Patent Application
20170122902
The present invention relates to a separation medium cassette for sample separation adsorption, and an analysis device for sample separation adsorption including the separation medium cassette for sample separation adsorption.
In recent years, direct blotting has been developed, which discharges samples separated in a separation medium by way of electrophoresis from an end face of the separation medium and transfers directly to a transfer membrane opposing the end face of the separation medium.
Patent Document 1 discloses a cassette that electrophoretically separates molecular fragments in solution. The cassette described in Patent Document 1 includes (a) a gelatinous substrate containing molecular fragments for separation at a first end thereof and configured sufficient to concentrate similar separated molecular fragments (sample) at a second end thereof; and (b) a containment means which supports the gelatinous substrate. The components separated in the gel cassette transfer to a moving membrane at a discharge port (slot 40 in Patent Document 1).
However, with the gel cassette according to Patent Document 1, since the moving membrane contacts the gel cassette, the cassette may warp. Since warping of the gel cassette greatly influences the test results, it is not possible to obtain accurate analysis results if the cassette warps. In other words, there are problems in that the gap between gel plates is completely eliminated and transfer is inhibited, and the width of the gap between both ends of the gel plate and the central part varies and the transfer rate is no longer constant in the width direction.
The present invention has been made taking account of the above-mentioned issues, and has a main object of providing technology for performing more accurate analysis in a sample separation adsorption method.
In order to solve the above-mentioned problem, a separation medium cassette for sample separation adsorption according to a first aspect of the present invention includes: a separation medium that separates sample by way of electrophoresis and discharges from an end face thereof; a first insulating portion and a second insulating portion that form a pair sandwiching the separation medium; and a warping prevention portion that is inserted between the first insulating portion and the second insulating portion, and suppresses the first insulating portion and the second insulating portion from warping so as to approach each other.
According to the present invention, it is possible to perform more accurate analysis in a sample separation adsorption method.
A separation medium cassette for sample separation adsorption according to the present invention includes: a separation medium that separates sample by way of electrophoresis and discharges from an end face thereof; a first insulating portion and a second insulating portion that form a pair sandwiching the separation medium; and a warping prevention portion that is inserted between the first insulating portion and the second insulating portion, and suppresses the first insulating portion and the second insulating portion from warping so as to approach each other.
In the present disclosure, sample separation adsorption method indicates technology for transferring a separated sample to a transfer membrane by arranging the transfer membrane so as to oppose an end face of a separation medium separating a sample by way of electrophoresis, positioned downstream in the running direction of electrophoresis, and then causing the sample separated by way of electrophoresis to discharge from this end face as is. In order to continuously perform transfer to the transfer membrane, the transfer membrane is relatively moved in relation to the end face. According to the sample separation adsorption method, it is possible to perform the separation of sample and transfer to the transfer membrane in few steps.
In the present disclosure, separation medium cassette for sample separation adsorption indicates a structure including a separation medium and an insulating portion that retains the separation medium, and is inserted in an analysis device for conducting the sample separation adsorption method.
In the present disclosure, sample is a substance to be analyzed by electrophoresis and transfer. As the sample, a preparation from biological material (e.g., biont, body fluid, cell strain, tissue culture, or tissue fragment) can be suitably used, and in particular, protein samples, DNA samples and RNA samples can be suitable used. These samples may be stained (immunostaining or fluorescent labeling) by a fluorescent sample or the like in advance. In addition, the sample is preferably subjected to pretreatment such as purification and SDS processing.
In the present disclosure, separation medium indicates a medium that can separate the sample by way of electrophoresis, and is not limited to these; however, for example, it is possible to exemplify gels such as polyacrylamide gel and agarose gel. In addition, it may be of a structure in which ultrafine posts (nano-pillars) are provided standing.
In the present disclosure, insulating portion is an insulating body, and retains the separation medium by sandwiching. As the insulating body, although glass having a hydrophilic surface can be suitably used, for example, it is not limited thereto, and it is possible to use an insulating material such as acrylic resin, polycarbonate resin, polystyrene resin, PET resin, vinyl chloride resin, and ceramics.
In the present disclosure, warping prevention portion indicates a structure inserted between a first insulating portion and second insulating portion, and suppressing the first insulating portion and second insulating portion from warping so as to approach each other.
As the substance constituting the warping prevention portion, for example, it is possible to use plastic resins such as polymethylmethacrylate (acrylic), polystyrene, polyethylene, polypropylene, polyethylene terephthalate (PET) and polyether ether ketone, glass or silicon; however, it is not limited to these, and it is possible to use a substance that is an insulating body. It should be noted that, in the case of forming the separation medium in a state in which the warping prevention portion is inserted between the first insulating portion and second insulating portion, it is preferable for the warping prevention portion not to inhibit formation of the separation medium (e.g., polymerization of gel), and to be a substance of high gas barrier property.
In addition, although the form of the warping prevention portion is not particularly limited, for example, it can be established in forms such as a column shapes like a cylinder, elliptical column, triangular column, square column, and polygonal columns of five or more sides, and wall-like.
Furthermore, the warping prevention portion may be integrated by adhesion or the like to at least one of the first insulating portion and second insulating portion, or may be inserted between the first insulating portion and second insulating portion when forming the separation medium.
Moreover, the analysis device for sample separation adsorption according to the present invention includes the above-mentioned separation medium cassette for sample separation adsorption, and a moving portion that causes the transfer membrane to move so as to pass through a position opposing an end face in the direction of electrophoresis of the separation medium.
In the present disclosure, transfer membrane is a membrane that catches (blots or transfers) the separated sample while retaining the separation pattern, and is also simply called a film, transfer membrane, membrane or filter, due to a membrane of thin sheet form being used generally. As the transfer membrane, for example, although nitrocellulose membranes, nylon membranes, polyvinylidene fluoride (PVDF) membranes, etc. can be exemplified, it is not limited thereto, and so long as being a membrane that can catch the separation pattern of a sample from the separation medium by way of a capillary method, electroblotting method, etc., it can be suitably employed.
In the present disclosure, analysis device for sample separation adsorption is a device for analyzing sample by way of a sample separation adsorption method, and indicates a device that performs from a sample being introduced until the transfer thereof to the transfer membrane with one device.
Hereinafter, an embodiment of the present invention will be explained in detail.
The gel cassette 22 has a structure in which a first insulating portion 13a and second insulating portion 13b form a pair to sandwich a gel (separation medium) 19.
Inside of the analysis device 1, the gel cassette 22 is arranged in a vertical direction, and the buffer solution tank 14a is arranged on an upstream side (vertically upper side) in the running direction D of electrophoresis of the gel cassette 22. The gel cassette 22 and buffer solution tank 14a are arranged so that an end face 19a of the gel 19 of the gel cassette 22 contacts an opening part of the buffer solution tank 14a provided at a bottom part. The cathode 10 is arranged inside of the buffer solution tank 14a, and buffer solution is filled so as to immerse the cathode 10 and the end face 19a of the gel 19 in the buffer solution tank 14a.
The buffer solution tank 14b is arranged so as to surround the gel cassette 22 on a downstream side (vertically lower side) in the running direction D of electrophoresis of the gel cassette 22, and the end face 19b of the gel 19 is exposed within the buffer solution tank 14b. The anode 11 is arranged inside of the buffer solution tank 14b, and buffer solution is filled so as to immerse the anode 11 and the end face 19b of the gel in the buffer solution tank 14b.
As the buffer solution filled in the buffer solution tank 14a and the buffer solution tank 14b, it is possible to use any buffer solution having electrical conductivity; however, as the buffer solutions usable in electrophoresis, for example, it is possible to use buffer solutions such as a Tris/glycine-based buffer solution, acetic acid buffer solution, sodium carbonate-based buffer solution, CAPS buffer solution, Tris/boric acid/EDTA buffer solution, Tris/acetic acid/EDTA buffer solution, MOPS, phosphoric acid buffer solution, and Tris/tricine-based buffer solution.
The cathode 10 and anode 11 are formed from materials having electrical conductivity such as metals. As the material forming the cathode 10 and anode 11, platinum is preferable from the view point of suppressing ionization of the electrodes, for example.
The transfer membrane 12 has a sheet form, and comes to move through a position opposing the end face 19b positioned on a downstream side in the running direction D of electrophoresis of the gel 19. The connecting part 23 consisting of resin or the like is provided at an end in the running direction F of the transfer membrane 12, and is connected to the arm 15, which is driven by the motor 16. Therefore, by the motor 16 driving the arm 15, it is possible to cause the transfer membrane 12 to relatively move in relation to the end face 19b of the gel 19. For example, in the cross-sectional view shown in
The motor 16 drives the arm 15 that is connected. The motor 16 is sufficient so long as being a motor that can drive the arm 15, so as to relatively move the transfer membrane 12 in relation to the end face of the gel cassette 22. As an example of a motor used as the motor 16, it is possible to exemplify a stepping motor having a 0.36° step angle, and 500 kHz maximum frequency.
The power unit 17 supplies electricity required in order for the motor 16, etc. to operate, as well as providing voltage to apply between
It should be noted that the voltage applied between the cathode 10 and anode 11 may be a constant voltage, may be a constant current, or may switch between these.
Operation of the analysis device 1 will be explained hereinafter.
The sample is introduced to the gel cassette 22, and electrophoresis starts by flowing current between the cathode 10 and anode 11. The sample introduced to the gel cassette 22 is separated by way of electrophoresis. In other words, each component contained in the sample undergoes phoresis at different speeds in the separation medium 19 according to the characteristics (e.g., molecular weight) of each component, and is separated into every component. Then, components having a fast phoretic velocity are discharged early from the end face 19b, and components having a slow phoretic velocity are discharged later from the end face 19b. The transfer membrane 12 is moved so as to pass through a position opposing the end face 19b by the motor 16 driving the arm 15. Each component of the sample discharged from the end face 19b is transferred consecutively at different sites on the transfer membrane 12 according to the order of discharging thereof. In the above way, it is possible to successfully transfer the separated sample to the transfer membrane. It should be noted that the transfer membrane to which the separated sample is transferred can be offered for further analysis such as immunostaining, for example.
At this time, a force causing to warp so as to approach the second insulating portion 13b may act on the first insulating portion 13a by the transfer membrane 12 moving. In particular, the first insulating portion 13a and second insulating portion 13b tend to easily warp even with slight force, since (i) it is necessary to form to be thin to some extent for heat dissipation, and (ii) in order to thin the bands of sample transferred to the transfer membrane 12, they may be formed in a tapering (tapered) shape towards the running direction D of electrophoresis. Herein, the gel cassette 22 according to the present embodiment includes a warping prevention part 20 that is inserted between the first insulating portion 13a and second insulating portion 13b, and suppresses warping such that the first insulating portion 13a and second insulating portion 13b approach each other, whereby it is possible to suppress the first insulating portion 13a and second insulating portion 13b from warping, and thus perform analysis more accurately. Hereinafter, the gel cassette 22 according to the present embodiment will be explained in detail.
As shown in
According to this configuration, even in a case of a force such that brings together is acting on the first insulating portion 13a and second insulating portion 13b, the warping prevention portion 20 inserted between the first insulating portion 13a and second insulating portion 13b works like a prop to suppress the first insulating portion 13a and second insulating portion 13b from warping. It is thereby possible to perform more accurate analysis. In addition, in order to be able to suppress the warping of the first insulating portion 13a and second insulating portion 13b by way of the warping prevention portion 20, it is possible to suitably use a material such as acrylic resin which has low cost and low rigidity compared to glass, etc., as the first insulating portion 13a and second insulating portion 13b.
It should be noted that the warping prevention portion 20 may be a shape connecting with both the first insulating portion 13a and second insulating portion 13b, in a state in which the first insulating portion 13a and second insulating portion 13b are not warping; however, it is may be a shape in which one among the first insulating portion 13a and second insulating portion 13b is not connected. In a state in which the first insulating portion 13a and second insulating portion 13b are not warping, in the case of the warping prevention portion 20 being a form that does not connect with one among the first insulating portion 13a and second insulating portion 13b, the first insulating portion 13a and second insulating portion 13b can warp so as to approach each other initially; however, by the first insulating portion 13a and second insulating portion 13b warping so as to approach each other, when the warping prevention portion 20 connects (abuts) with both of the first insulating portion 13a and second insulating portion 13b, the warping prevention portion 20 acts like a prop as mentioned above, and can suppress the first insulating portion 13a and second insulating portion 13b from warping any more than this.
In addition, as shown in
The sample is introduced to the gel 19 from the sample introduction port 18. The method of introducing sample is not particularly limited; however, it can be introduced by determining the quantity with a Pipetman, for example. In addition, in the case of performing electrophoresis in a second dimension in two-dimensional electrophoresis, the sample may be introduced by plugging an IPG gel after performing isoelectric focusing into the sample introduction port 18.
The sample introduced to the sample introduction port 18 is separated by way of electrophoresis, and is discharged from the end face 19b of the gel 19 positioned downstream in the running direction D. In the present disclosure, the path on which a sample separated by electrophoresis flows is called a lane.
As shown in
In addition, as shown in
In addition, as shown in in
It should be noted that the gel cassette 22 may assume a form for storage over a long time in a state not inserted into the analysis device 1.
For example, it may include a sample introduction port lid (not illustrated) that covers the sample introduction port 18. As the material of the sample introduction port lid, for example, although an acrylic resin for which processing is easy can be suitably used, it is not limited thereto, and it is possible to use insulating materials such as acrylic resin, polycarbonate resin, polystyrene resin, PET resin, vinyl chloride, and ceramic.
In addition, a package for wrapping the entirety of the gel cassette 22 may be included. Inside this package, a reinforcing material for supplementing the strength of the gel cassette 22, buffer solution for preventing drying of the gel 19, etc. may be placed.
The analysis device 1 may further include a guide mechanism (roller, etc.) for defining the movement path of the transfer membrane 12.
In addition, in the analysis device 1, although the gel cassette 22 is arranged in the vertical direction, the present embodiment is not limited thereto. The gel cassette 22 may be arranged in the horizontal direction, for example.
Another embodiment (second embodiment) of the present invention is as follows when explaining based on
As shown in
According to this configuration, in the lanes on both sides of the warping prevention parts 20a to 20g having the shape thicker at one end along the electrophoresis direction D (e.g., lanes 21a to 21f), the width of the lane narrows as advancing in the electrophoresis direction D (end face 19b side). For example, in the lanes 21a and 21b, which are lanes on both sides of the warping prevention part 20b, the width of the lane narrows as advancing to the side of the end face 19b. Therefore, the sample is led into a narrower space as advancing in the lane, to be concentrated. The distance between this concentrated sample and samples flowing in adjacent lanes (e.g., lanes 21a and 21c for lane 21b) is thereby ensured. In the above way, it is possible to concentrate the sample and prevent the mixing of samples between lanes, and thus perform more detailed analysis. In other words, as the phoresis distance of sample lengthens, the sample scatters and the band of the sample transferred to the transfer membrane 12 widens in the width direction W, and in the case of the bands of samples flowing in adjacent lanes overlapping, the quantitative analysis of results may become difficult; however, according to the present embodiment, it is possible to successfully segregate between bands by narrowing the width of bands of samples.
In addition, with the gel cassette 22 according to the present embodiment, similarly to the gel cassette 22 of the first embodiment, the warping prevention portion 20 is inserted between the ends in the electrophoresis direction D of the first insulating portion 13a and second insulating portion 13b. According to this configuration, since it is possible to suitably suppress deformation of the end face 19b of the gel 19, it is possible to further reduce the negative influence on discharging of sample from the end face 19b, and thus perform more accurate analysis.
In addition, with the gel cassette 22 according to the present embodiment, similarly to the gel cassette 22 of the first embodiment, at least a part of the warping prevention parts 20a to 20g (warping prevention parts 20c, 20d and 20e) is inserted at a central part in the width direction W that is orthogonal to the electrophoresis direction D. According to this configuration, it is possible to suitably suppress the first insulating portion 13a and second insulating portion 13b from warping by reinforcing the central parts of the first insulating portion 13a and second insulating portion 13b, which easily warp. It is thereby possible to perform more accurate analysis.
Another embodiment (third embodiment) of the present invention is as follows when explaining based on
As shown in
According to this configuration, the sample is separated and discharged without crossing between lanes. It is thereby possible to perform more detailed analysis by preventing the mixing of samples between lanes. In other words, it is possible to suppress bands of samples flowing between adjacent lanes from overlapping, and quantitative analysis from being hindered, as mentioned above.
In addition, with the gel cassette 22 according to the present embodiment, at least a part of the warping prevention portion 20 is inserted between the ends in the electrophoresis direction D of the first insulating portion 13a and second insulating portion 13b. According to this configuration, since it is possible to suitably suppress deformation of the end face 19b of the gel 19, it is possible to further reduce the negative influence on discharging of sample from the end face 19b, and thus perform more accurate analysis.
In addition, with the gel cassette 22 according to the present embodiment, similarly to the gel cassette 22 of the first and second embodiments, at least a part of the warping prevention parts 20a to 20g (warping prevention parts 20c and 20d) is inserted at the central part in the width direction W that is orthogonal to the electrophoresis direction D. According to this configuration, it is possible to suitably suppress the first insulating portion 13a and second insulating portion 13b from warping by reinforcing the central parts of the first insulating portion 13a and second insulating portion 13b, which easily warp. It is thereby possible to perform more accurate analysis.
Another embodiment (fourth embodiment) of the present invention is as follows when explaining based on
As shown in
According to this configuration, by the warping prevention portion 20 being inserted at the central part in the width direction D that is orthogonal to the electrophoresis direction D, it is possible to suitably suppress the first insulating portion 13a and second insulating portion 13b from warping by reinforcing the central parts of the first insulating portion 13a and second insulating portion 13b, which easily warp. It is thereby possible to perform more accurate analysis.
In addition, with the gel cassette 22 according to the present embodiment, similarly to the gel cassette 22 of the third embodiment, at least a part of the warping prevention portion 20 is inserted between the ends in the electrophoresis direction D of the first insulating portion 13a and second insulating portion 13b. According to this configuration, since it is possible to suitably suppress deformation of the end face 19b of the gel 19, it is possible to further reduce the negative influence on discharging of sample from the end face 19b, and thus perform more accurate analysis.
A separation medium cassette for sample separation adsorption according to a first aspect of the present invention includes: a separation medium that separates sample by way of electrophoresis and discharges from an end face thereof; a first insulating portion and a second insulating portion that form a pair sandwiching the separation medium; and a warping prevention portion that is inserted between the first insulating portion and the second insulating portion, and suppresses the first insulating portion and the second insulating portion from warping so as to approach each other.
According to the above-mentioned configuration, a separation medium cassette for sample separation adsorption is provided. With the sample separation adsorption method, in order to continuously transfer the sample discharged from the end face of the separation medium to the transfer membrane, the transfer membrane is moved so as to pass through a position opposing the end face of the separation medium. At this time, for movement of this transfer membrane, force is applied to the insulating portions retaining the separation medium, and the insulating portions may warp. If the insulating portions warp, the shape of the separation medium will also change, and there is concern over influencing the separation and discharge of sample.
In contrast, according to the above-mentioned configuration, even in a case of a force such that brings together is acting on the first insulating portion and second insulating portion, the warping prevention portion inserted between the first insulating portion and second insulating portion works like a prop to suppress the first insulating portion and second insulating portion from warping. It is thereby possible to perform more accurate analysis.
According to a second aspect of the present invention, in the separation medium cassette for sample separation adsorption of the first aspect, at least a part of the warping prevention portion may be inserted between ends in a direction of the electrophoresis of the first insulating portion and the second insulating portion.
According to the above-mentioned configuration, since the warping prevention portion is inserted at the ends of the first insulating portion and second insulating portion, it is possible to more suitably suppress the end face of the separation medium from deforming. It is thereby possible to further reduce the negative influence on separation of the sample in the separation medium and discharging of sample from the end face of the separation medium, and thus perform more accurate analysis.
According to a third aspect of the present invention, in the separation medium cassette for sample separation adsorption of the first or second aspect, at least a part of the warping prevention portion may be inserted at a central part in a width direction that is orthogonal to the direction of the electrophoresis.
According to the above-mentioned configuration, by at least a part of the warping prevention portion being inserted at a central part in the width direction that is orthogonal to the electrophoresis direction, it is possible to suitably suppress the first insulating portion and second insulating portion from warping by reinforcing the central parts of the first insulating portion and second insulating portion, which easily warp. It is thereby possible to perform accurate analysis.
According to a fourth aspect of the present invention, in the separation medium cassette for sample separation adsorption of the first to third aspects, the separation medium may have a plurality of lanes, and separates the sample into each of the lanes, and the warping prevent portion may be inserted between lanes that are adjacent.
According to the above-mentioned configuration, the warping prevention portion is inserted at a position not inhibiting the separation of sample flowing in the lanes or the discharge of separated sample. It is thereby possible to perform accurate analysis.
According to a fifth aspect of the present invention, in the separation medium cassette for sample separation adsorption of the fourth aspect, the warping prevention portion may have a shape thicker at one end along the direction of the electrophoresis.
According to the above-mentioned configuration, with the lanes at both sides of the warping prevention portion having a shape thicker at one end along the electrophoresis direction, the width of the lane narrows as advancing in the electrophoresis direction. Therefore, the sample is led to a narrower space as advancing within the lane, and concentrated. The distance from the samples flowing in adjacent lanes is thereby ensured. In the above way, it is possible to concentrate the sample and prevent the mixing of samples between lanes, and thus perform more detailed analysis.
According to a sixth aspect of the present invention, in the separation medium cassette for sample separation adsorption of the fourth or fifth aspect, the warping prevention portion may be provided so as to divide between the lanes that are adjacent.
According to the above-mentioned configuration, the sample is separated and discharged without crossing between lanes. It is thereby possible to perform more detailed analysis by preventing the mixing of sample between lanes.
An analysis device for sample separation adsorption according to a seventh aspect of the present invention includes: the separation medium cassette for sample separation adsorption according to any one of the first to sixth aspects; and a moving part that causes a transfer membrane to move so as to pass through a position opposing an end face in the direction of the electrophoresis of the separation medium.
Similar effects as the above-mentioned first to sixth aspects are exerted according to the above-mentioned configuration.
The present invention is not to be limited the aforementioned embodiment, with various modifications being possible within the scope indicated by the claims, and embodiments obtained by appropriately combining the technical means disclosed in each of the different embodiments are also included in the technical scope of the present invention. Furthermore, it is possible to form novel technical features by combining the technical means disclosed in each of the respective embodiments.
The present invention can be widely applied in fields that separate and analyze samples.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2014-074022 | Mar 2014 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2015/057557 | 3/13/2015 | WO | 00 |
