JP4947373B2 - Electrode and transfer apparatus and transfer method using the same - Google Patents

Description

  The present invention relates to an electrode for passing an electric current through an electrolytic solution, and a transfer apparatus and transfer method for transferring a sample from a specific medium to another medium using the electrode.

Conventionally, in the field of life science, in particular, a current is passed through an electrolytic solution. Specifically, an electric current is passed through a medium (eg, polyacrylamide gel, agarose gel, etc.) containing a charged sample (eg, protein denatured by SDS, DNA, etc.) via a buffer solution. Thus, electrophoresis of the sample is performed. Further, the medium containing the sample is overlaid with a medium (membrane made of nitrocellulose, PVDF, etc.) to which the sample is to be transferred, and similarly, the sample is electrophoresed by passing an electric current through a buffer solution. In addition, a sample is also transferred (see Non-Patent Document 1, etc.).
Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd Ed. , Cold Spring Harbor Laboratory (2001)

  However, when an electric current is passed through the electrolytic solution as described above, the electrolyte may be electrolyzed at the electrode, and bubbles may be generated. When the flowing current increases, the amount of generated bubbles also increases, which causes adverse effects such as distortion in the surrounding potential distribution.

  For example, in a transfer device in which an electric current is passed through a gel containing a protein sample or the like via an electrolytic solution and the sample is transferred to a membrane disposed in contact with the gel, conventionally, a filter paper is used between the electrode and the gel. The filter paper, which was sandwiched between them, absorbed bubbles generated in the electrode, but was insufficient.

  Further, when combining the filter paper and the gel, bubbles are taken in, which may cause the potential to be disturbed. FIG. 3 is a diagram illustrating a transfer result of a transfer device using a filter paper according to the related art. As shown in FIG. 3 (a), it is possible to combine the filter paper and the gel well without introducing bubbles. However, as shown in FIGS. In some cases, it was disturbed. Therefore, a method for blocking or absorbing the bubbles without using filter paper has been demanded.

  Furthermore, until now, there has been no study on an electrode for allowing a current to flow through an electrolytic solution that can remove the generated bubbles.

  This invention is made | formed in view of the said subject, and it aims at providing the electrode for flowing an electric current through electrolyte solution which can remove the produced | generated bubble.

  In order to solve the above problems, an electrode according to the present invention is an electrode for passing a current through an electrolytic solution, and includes an electrode layer and an insulating layer provided below the electrode layer. The electrode layer is provided with a gap penetrating the electrode layer, and the insulating layer is made of a porous material.

  According to the above configuration, since the gap penetrating the electrode layer is provided in the electrode layer, bubbles generated in the electrode layer when an electric current flows through the electrolytic solution pass through the gap. It can move to the insulating layer. And since the said insulating layer consists of a porous substance, it can absorb the bubble which has moved. As described above, according to the above configuration, the electrode according to the present invention can remove bubbles generated in the electrode.

  In the above electrode, the porous substance includes silicone resin, α-olefin copolymer, polytetrafluoroethylene resin, fluorine resin, polyurethane resin, polyvinyl alcohol resin, polyvinyl chloride resin, chlorinated polyethylene resin, polyethylene resin, Polypropylene resin, ethylene-vinyl acetate copolymer, polymethyl methacrylate resin, polystyrene resin, styrene-butadiene-acrylonitrile copolymer, polyvinyl formal resin, epoxy resin, phenol resin, urea resin, acrylic rubber, acrylonitrile-butadiene rubber, Styrene-butadiene rubber, butadiene rubber, isoprene rubber, chloroprene rubber, polystyrene elastomer, polyolefin elastomer, polyester elastomer, polyamide elastomer, Mikyuraito, mica, glass fiber, rock wool, be carbon fibers, and a material selected from the group consisting of ceramic fibers preferred.

  Since the above-described substances are porous and can easily take in gas, they can be suitably used for forming the insulating layer.

  In the electrode, the gap may be provided in the electrode layer in a stripe shape or a mesh shape.

  According to said structure, the bubble which penetrated this electrode layer is provided in the said electrode layer in stripe form or mesh shape, and the bubble produced | generated in this electrode layer is permeate | transmitted to the said insulating layer efficiently. Can do.

  In the electrode, the ratio of the voids in the electrode layer is preferably 1/20 or more in area.

  According to said structure, the bubble produced | generated in this electrode layer can be efficiently permeate | transmitted to the said insulating layer by providing the said space | gap in the said electrode layer in the ratio of 1/20 or more in an area.

  A transfer device according to the present invention is a transfer device that transfers current to a first medium including a sample by passing an electric current through an electrolyte and transferring the sample to a second medium disposed in contact with the first medium, A first voltage application unit for supplying the current is provided, and the first voltage application unit includes the electrode according to the present invention.

  According to the above configuration, as described above, the electrode according to the present invention can remove bubbles generated when an electric current is passed through the electrolytic solution. Therefore, in the transfer device, the electrode includes a first medium including a sample. Bubbles generated when an electric current is passed through the electrolytic solution can be suppressed, and potential disturbance caused by the bubbles can be suppressed.

  In the transfer device, the first medium may be a gel.

  According to said structure, a 1st medium is a gel. Since the gel is generally soft, it may be damaged by bubbles generated in the electrode. However, according to the above configuration, since the electrode according to the present invention is used, the bubbles generated in the electrode are removed and the gel is gel. Damage to the first medium can be suppressed.

  In the transfer device according to the present invention, it is preferable that the first voltage applying unit causes the current to flow along a first direction from the second medium toward the first medium or in the opposite direction.

  According to the above configuration, if the sample has a negative charge, the sample is suitably transferred from the first medium to the second medium by flowing the current in the first direction from the second medium toward the first medium. Can be electrophoresed. If the sample has a positive charge, the sample can be suitably electrophoresed from the first medium to the second medium by flowing the current in the direction opposite to the first direction. As described above, according to the above configuration, the sample can be suitably transferred from the first medium to the second medium.

  The transfer device according to the present invention further includes a second voltage applying means for causing a current to flow along a second direction orthogonal to the first direction, and the electrode layers are insulated from each other and along the second direction. It may consist of a plurality of electrode regions arranged side by side.

  According to the above configuration, since the transfer device includes the second voltage applying unit, the sample in the first medium is electrophoresed before the sample is transferred from the first medium to the second medium. Can be separated. At this time, when a flat plate electrode or the like is used as an electrode included in the first voltage application unit, the electrode may interfere with the separation. However, in the transfer device, the electrode layer of the electrode included in the first voltage application unit. However, the electrodes do not interfere with the separation because they are composed of a plurality of electrode regions that are insulated from each other and arranged in the second direction. Therefore, according to said structure, both electrophoresis and transcription | transfer can be implemented suitably.

  In the transfer device, the electrode region is preferably linear.

  According to said structure, since the said electrode area | region is linear, when it arranges along a 2nd direction, a plane is filled easily and the said sample in a 1st medium is efficiently transferred to a 2nd medium. can do.

  In the transfer device, it is preferable that the plurality of electrode regions are arranged in parallel to each other perpendicular to the second direction.

  Since the linear electrode regions arranged orthogonally to the second direction have little influence on the voltage applied in the second direction, the sample in the first medium is preferably separated. Can do.

  The transfer method according to the present invention is a transfer method in which an electric current is passed through a first medium containing a sample via an electrolytic solution to transfer the sample to a second medium disposed in contact with the first medium, At least one of the positive electrode and the negative electrode for applying the voltage is an electrode according to the present invention.

  According to the above configuration, as described above, the electrode according to the present invention can remove bubbles generated when an electric current is passed through the electrolytic solution. Therefore, in the above transfer method, the first medium including the sample is applied to the first medium. Bubbles generated when an electric current is passed through the electrolytic solution can be suppressed, and potential disturbance caused by the bubbles can be suppressed.

  The electrode according to the present invention includes an electrode layer that can permeate air bubbles and an insulating layer that is provided below the electrode layer and can absorb the air bubbles. Bubbles generated at the time can be removed.

(electrode)
The present invention provides an electrode for passing an electric current through an electrolytic solution. In particular, the electrode can be suitably used for an electrophoresis apparatus, a transfer (blotting) apparatus, or the like that utilizes an electrophoresis phenomenon caused by passing an electric current through a buffer solution. In addition, although the use aspect of the electrode for flowing an electric current through electrolyte solution may be in the state immersed in this electrolyte solution, it is not restricted to this, The state etc. which contacted the medium containing this electrolyte solution, etc. possible. That is, it is only necessary that current flows while the electrolyte is in contact with the electrode, and the way of supplying the electrolyte is not particularly limited.

  The electrolyte solution may be appropriately selected depending on the application, but it is preferable that, for example, a highly conductive reagent having a buffering effect such as Tris, CAPS, or carbonate is included. The electrolytic solution may also contain a pH adjuster, a modifier, a surfactant, alcohol, and the like. For example, when a polypeptide is transcribed, an alcohol is preferably included to promote the binding between the polypeptide and a transfer medium, and SDS is preferably included to denature the polypeptide. . In addition, when a polynucleotide is transferred, an alkali salt such as NaOH can be used as a denaturing agent. Specific examples include, but are not limited to, Tris / glycine buffer solution, acetate buffer solution, sodium carbonate buffer solution, CAPS buffer solution, Tris / boric acid / EDTA buffer solution, Tris / acetic acid solution. / EDTA buffer solution, MOPS, phosphate buffer solution, Tris / Tricine buffer solution and other buffer solutions can be used.

  Fig.2 (a) is a schematic diagram which shows the structure in one Embodiment of the electrode which concerns on this invention. As shown in FIG. 2A, the electrode 10 according to the present invention includes an electrode layer 11 and an insulating layer 12 provided below the electrode layer 11. In FIG. 2A, the length is written, but this is only an example, and does not limit the scope of the present invention.

  The electrode layer 11 only needs to be formed of a conductive material, and is preferably a material that does not deteriorate even when brought into contact with the electrolytic solution used. Specifically, the electrode layer 11 is not limited to these, but platinum, copper , Zinc or the like.

  Further, as shown in FIG. 2A, the electrode layer 11 is provided with a gap that penetrates the electrode layer 11. Thereby, bubbles generated in the electrode layer 11 can be transmitted to the insulating layer 12.

  The voids need only penetrate the electrode layer 10, but are preferably uniformly present in the entire electrode layer, but are not limited thereto, but are, for example, striped or meshed Can be provided.

  In one embodiment, the gap is provided in the electrode 10 in a stripe shape. FIG. 2B is a top view of the electrode 10. In the top view, the gap is provided at a position where the insulating layer 12 is exposed. As shown in FIG. 2B, the stripe shape is intended to be a shape in which a plurality of linear voids are arranged at arbitrary intervals. The said space | interval can be 50-200 micrometers, for example, and the line | wire width of the said line can be 50-200 micrometers, for example.

  In another embodiment, the electrode 20 is provided with the voids in a mesh shape. FIG. 2C is a top view of the electrode 20. In the top view, the gap is provided at a position where the insulating layer 22 is exposed. As shown in FIG. 2C, the net shape is intended to be a shape in which a plurality of the above-mentioned voids having a specific shape are arranged at arbitrary intervals. The said shape can be made into a square, a circle etc., for example, and the said space | interval can be 50-200 micrometers, for example.

  Further, a part may be a stripe shape, a part may be a mesh shape, or a part of another shape. Furthermore, by making the ratio of the voids in the electrode layer preferably 1/20 or more in area, more preferably 1/10 or more, and even more preferably 1/3 or more, air bubbles can be more efficiently transmitted to the insulating layer. Can be made.

  The insulating layer 12 (22) is made of a porous material. Thereby, the said bubble which permeate | transmitted the electrode layer 11 (21) can be absorbed. Examples of the porous substance include silicone resin, α-olefin copolymer, polytetrafluoroethylene resin, fluorine resin, polyurethane resin, polyvinyl alcohol resin, polyvinyl chloride resin, chlorinated polyethylene resin, polyethylene resin, and polypropylene. Resin, ethylene-vinyl acetate copolymer, polymethyl methacrylate resin, polystyrene resin, styrene-butadiene-acrylonitrile copolymer, polyvinyl formal resin, epoxy resin, phenol resin, urea resin, acrylic rubber, acrylonitrile-butadiene rubber, styrene -Butadiene rubber, butadiene rubber, isoprene rubber, chloroprene rubber, polystyrene elastomer, polyolefin elastomer, polyester elastomer, polyamide elastomer, etc. Synthetic resins, or vermiculite, mica, glass fiber, rock wool, carbon fibers, can be suitably used inorganic porous material such as ceramic fibers. Since these substances are porous and can easily take in a gas, they can be suitably used for forming the insulating layer.

  In one embodiment of the present invention, the electrode 10 may further include a substrate 13 for supporting the insulating layer 12 below the insulating layer 12, as shown in FIG. The material for forming the substrate 13 is not particularly limited, and glass, silicon, metal, or the like can be used.

  The electrode 10 can be produced using, for example, a well-known and commonly used thin film production technique. That is, using a substrate made of a porous material, the substrate is used as the insulating layer 12 as it is, or a layer made of a porous material is formed on the substrate 13 using a sputtering device, a spin coating device, or the like. By forming the film, the insulating layer 12 is formed. Then, the electrode layer 11 is similarly formed on the insulating layer 12 by using a well-known and conventional thin film forming technique. Finally, a gap that penetrates the electrode layer 11 is created using a dicing saw, a laser engraver, a photolithography technique, or the like. In addition, the electrode 20 according to another embodiment can be manufactured in the same manner as the electrode 10.

(Transfer device and method)
The present invention also provides a transfer apparatus and a transfer method in which an electric current is passed through a first medium containing a sample via an electrolytic solution to transfer the sample to a second medium disposed in contact with the first medium.

  FIG. 4 is a schematic diagram showing an outline of the transfer apparatus 100 according to an embodiment of the present invention. As shown in FIG. 4, the transfer apparatus 100 according to the present embodiment includes a negative electrode composed of substrates 101 and 102, an electrode layer 103 and an insulating layer 104, a positive electrode composed of an electrode layer 105 and an insulating layer 106 (the negative electrode and the negative electrode). The positive electrode constitutes a first voltage applying means), and a wire connecting device 109, and the electrode layers 103 and 105 hold the first medium 107 and the second medium 108.

  The first medium 107 is not particularly limited as long as it contains a sample. For example, the first medium 107 is a gel generally used for electrophoresis, such as an agarose gel or a polyacrylamide gel, and contains the sample. It can be used suitably. The sample is not particularly limited, but a preparation from a biological material (for example, an individual organism, a body fluid, a cell line, a tissue culture, or a tissue fragment) can be used, and more preferably a polypeptide or a polypeptide. Nucleotides can be used.

  The second medium 108 may be any material that can fix the material to be transferred, and the shape is not particularly limited, but a thin film is preferable. Specifically, as the second medium 108, a nitrocellulose membrane, a PVDF membrane, a nylon membrane, or the like used for biopolymer analysis techniques such as a well-known western blotting method can be suitably used. The second medium 108 is held so as to be in contact with the first medium 107.

  It is preferable that the first medium 107 and the second medium 108 are immersed in a buffer solution that is an electrolytic solution or contain the buffer solution. Thereby, a buffer solution can be supplied between the electrode layer 103 and the electrode layer 105. The method of supplying the buffer solution is not limited to the above, and for example, a filter paper containing the buffer solution may be further sandwiched.

  The wire connection device 109 is a member for applying a potential to the electrode layers 103 and 105, as long as it has a portion formed of a conductive material. For example, the wire connection device 109 has a mesh-like copper electrode. It can be used suitably. In one embodiment, the wire connection device 109 has a mesh-like copper electrode having a potential of − and +, and the copper electrode is inserted between the electrode layers 103 and 105 so that each of the copper electrodes has an electrode layer. A potential is applied to the electrode layers 103 and 105 by being in contact with 103 and 105, respectively. As the potential difference to be applied, for example, 1 to 500 V can be used.

  Since the buffer solution is supplied between the electrode layer 103 and the electrode layer 105 as described above, a current is caused to flow between the electrode layers via the electrolytic solution by applying a voltage between the electrode layers. Can do. Since the current flows in the first medium 107 and the second medium 108 in the direction from the second medium 108 toward the first medium 107 (first direction), the sample in the first medium having a negative charge is electrophoresed. Then, it can be transferred to the second medium. At this time, the electrode layer 103 and the insulating layer 104, and the electrode layer 105 and the insulating layer 106 constitute an electrode according to the present invention. Therefore, bubbles generated in the electrode layers 103 and 105 can be removed. Thereby, in the transfer device 100 according to the present invention, it is possible to suppress the disturbance of the potential distribution caused by the bubbles and the diffusion and disturbance of the sample after the transfer. In particular, when a gel is used as the first medium, it is possible to prevent cracks caused by the bubbles passing through the gel. When the sample has a positive charge, a current may be passed in the direction opposite to the first direction.

  Moreover, a wire connection instrument can also be made into another form. FIG. 5 is a schematic diagram showing an outline of a transfer apparatus 200 according to another embodiment of the present invention. Descriptions of the transfer device 200 other than the substrates 201 and 202 and the connection device 209 are the same as those of the transfer device 100.

  As shown in FIG. 5, the wire connection device 209 is a conductor that connects the electrode layer 203 and the substrate 201 having a negative potential, and the electrode layer 205 and the substrate 202 having a positive potential. Thus, the potential is applied to the electrode layers 203 and 205 by the wire connection device 209.

  As described above, in the transfer device according to the present invention, the structure of the wire connection device is not particularly limited as long as a voltage can be applied between the electrodes.

  Further, in the transfer apparatus according to the present invention, both the positive electrode and the negative electrode need not be the electrodes according to the present invention. That is, if any one of the positive electrode and the negative electrode is an electrode according to the present invention, bubbles in the one electrode can be removed, so that the potential distribution caused by the bubbles is disturbed, and the sample after transfer Diffusion and turbulence can be suppressed. In particular, if the electrode on the side in contact with the first medium is an electrode according to the present invention, it is more preferable because the bubbles can be prevented from cracking through the first medium.

(Electrophoresis and transfer device)
In one embodiment, the transfer device according to the present invention can also be used as an electrophoresis and transfer device.

  FIG. 6 is a diagram showing a schematic configuration of the transfer apparatus 300 according to the present embodiment. As shown in FIG. 6, the transfer apparatus 300 includes substrates 301 and 302, a negative electrode composed of an electrode layer 303 and an insulating layer 304, and a positive electrode composed of an electrode layer 305 and an insulating layer 306. The negative electrode and the positive electrode constitute first voltage applying means, and are divided into regions of a separation unit 310 and a connection unit 311. In the separation unit 310, the electrode layers 303 and 305 hold the first medium 307 and the second medium 308. Moreover, in the connection part 311, the connection device 309 switches connection and disconnection to the power sources of the electrode layers 303 and 305.

  The transfer device 300 also includes a buffer solution tank 312 provided with an electrode 314 and a buffer solution tank 313 provided with an electrode 315. The electrodes 314 and 315 constitute second voltage applying means. As shown in FIG. 6, the direction in which the voltage of the second voltage applying unit is applied (second direction) is the direction in which the voltage of the first voltage applying unit is applied (first direction, first from the second medium 308). And the direction toward the medium 307).

  FIG. 7 is a schematic diagram for explaining the operation of the transfer apparatus 300. As described above, since the transfer apparatus 300 includes the first voltage application unit and the second voltage application unit in which the direction in which the voltage is applied is orthogonal, the connection to each power source is controlled, and the second After the sample in the first medium 307 is separated in the second direction using only the voltage application means (FIG. 7A), the separated sample in the first medium 307 is obtained using only the first voltage application means. The image can be transferred to the second medium 308 (FIG. 7B).

  Here, the electrode layers 303 and 305 are composed of a plurality of electrode regions that are insulated from each other and arranged in the second direction. Therefore, even when the second voltage applying means applies a voltage between the electrodes 314 and 315, the plurality of electrode regions are insulated from each other, and therefore can have different potentials. Since they are arranged along the direction, a potential gradient can be formed in the second direction. Thus, the electrode layers 303 and 305 do not disturb the electrophoresis in the second direction. For example, when the electrode layers 303 and 305 are flat electrodes or the like, the inside of the electrode layers is equipotential, so a potential gradient is not formed and the electrophoresis in the second direction is hindered.

  In addition, it is preferable that the shape of the said several electrode area | region is linear so that it may arrange suitably so that a plane may be filled along a 2nd direction. Further, it is preferable that the plurality of electrode regions are arranged in parallel to each other perpendicular to the second direction. Such an electrode region has little influence on the voltage applied in the second direction. As the electrode layers 305 and 306 satisfying the above conditions, for example, a layer provided with a stripe-shaped gap as shown in FIG. 2B can be suitably used.

  Note that the shapes of both the electrode layers 303 and 305 are not necessarily those described above. In one aspect, at least the electrode layer 303 includes a plurality of electrode regions that are insulated from each other and arranged in the second direction, and the substrate 302, the electrode layer 305, and the insulating layer 306 are formed to be removable. do it. FIG. 8 is a schematic diagram illustrating a modified example of the operation of the transfer device 300. As shown in FIG. 8A, when the separation in the second direction is performed, the substrate 302, the electrode layer 305, and the insulating layer 306 are removed, and as shown in FIG. When transferring to the electrode, separation and transfer can be suitably performed by combining the electrode layer 305 and the like, even if the electrode layer 305 is a flat plate electrode.

  Further, since at least one of the electrodes of the first voltage applying means is an electrode according to the present invention, the generation of bubbles is suppressed. That is, by using the present invention, it is possible to provide an electrophoresis and transfer apparatus in which bubble generation is suppressed.

  The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope shown in the claims, and embodiments obtained by appropriately combining technical means disclosed in different embodiments. Is also included in the technical scope of the present invention.

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

[Example 1: Production of electrode substrate]
(I) Preparation of platinum-deposited glass substrate After depositing 500 ５ Cr on a 6 cm x 5 cm glass plate using a sputtering device (CFS-4EP-LL, Shibaura Mechatronics), a further 3000 Ｐ Pt was formed. did. In addition, each film thickness was measured using the stylus type surface shape measuring device (Dektak6M, ULVAC-ES).

(Ii) Production of Striped Electrode Glass Substrate As in (i), about 0.3 μm of Pt was formed on the glass substrate. Subsequently, the Pt thin film was cut using a dicing saw (DAD522, disco) so that both the Pt electrode width and the interelectrode width were 100 μm.

(Iii) Stripe electrode silicone resin substrate A stripe electrode silicone resin substrate (electrode 10) having the structure shown in FIG. 2 (a) was prepared as follows. 10 g of SILPOT 184 (Dow Corning Asia Co., Ltd.) and 0.1 g of CATALYST SILPOT 184 (Dow Corning Asia Co., Ltd.) were mixed with a spoon after weighing. Next, 0.8 g of the obtained mixture was dropped onto the center of a 6 cm × 5 cm glass plate (substrate 13). Then, using a spin coater, at 500 rpm for 10 seconds (if shorter than this, the silicone resin may be scattered), at 1000 rpm for 30 seconds (if shorter than this, the silicone resin may not spread uniformly), at 150 rpm Application for 20 seconds was performed. Finally, polymerization was carried out for 1 hour in an incubator at 80 ° C. to form a silicone resin thin film (insulating layer 12) having a thickness of about 100 μm.

  Next, as in (i), a Pt film (electrode layer 11) having a thickness of about 3000 mm was formed on the silicone resin thin film using a sputtering apparatus. Subsequently, as shown in FIG. 2B, voids were formed in a stripe shape. Specifically, the Pt film and the silicone resin thin film below the Pt film and the lower part thereof are 70 μm deep as shown in FIG. 2A using a laser engraving machine so that both the Pt electrode width and the interelectrode width are 100 μm. (The space | gap which penetrated the electrode layer 11 was created).

  FIG. 1A shows a photograph of the obtained stripe electrode silicone resin substrate, and FIG. 1B shows an enlarged photograph of the substrate.

[Example 2: Preparation of gel (first medium)]
A 6 cm × 5 cm gel plate (glass plate) was combined with a 1 mm spacer in between and placed in a gel preparation container. A separation gel solution (13% acrylamide mix (acrylamide: bisacrylamide = 29.2: 0.8), 375 mM Tris-HCl (pH 8.8), 0.05% APS, 0.1% TEMED) was placed 7 mm from the tip. After adding to the position, water was overlaid. After the separated gel solution is polymerized, water is removed, and the concentrated gel solution (4% acrylamide mix (acrylamide: bisacrylamide = 29.2: 0.8), 125 mM Tris-HCl (pH 8.8), 0.05% APS, 0.2% TEMED) was added and a sample comb was set.

  As a sample, SeeBlue plus2 M. W. Marker (Invitrogen) and fluorescently labeled DyLight fluorescent protein molecular weight markers (PIERCE) were used. Any of the above samples was mixed with an equal amount of 0.5% agarose gel in a dissolved state, dropped into a sample well, and subjected to SDS-PAGE after coagulation. The cathode electrophoresis buffer composition was 25 mM Tris, 192 mM glycine and 0.1% SDS, and the anode electrophoresis buffer composition was 150 mM Tris-HCl (pH 8.8).

  As described above, a gel (first medium) containing a protein (sample) band was prepared.

[Example 3: Transfer using stripe electrode only on one side]
First, the filter paper and the PVDF membrane were cut so as to have the same size as the gel. Next, the PVDF membrane was activated with methanol and then immersed in a buffer solution (transfer buffer, 190 mM Tris, 5% methanol). Similarly, the filter paper was immersed in the buffer solution. The gel was also replaced with the buffer solution.

(I) Transfer Using Striped Electrode Silicone Resin Substrate On the striped electrode silicone resin substrate (cathode) produced in Example 1, the gel (first medium), the PVDF film (second medium), and two sheets After the filter papers were stacked in this order, a flat plate-like anode was installed.

The upper limit of the current value is 0.8 mA / cm ² , a voltage of 2V is applied for 10 minutes, a voltage of 3V is applied for 10 minutes, and a voltage of 5V is further applied for 40 minutes, and the gel is applied to the PVDF film (second medium). The protein band was transcribed. A result is shown to Fig.9 (a). In addition, the detection of DyLight fluorescent protein molecular weight markers was performed using Exy using Tyhoon Trio (GE Healthcare). 615 nm, Em. Performed at 680 nm.

(Ii) Transfer using stripe electrode glass substrate Transfer was performed in the same manner as in (i) except that the stripe electrode glass substrate prepared in Example 1 was used instead of the stripe electrode silicone resin substrate. The result is shown in FIG.

  As shown in FIG. 9B, when a stripe electrode glass substrate is used, a large amount of bubbles are generated between the stripe electrode and the gel immediately after transfer, and the bubbles pass through the gel. A fine laceration of the gel that appears to have occurred was observed on the entire surface. On the other hand, as shown in FIG. 9 (a), when a striped electrode silicone resin substrate is used, no gas is seen between the striped electrode and the gel, and the gas is formed through the gel. There were no scratches.

  Furthermore, as can be seen by comparing the PVDF film shown in FIG. 9 (a) and the PVDF film shown in FIG. 9 (b), when the stripe electrode silicone resin substrate is used, compared to when the stripe electrode glass substrate is used. The transfer result with higher resolution was obtained.

[Example 4: Transfer using stripe electrodes on both sides]
We studied transfer using stripe electrodes on both sides.

  (I) First, as a negative control, a case where a gel (first medium) and a PVDF film (second medium) were sandwiched between flat plate electrodes (Pt) was examined. The transfer result is shown in FIG. The gel and PVDF membrane were prepared in the same manner as in Example 3.

  (Ii) The case where the gel (first medium) and the PVDF film (second medium) were sandwiched between the stripe electrode silicone resin substrates prepared in Example 1 was examined. For the energization of the both stripe electrode silicone resin substrates, a wire connection device (wire connection device A) inserted between both the stripe electrodes as shown in FIG. 4 was used. The transfer result is shown in FIG.

  (Iii) Same as (ii), except that the stripe electrode silicone resin substrate was energized using a wire connection device (wire connection device B) having a power source connected to both stripe electrodes as shown in FIG. went. The transfer result is shown in FIG.

  As shown in FIG. 10 (a), when flat plate electrodes were used on both sides, the transferred protein band was extremely disturbed. On the other hand, as shown in FIGS. 10B and 10C, when the stripe electrode silicone resin substrate was used on both surfaces, protein transfer with less disturbance was possible.

  In addition, when the connection device A was used, good transfer was possible for all samples. It was suggested that this was because the connection was easier than the connection device B, and all regions of the electrode could be connected.

  On the other hand, when the connection device B was used, the value of the flowing current was about 10 times that when the connection device A was used, and the amount of protein remaining in the gel after transfer was small (FIG. 10 (c)). ). It was suggested that this is because the connection device B has few connection failures and the transfer efficiency is hardly decreased.

  The present invention can be used, for example, in the field of manufacturing instruments for life science research.

FIG. 1A is a photograph of an electrode according to an embodiment of the present invention, and FIG. 1B is an enlarged photograph of an electrode according to an embodiment of the present invention. FIG. 2A is a cross-sectional view illustrating an outline of an electrode according to an embodiment of the present invention, and FIG. 2B is a top view illustrating an outline of an electrode according to an embodiment of the present invention. FIG. 2C is a top view schematically showing an electrode according to another embodiment of the present invention. FIG. 3 is a photograph showing a transfer result of a transfer device according to the prior art. FIG. 4 is a schematic diagram showing an outline of a transfer apparatus according to an embodiment of the present invention. FIG. 5 is a schematic diagram showing an outline of a transfer apparatus according to an embodiment of the present invention. FIG. 6 is a diagram showing a schematic configuration of a transfer apparatus (electrophoresis / transfer apparatus) according to an embodiment of the present invention. FIG. 7A is a schematic diagram for explaining the operation during separation of the transfer device (electrophoresis and transfer device) according to one embodiment of the present invention, and FIG. 7B is one embodiment of the present invention. It is a schematic diagram explaining the operation | movement at the time of transcription | transfer of the transfer apparatus (electrophoresis and transfer apparatus) which concerns on. FIG. 8A is a schematic diagram for explaining a modification example of the operation at the time of separation of the transfer device (electrophoresis and transfer device) according to one embodiment of the present invention, and FIG. It is a schematic diagram explaining the modification of the operation | movement at the time of transcription | transfer of the transfer apparatus (electrophoresis and transfer apparatus) which concerns on one Embodiment. FIG. 9A is a photograph showing the transfer result of the transfer apparatus according to the embodiment of the present invention, and FIG. 9B is a photograph showing the transfer result of the transfer apparatus as a comparative example. 10A is a photograph showing a transfer result of a transfer apparatus as a comparative example, and FIG. 10B is a photograph showing a transfer result of the transfer apparatus according to an embodiment of the present invention. (C) is a photograph showing a transfer result of a transfer device according to another embodiment of the present invention.

Explanation of symbols

10, 20 Electrode 11, 21 Electrode layer 12, 22 Insulating layer 100, 200, 300 Transfer device 101, 102, 201, 202, 301, 302 Substrate 103, 105, 203, 205, 303, 305 Electrode layer 104, 106, 204, 206, 304, 306 Insulating layer 107, 207, 307 First medium 108, 208, 308 Second medium 109, 209, 309 Connecting device 310 Separating section 311 Connecting section 312, 313 Buffer tank 314, 315 Electrode

Claims (9)

A transfer device for transferring an electric current to a first medium including a sample via an electrolytic solution and transferring the sample to a second medium disposed in contact with the first medium,
An electrode for passing an electric current through the electrolytic solution;
The electrode includes an electrode layer and an insulating layer provided below the electrode layer,
The electrode layer is provided with a gap penetrating the electrode layer,
The transfer device, wherein the insulating layer is made of a porous material. The porous material is silicone resin, α-olefin copolymer, polytetrafluoroethylene resin, fluorine resin, polyurethane resin, polyvinyl alcohol resin, polyvinyl chloride resin, chlorinated polyethylene resin, polyethylene resin, polypropylene resin, ethylene - acetic acid vinyl copolymer, polymethyl methacrylate resin, polystyrene resin, styrene - butadiene - acrylonitrile copolymer, polyvinyl formal resins, epoxy resins, phenol resins, urea resins, acrylic rubber, acrylonitrile - butadiene rubbers, styrene - butadiene Rubber, Butadiene rubber, Isoprene rubber, Chloroprene rubber, Polystyrene elastomer, Polyolefin elastomer, Polyester elastomer, Polyamide elastomer, Bamiki Light, mica, glass fiber, rock wool, carbon fibers, and transfer device according to claim 1, characterized in that one or more substances selected from the group consisting of ceramic fibers.   The transfer device according to claim 1, wherein the gap is provided in the electrode layer in a stripe shape or a mesh shape.   The transfer device according to claim 1, wherein a ratio of the voids in the electrode layer is 1/20 or more in area. First voltage applying means for flowing the current,
The transfer apparatus according to claim 1, wherein the first voltage applying unit includes the electrode.   The transfer device according to claim 5, wherein the first medium is a gel.   7. The transfer device according to claim 5, wherein the first voltage applying unit causes the current to flow along a first direction from the second medium toward the first medium or in the opposite direction. 8. A second voltage applying means for passing a current along a second direction orthogonal to the first direction;
The transfer device according to claim 7, wherein the electrode layer includes a plurality of electrode regions that are insulated from each other and arranged in the second direction. A transfer method in which an electric current is passed through a first medium containing a sample through an electrolytic solution to transfer the sample to a second medium disposed in contact with the first medium,
At least one of the positive electrode and the negative electrode for flowing the current includes an electrode layer and an insulating layer provided below the electrode layer,
The electrode layer is provided with a gap penetrating the electrode layer,
The transfer method, wherein the insulating layer is made of a porous material.