filled can be appropriately selected as necessary. Moreover, what is necessary is just to increase / decrease the number of the some reagent tanks 11 * 12 according to a required process process.

  In a specific embodiment, the plurality of reagent tanks 11 and 12 are provided with a peelable and removable sheet-like sealing material for sealing the filled reagent (see FIG. 13). By providing the sheet-like sealing material, scattering of the reagent contained can be avoided, and the first separation unit 10 containing the reagent can be easily stored.

  In one aspect, the sheet-like sealing material can be peeled and removed by a sheet peeling means (not shown) provided in the automated two-dimensional electrophoresis apparatus 100. The sheet peeling unit is preferably driven by the driving unit 4. In this case, even if the first separation unit 10 is installed in the automated two-dimensional electrophoresis apparatus 100 with the sheet-like sealing material provided, it can be successfully peeled and removed under the control of the control means. In addition, in FIG. 13, the arm 31 of the holding means 3 has shown the form which is a sheet | seat peeling means.

  In another aspect, the sheet-like sealing material can be pierced by a pressing external force from the electrophoresis gel chip 56. Further, the support 51 may be provided with an auxiliary tool for drilling in order to facilitate drilling. In this case, the sheet-shaped sealing material can be perforated by the normal operation of the electrophoretic gel chip 56 in the first separation unit without providing additional means such as the sheet peeling means.

  In a further embodiment, the first separation unit 10 has an on-demand adjustment reagent tank, and the on-time adjustment reagent tank is in a single reagent tank containing a plurality of reagents by a sheet-like sealing material, When the sheet-like sealing material is peeled off or perforated, a plurality of reagents are mixed. Even when a reagent that cannot be adjusted in advance and needs to be adjusted as needed is used in the process executed by the automated two-dimensional electrophoresis apparatus 100, the automated two-dimensional electrophoresis apparatus according to the present embodiment is used. If 100 is used, complicated work can be avoided.

  The automated two-dimensional electrophoresis apparatus 100 including the first separation unit 10 having the reagent tanks 11 and 12 as described above includes reagent injection means (not shown) for injecting the reagent into the reagent tanks 11 and 12. It is still more preferable that it is characterized. This reagent injection means is preferably configured to operate in conjunction with the holding means and the driving means.

  Although the present invention has been described using an embodiment in which the insulator 20a, the first buffer solution tank 28a, and the second buffer solution tank 28b are integrally formed in the second separation unit 20, these are configured separately. It may be.

  In another embodiment of the second separation unit 20, as shown in FIG. 14, the second separation unit 20 includes a lower insulating plate 21 and an upper insulation sandwiching a second medium storage unit 24 ′ that stores the second medium 24. It comprises an insulator 20a formed from a plate 22, and an electrophoresis tank 30 having an insulator receiver 27 that receives the insulator 20a, a first buffer tank 28a, and a second buffer tank 28b. In the insulator 20a, the lower insulating plate 21 and the upper insulating plate 22 have a first opening 25 and a second opening for the second medium 24 to contact the buffer solution in the first buffer solution tank 28a and the second buffer solution tank 28b, respectively. An opening 26 is provided. In the migration tank 30, the insulator receiving portion 27 has a flat surface that can receive the insulator 20a (particularly the lower insulating plate 21) without any gap.

  In the present embodiment, the materials constituting the insulator 20a and the migration tank 30 may be the same or different. The migration tank 30 is preferably made of a highly waterproof material from the viewpoint of being filled with a buffer solution. The insulator 20a and the electrophoresis tank 30 are preferably fixed, but more preferably detachable by a vacuum suction mechanism, a pinching and fixing mechanism, a magnetic force fixing mechanism, and an electrostatic suction mechanism. Furthermore, the migration tank 30 may be provided with the insulator receiving portion 27, the first buffer solution tank 28a, and the second buffer solution tank 28b separately. In this case, the first buffer solution does not leak without being leaked. As long as it has a form that can be closely attached so as to be supplied to the opening 25 and the second opening 26, it is possible to employ a vacuum suction mechanism, a pinch fixing mechanism, a magnetic force fixing mechanism, an electrostatic suction mechanism, and the like. it can.

  It should be noted that the specific embodiments made in the section of the best mode for carrying out the invention and the following examples are merely to clarify the technical contents of the present invention, and to such specific examples. It is not to be construed as limiting in any way whatsoever, and those skilled in the art can implement the invention within the spirit of the invention and the scope of the appended claims.

  Moreover, all the academic literatures and patent literatures described in this specification are incorporated herein by reference.

  The present invention can be used in the field of manufacturing electrophoresis gel chips for analyzing biological samples and the like.

50 Electrophoretic Gel Chip 51 Support 52 Adhesive Layer 52a Chip Adhesive Layer 52b Gel Adhesive Layer 53 Electrophoretic Gel 54 Protective Film (Protective Film)
55 Electrophoretic gel in sheet form 55a Electrophoretic gel in strip form 60 Cutting device 61 Movable stage (driving means, automatic feeding device, fixing instrument, fixing means)
62 Blade drive part (drive means)
62a Blade 63 Frame and stopper (fixing device, fixing means)
63a Fastening device (fixing device, fixing means)
64a Adhesive tape 70 Fixing jig 70a Groove 100 Automated two-dimensional electrophoresis apparatus

Claims (18)

Electrophoresis comprising a support made of an insulator, a strip-shaped electrophoresis gel fixed to the support, and a protective film covering a surface of the strip-shaped electrophoresis gel opposite to the support A method for producing a gel chip, comprising:
A cutting step of cutting the sheet-like electrophoresis gel whose one surface is covered with a protective film to separate the band-shaped electrophoresis gel, and the band-shaped electrophoresis gel separated in the cutting step A method for producing an electrophoresis gel chip, comprising a fixing step of fixing to a gel.   2. The electrophoresis according to claim 1, wherein in the cutting step, the blade is moved relative to the sheet-like electrophoresis gel so that the blade crosses the sheet-like electrophoresis gel. Gel chip manufacturing method. In the cutting process,
After separating the one band-shaped electrophoresis gel from the sheet-shaped electrophoresis gel,
Adjusting the relative position of the blade and the sheet-like electrophoresis gel in a second direction perpendicular to the first direction traversed by the blade;
The electrophoresis gel chip according to claim 2, wherein the blade is moved relative to the sheet-like electrophoresis gel so that the blade crosses the sheet-like electrophoresis gel again. Manufacturing method.   In the said cutting process, an automatic feeder adjusts the said relative position in a 2nd direction, The manufacturing method of the electrophoresis gel chip | tip of Claim 3 characterized by the above-mentioned.   The said cutting process WHEREIN: The said relative position in a 2nd direction is adjusted in the state which the said blade and the said sheet-like electrophoresis gel separated, The manufacture of the electrophoresis gel chip | tip of Claim 3 or 4 characterized by the above-mentioned. Method.   6. The cutting process according to claim 1, wherein in the cutting step, the sheet-like electrophoresis gel is cut in a state in which a fixing device is sandwiched and fixed with the sheet-like electrophoresis gel interposed therebetween. The manufacturing method of the electrophoresis gel chip of description.   In the cutting step, the sheet-like electrophoresis gel is cut together with the adhesive tape in a state where the adhesive tape is attached to the sheet-like electrophoresis gel. 2. A method for producing an electrophoresis gel chip according to item 1.   The said fixing step WHEREIN: The said strip | belt-shaped electrophoresis gel is fixed to the said support body in the state inserted in the groove | channel provided in the fixing jig, The said strip | belt-shaped electrophoresis gel is fixed to the said support body. The manufacturing method of the electrophoresis gel chip | tip of any one. The support is a plate-like insulator,
The method for producing an electrophoresis gel chip according to claim 1, wherein in the fixing step, the belt-shaped electrophoresis gel is fixed to a side end surface of the support. The length of the strip-shaped electrophoresis gel separated in the cutting step is longer than the length of the region for fixing the strip-shaped electrophoresis gel in the support,
The electrophoresis gel chip according to any one of claims 1 to 9, further comprising a removal step of removing a portion of the belt-shaped electrophoresis gel that protrudes from the support after the fixing step. Manufacturing method. Electrophoresis comprising a support made of an insulator, a strip-shaped electrophoresis gel fixed to the support, and a protective film covering a surface of the strip-shaped electrophoresis gel opposite to the support A gel chip manufacturing kit,
A kit for producing an electrophoresis gel chip, comprising a cutting device for cutting a sheet-like electrophoresis gel whose one surface is covered with a protective film and separating the belt-like electrophoresis gel. The cutting device includes a blade and a driving means,
12. The drive means according to claim 11, wherein the blade is moved relative to the sheet-like electrophoresis gel so that the blade traverses the sheet-like electrophoresis gel. Production kit for electrophoresis gel chip.   The drive means separates the band-shaped electrophoresis gel from the sheet-like electrophoresis gel, and then the blade and the sheet-like shape in a second direction perpendicular to the first direction across the blade. The relative position with respect to the electrophoresis gel is adjusted, and the blade is moved relative to the sheet-like electrophoresis gel so that the blade crosses the sheet-like electrophoresis gel again. An electrophoresis gel chip manufacturing kit according to claim 12.   The electrophoretic gel chip manufacturing kit according to claim 13, wherein the driving unit automatically adjusts the relative position in the second direction.   The electrophoretic gel chip according to claim 13 or 14, wherein the driving means adjusts the relative position in the second direction in a state where the blade and the sheet-like electrophoretic gel are separated from each other. Production kit.   The electrophoretic gel chip manufacturing kit according to any one of claims 11 to 15, wherein the cutting device further includes fixing means for fixing the sheet-like electrophoretic gel with the sheet-like electrophoretic gel interposed therebetween.   The kit for producing an electrophoresis gel chip according to any one of claims 11 to 16, further comprising a fixing jig having a groove for inserting the band-shaped electrophoresis gel. A support made of an insulator;
A strip-shaped electrophoresis gel fixed to the support;
An electrophoresis gel chip comprising a protective film covering a surface of the belt-shaped electrophoresis gel opposite to the support.

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