Patent Grant
8449748
This application is the U.S. national phase of International Application No. PCT/JP2009/059992 filed 1 Jun. 2009, which designated the U.S. and claims priority to JP Application No. 2008-171272 filed 30 Jun. 2008, the entire contents of each of which are hereby incorporated by reference.
The present invention relates to a sample separation/adsorption appliance that separates a biological or chemical sample in which different kinds of components are mixed into these components and then causes these components to be adsorbed to adsorption members.
Various kinds of methods have been developed by which samples such as proteins and nucleic acids are separated and detected, taking advantage of their individual properties. Also, apparatuses used for the methods have been developed. Examples of the separation methods include gel electrophoresis, capillary electrophoresis, and liquid chromatography. Among these methods, gel electrophoresis has been widely used from the viewpoint of its easiness and realization of high resolution separation.
Gel electrophoresis is carried out in the following modes: electrophoresis by which a sample is separated in only one direction; and two-dimensional electrophoresis by which a sample is separated in two directions. The two-dimensional electrophoresis is generally carried out for analysis of proteins.
In a case where a protein is separated by two-dimensional electrophoresis, isoelectric focusing electrophoresis that uses a gel strip having a given pH gradient is used for separation in the first-dimensional direction. The isoelectric focusing electrophoresis is a method of applying a voltage across the gel strip to perform separation, taking advantage of differences in isoelectric points specific to proteins. Further, for the separation in the second-dimensional direction, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using sodium dodecyl sulfate (SDS), which is an anionic surfactant, is widely used. The two-dimensional electrophoresis is widely used for proteome analysis because it enables separation of many proteins at once and comprehensive analysis of the proteins. Proteome is the term that indicates an entire protein into which translation is carried out in a particular cell, organ, etc. Examples of studies for the proteome include profiling, functional analysis, etc. of proteins.
Further, it is known that a protein synthesized in vivo is adjusted for its function by being subjected to chemical modification called post-translational modification, such as phosphorylation. The above-described electrophoresis can only separate a protein into different components. Therefore, only separation analysis is difficult to obtain information regarding post-translational modification of the components.
In proteome analysis, as a method for detecting a sample protein separated by electrophoresis, there is a method known as transfer (blotting) by which a sample separated in a gel is adsorbed and fixed to a transfer film. For the transfer film, a nitrocellulose film, a PVDF (Polyvinylidene difluoride) film, or the like film which is easy to be bound to a sample and is highly hydrophobic is used.
The sample having been transferred onto the film is detected with use of a fluorescent-labeled antibody, probe, or the like. As a method of detecting a protein with use of an antibody, Western blotting is known. By overlaying a particular antibody on a film having a protein transferred thereon, it becomes possible to specify a protein to some extent on the basis of antigen-antibody reaction. As to phosphorylation, which is biologically important post-translational modification, it is possible to detect the presence or absence of a phosphorylated protein, the differences between phosphorylation sites, and the like by overlaying an anti-phosphorylated antibody on the film subjected to protein transfer.
Thus, a combined use of electrophoresis and Western blotting is an extremely effective method for proteome analysis (for example, see Non-Patent Literature 1).
Conventionally, electrophoresis and Western blotting are carried out manually by researchers, using separate apparatuses. More specifically, it is general that after SDS-PAGE is carried out in an electrophoresis apparatus, a gel is taken out of the apparatus and moved in a transfer (blotting) apparatus, and then transfer (blotting) is carried out with a transfer film set on the transfer (blotting) apparatus. These operations are complicated and require some experience because the gel is a very soft material and therefore difficult to handle. However, there exists a technique (Patent Literature 1) that automates these operations. Patent Literature 1 discloses a technique of automating a series of operations from electrophoresis to Western blotting.
Patent Literature 1
Non-Patent Literature 1
In many cases, samples for use in analysis, such as proteins for use in proteome analysis and antibodies for use in immunoreaction, are generally separated or extracted from cells and tissues at much expense in time and effort. Therefore, in a sample separation/adsorption appliance for continuously performing a series of operations from electrophoresis to Western blotting, there is a demand for a technique for more efficiently transferring a sample while minimizing waste.
The present invention has been attained in view the above problems, and a main object of the present invention is to provide a sample separation/adsorption appliance that enables efficient transfer of a sample.
The inventors of the present invention diligently worked and found the following two problems of the technique disclosed in Patent Literature 1. One of the problems is that (a) since transfer (blotting) of the sample is carried out in a buffer solution, the buffer solution can easily flow into a clearance between a sample separation section and a sample adsorption member during transfer of the sample, and the sample flows out into the buffer solution in the clearance by self-diffusion. This decreases transfer efficiency of the sample. The other problem is that (b) when bubbles generated from electrodes by electrochemical reaction at the time of electrophoresis and transfer (blotting) comes into contact with the sample adsorption member during transfer of the sample, the sample adsorption member has an area where the sample is not transferred because no electric current passes through the area. This causes an uneven transfer pattern with spots. After a more thorough study, the inventors of the present invention accomplished the present invention.
More specifically, in order to solve the above problems, a sample separation/adsorption appliance according to the present invention comprises: a first buffer solution tank equipped with a first electrode and used for holding a first buffer solution; a second buffer solution tank equipped with a second electrode and used for holding a second buffer solution; a sample separation section that holds a sample separation medium for separating a sample; and a conductive section that holds a conductive medium, wherein: a sample adsorption member for adsorbing the sample is installed at a position between the sample separation medium and the conductive medium; when the sample adsorption member is installed at the position, the sample separation medium and the conductive medium come into contact with the sample adsorption member; an end of the sample separation medium opposite to an end being in contact with the sample adsorption member is connected to an inside of the first buffer solution tank; and an end of the conductive medium opposite to an end being in contact with the sample adsorption member is connected to an inside of the second buffer solution tank.
According to the above arrangement, the first buffer solution is held in the first buffer solution tank, and the second buffer solution is held in the second buffer solution tank. This causes the first and second electrodes to be electrically connected to each other via the first buffer solution, the sample separation medium, the sample adsorption member, the conductive medium, and the second buffer solution. Consequently, it is possible to separate the sample in the sample separation medium by electrophoresis. Also, it is possible to move the separated sample by electrophoresis from the sample separation medium to the sample adsorption member being in contact with the sample separation medium, so that the sample can be adsorbed to the sample adsorption member.
Unlike the conventional technique, movement of the sample from the sample separation medium to the sample adsorption member is carried out outside the second buffer solution tank. Therefore, it is possible to prevent self-diffusion (outflow) of the sample into the buffer solution. Also, it is possible to prevent the bubbles generated from the electrodes from reaching the sample adsorption member via the buffer solution. This enables efficient transfer of the sample. This in turn, it is possible to prevent the occurrence of uneven adsorption of the sample, to produce a sharper pattern, and to detect the sample with accuracy and high sensitivity.
A sample separation/adsorption appliance according to the present invention is arranged such that at the end of the sample separation medium being in contact with the sample adsorption member, the sample separation medium has a cut preferably on at least one of upper and lower surfaces thereof, more preferably on both of the upper and lower surfaces thereof. The cut is provided to make the leading edges thinner. The shape of the cut is not particularly limited as long as the sample separation medium and the sample separation section are tapered down toward their leading edges. Further, a length-to-height ratio of the cut is preferably in a range from ½ to 4.
According to the above arrangement, the sample separation medium has such a shape that at the end being in contact with the sample adsorption member, tapering of the sample separation medium starts from a certain point. According to the later-described findings by the inventors of the present invention, such a shape of the sample separation medium can curb the influence of the wall of the container (the sample separation section) that immobilizes the sample separation medium.
In a case where the cuts are provided on both of the upper and lower surfaces of the sample separation medium, the sample separation medium has such a shape that it is sharply pointed in a centerward direction of its one end. According to the later-described findings by the inventors of the present invention, such a shape of the sample separation medium can further curb the influence of the wall.
Further, when the length-to-height ratio of the cut is preferably in the range from ½ to 4, it is possible to further curb the influence of the wall, as described later.
A sample separation/adsorption appliance according to the present invention is preferably arranged such that at an end of the sample separation section being in contact with the sample adsorption member, the sample separation section has a cut on at least one of upper and lower surfaces thereof.
According to the above arrangement, it is possible to curb outflow of the sample caused by a capillary phenomenon that occurs in a clearance between the sample separation section and the sample adsorption member.
A sample separation/adsorption appliance according the present invention is preferably arranged such that a film impregnated with the sample separation medium is attached to an end face of the sample separation section being in contact with the sample adsorption member.
According to the above arrangement, the film provided at the leading edge of the sample separation section is impregnated with the sample separation medium, and the sample separation medium with which the film is impregnated comes into contact with the sample adsorption member. This makes it possible to bring the sample adsorption member into close contact with the sample separation medium without causing a clearance. Thus, it is possible to prevent outflow of the sample from the clearance and to secure a stably energized state.
A sample separation/adsorption appliance according the present invention is preferably arranged such that a film impregnated with the conductive medium is attached to an end face of the conductive section being in contact with the sample adsorption member.
According to the above arrangement, the film provided at the leading edge of the conductive section is impregnated with the conductive medium, and the conductive medium with which the film is impregnated comes into contact with the sample adsorption member. This makes it possible to bring the sample adsorption member into close contact with the conductive medium without causing a clearance. Thus, it is possible to secure a stably energized state.
A sample separation/adsorption appliance according the present invention is preferably arranged such that at an end of the conductive medium being in contact with the sample adsorption member, the conductive medium has a cut on at least one of upper and lower surfaces thereof.
Lines of electric force produced on the opposite sides of the sample adsorption member are formed between the sample separation medium and the conductive medium. According to the above arrangement, the conductive medium is tapered down. This makes it possible to make the lines of electric force dense. Thus, it is possible to prevent electrical diffusion of the sample at the transfer of the sample and to realize adsorption of the sample with high resolution.
A sample separation/adsorption appliance according the present invention is preferably arranged such that at an end of the conductive section being in contact with the sample adsorption member, the conductive section has a cut on at least one of upper and lower surfaces thereof.
According to the above arrangement, it is possible to curb outflow of the sample caused by a capillary phenomenon that occurs in a clearance between the conductive section and the sample adsorption member.
A sample separation/adsorption appliance according the present invention is preferably arranged to further comprise pressing means that presses at least one of the sample separation medium and the conductive medium against the sample adsorption member.
According to the above arrangement, it is possible to bring the sample separation medium, the sample adsorption member, and the conductive medium into close contact with each other. Thus, it is possible to prevent outflow of the sample from the clearance and to secure a stably energized state.
A sample separation/adsorption appliance according the present invention is preferably arranged to further comprise a third buffer solution tank for holding a third buffer solution, wherein the sample adsorption member is immersed in the third buffer solution.
According to the above arrangement, the sample adsorption member is immersed in the buffer solution. This makes it possible to secure a more stably energized state.
A sample separation/adsorption appliance according the present invention is preferably arranged such that the third buffer solution has a pH in a range from 6.0 to 8.5, and more preferably arranged such that each of the sample separation medium, the conductive medium, the first buffer solution, and the second buffer solution also has a pH in a range from 6.0 to 8.5.
If the third buffer solution is in an acidic pH range or in an alkaline pH range, electroosmotic flow may be generated in the sample adsorption member. If the electroosmotic flow is generated, the buffer solution is flown strongly toward the sample adsorption member. This inhibits high-resolution adsorption of the sample to the sample adsorption member. Further, decrease of the buffer solutions causes an unstable electrophoresis. According to the above arrangement, a pH of the sample adsorption member is kept in the neutral range. This makes it possible to curb the electroosmotic flow. Thus, it is possible to carry out high-resolution adsorption of the sample and to maintain stable electrophoresis.
Note that the electroosmotic flow may occur even in tapered parts of the sample separation medium and the conductive medium. In view of this, the sample separation medium, the conductive medium, and the first and second buffer solutions are adjusted to be in a neutral pH range, so that electroosmotic flows at the tapered parts can be curbed.
A sample separation/adsorption appliance according the present invention is preferably arranged to further comprise: sample adsorption member moving means that moves the sample adsorption member in a direction orthogonal to a direction from the sample separation medium to the conductive medium.
According to the above arrangement, a contact point of the sample adsorption member in contact with the sample separation medium changes to another points as the sample adsorption member moves. Therefore, the sample is continuously adsorbed to the sample adsorption member, and an area of the sample adsorption on the sample adsorption member varies depending upon a traveling speed of each sample in the sample separation medium. By taking advantage of this, it is possible to suitably perform various analyses.
A sample separation/adsorption appliance according the present invention is preferably arranged to further comprise: voltage detecting means that measures a voltage between the first electrode and the second electrode; and sample position detecting means that detects movement of the sample to such a position that the sample separation medium is in contact with the sample adsorption member, on the basis of the voltage measured by the voltage detecting means, wherein when the sample position detecting means detects movement of the sample to the position, the sample adsorption member moving means initiates movement of the sample adsorption member in the direction orthogonal to the direction from the sample separation medium to the conductive medium.
After initiation of the electrophoresis in the sample separation medium, a sample is not adsorbed until a piece in the sample which piece travels at the fastest speed reaches the leading edge of the sample separation section (the position at which the sample separation section comes into contact with the sample adsorption member). Therefore, it is useless to move the sample adsorption member before initiation of the adsorption of the sample.
According to the above arrangement, detection of the sample passing through the sample separation section is carried out by monitoring a value of voltage between the electrodes, so that movement of the sample adsorption member can be initiated after initiation of adsorption of the sample to the sample adsorption member. More specifically, when the sample reaches the position corresponding to the edge of the tapered part of the sample separation section by electrophoresis, electroconductivity decreases at the edge of the tapered part. This increases a value of resistance. Therefore, there occurs the phenomenon that the voltage value increases under the conditions of constant current (Under the conditions of constant voltage, a value of a current decreases). In view of this, by monitoring the increase in voltage, it is possible to easily detect initiation of adsorption of the sample to the sample adsorption member. By initiating movement of the sample adsorption member on the basis of the above principle, it is possible to efficiently perform adsorption of the sample to the sample adsorption member.
A sample separation/adsorption appliance of the present invention is arranged such that a position where the sample separation section comes into contact with the sample adsorption member is outside the buffer solution tanks in which the electrodes are provided. Therefore, it is possible to efficiently transfer the sample.
Additional objects, features, and strengths of the present invention will be made clear by the description below. Further, the advantages of the present invention will be evident from the following explanation in reference to the drawings.
As shown in
The sample separation medium 131 and the conductive medium 133 are in contact with a sample adsorption member 132 for adsorbing a sample, respectively, on the opposite sides of the sample adsorption member 132. The sample separation medium 131 is in contact with the sample adsorption member 132 at an end corresponding to a leading edge 112 of the sample separation section 110. The conductive medium 133 is in contact with the sample adsorption member 132 at an end corresponding to a leading edge 122 of the conductive section 120.
At a connection section 111 of the sample separation section 110, an end of the sample separation medium 131 opposite to an end being in contact with the sample adsorption member 132 is connected to the inside of the first buffer solution tank 103. At a connection section 121 of the conductive section 120, an end of the conductive medium 133 opposite to an end being in contact with the sample adsorption member 132 is connected to the inside of the second buffer solution tank 104.
When buffer solutions are poured into the first buffer solution tank 103 and the second buffer solution tank 104, the sample separation medium 131, the sample adsorption member 132, and the conductive medium 133 are electrically connected via the buffer solutions to the cathode 101 provided inside the first buffer solution tank 103 and the anode 102 provided inside the second buffer solution tank 104. Note that the sample separation/adsorption appliance 100 according to the present embodiment includes a third buffer solution tank 105 that accommodates the sample adsorption member 132 and, is used for holding a third buffer solution. Holding the buffer solution in the third buffer solution tank makes it possible to prevent drying of the sample adsorption member 132 and to more suitably establish electric connection between the cathode 101 and the anode 102.
Electric connection between the cathode 101 and the anode 102 enables (i) separation of a sample inside the sample separation medium 131 by electrophoresis and (ii) transfer of the separated sample to the sample adsorption member 132 by electrophoresis. In the present embodiment, unlike the conventional technique, transfer of the sample from the sample separation medium 131 to the sample adsorption member 132 is carried out outside the second buffer solution tank. This makes it possible to prevent self-diffusion (outflow) of the sample into the buffer solution and to prevent bubbles generated at the electrodes from reaching the sample adsorption member 132 by way of the buffer solutions. Thus, it is possible to efficiently transfer the sample while cutting waste caused by outflow of the sample into the buffer solution and reducing an uneven transfer pattern of the sample caused by the bubbles. Note that electrophoresis and transfer can be also carried out with the arrangement in which the sample adsorption member 132 is in contact with the sample separation medium 131 and the conductive medium 133 in the buffer solution contained inside the third buffer solution tank 105. In this case, the presence of the sample separation medium 131 and the conductive medium 133 makes it possible to prevent the bubbles generated at the electrodes from reaching the sample adsorption member 132 by way of the buffer solutions.
The sample separation section 110, which is a member for holding the sample separation medium 131, is fixed onto a stage 140, as shown in
The sample separation medium 131 is a medium for separating a sample by electrophoresis. Examples of the sample separation medium 131 include media generally used in electrophoresis, such as polyacrylamide gel and agarose gel. Apart from this, the sample separation medium 131 may be nanoscale pillars, the so-called nanopillars, stood up inside the sample separation section 110.
The conductive section 120, which is a member for holding the conductive medium 133, is provided so as to move over the stage 140 in a horizontal direction, as shown in
The conductive medium 133 is not particularly limited as long as it causes only electrolytes to pass through without outflow of the buffer solution so that electrical conductivity is ensured. For example, a structure such as polyacrylamide gel, agarose gel, or nanopillar may be filled in the conductive section 120.
The sample adsorption member 132 is a member for adsorbing a sample. In the present embodiment, as shown in
The sample separation/adsorption appliance may be offered in a state where the sample adsorption member 132 is contained therein. Alternatively, the sample separation/adsorption appliance may be arranged such that the sample adsorption member 132 can be filled later by the user thereof. The sample adsorption member 132 is preferably made from a material that ensures strength. For example, if the sample is a protein, a PVDF (Polyvinylidene difluoride) film or the like may be used as the sample adsorption member 132. Note that the PVDF film is preferably subjected to pretreatment for hydrophilicity with methanol or the like. Alternatively, nylon, nitrocellulose, or conventionally-used film that is prone to binding to nucleic acids or proteins may be used as the sample adsorption member 132.
The buffer solutions held in the first buffer solution tank 103, the second buffer solution tank 104, and the third buffer solution tank 105 may be anything as long as they ensure electrical conductivity. Particularly, the buffer solutions are preferably buffer solutions in a neutral pH range. The buffer solutions in a neutral pH range make it possible to curb generation of electroosmotic flow that results in movement of the buffer solutions. Particularly, if the buffer solution held in the third buffer solution tank 105 is in an acidic pH range or in an alkaline pH range, electroosmotic flow may be generated in the sample adsorption member 132. If the electroosmotic flow is generated, the buffer solution is flown strongly toward the sample adsorption member 132. This inhibits high-resolution adsorption of the sample to the sample adsorption member 132. Further, decrease of the buffer solutions causes an unstable electrophoresis. Adjustment of the buffer solution held in the third buffer solution tank 105 in the neutral pH range makes it possible to curb the electroosmotic flow. Thus, it is possible to carry out high-resolution adsorption of the sample and stable electrophoresis.
Note that the electroosmotic flow may occur even in tapered parts of the sample separation medium 131 and the conductive medium 133. In view of this, the sample separation medium 131, the conductive medium 133, and the buffer solutions held in the first buffer solution tank 103 and, the second buffer solution tank 104 are adjusted to be in a neutral pH range, so that electroosmotic flows at the tapered parts can be curbed.
The cathode 101 and the anode 102 may be made from any conductive material, but are preferably made from platinum in order to prevent the electrodes from being ionized.
At the end being in contact with the sample adsorption member 132 (in the vicinity of the leading edge 112 of the sample separation section 110), the sample separation medium 131 preferably has a cut on at least one of an upper surface and a lower surface thereof. Note that an upward direction is a direction orthogonal to a direction from the sample separation medium 131 to the sample adsorption member 132. Such a cut causes the sample separation medium 131 to have such a shape that at the end being in contact with the sample adsorption member 132, tapering of the sample separation medium 131 suddenly starts (the sample separation medium 131 is getting thinner) from a certain point of the sample separation medium 131. Note that the cut is provided to make the leading edge thinner, and the shape of the cut is not particularly limited as long as the sample separation medium 131 is tapered down toward the leading edge.
That is, the sample separation medium 131 is tapered down only at an end part that comes into contact with the sample adsorption member 132. After the sample is separated by electrophoresis in the sample separation medium 131, the sample is let out from an end face of the sample separation medium 131 and adsorbed (transferred) onto the sample adsorption member 132. A smaller contact part between sample separation medium 131 and the sample adsorption member 132 makes a sharper pattern of a transferred sample, which allows the appliance according to the present embodiment to perform detection of the sample with higher resolution. However, as shown in (a) of
As shown in (b) of
The shapes of the leading edge of the sample separation medium 131 will be more specifically described with reference to
For example, the shape of the sample separation medium 131 as shown in (a) of
The following shows the results of simulations with variously changed length-to-height ratios of the cut. In (c) of
Further, the following will show comparison between the arrangement shown in (g) of
Further, it is preferable that in the vicinity of the leading edge 112, the shape of the sample separation section 110 itself is also tapered. More specifically, it is preferable that at the leading edge 112, a cut is provided on at least one of the upper and lower surfaces of the sample separation section 110.
Note that as in the case with the sample separation section 110, it is also preferable that a contact part where the conductive section 120 is in contact with the sample adsorption member 132 has a small area so that the capillary phenomenon is curbed, and it is preferable that at the leading edge 122 of the conductive section 120, a cut is provided on at least one of the upper and lower surfaces of the conductive section 120.
Still further, it is preferable that at the end of the conductive medium 133 being in contact with the sample adsorption member 132, the conductive medium 133 has a cut on at least one of the upper and lower surfaces of the conductive medium 133. The sample adsorption member 132 is positioned between the sample separation medium 131 and the conductive medium 133. If both the sample separation medium 131 and the conductive medium 133 are tapered down, it is possible to make lines of electric force formed on the opposite sides of the sample separation medium 131 denser. This makes it possible to prevent electrical diffusion of the sample at the emission of the sample and to realize adsorption of the sample with high resolution.
As previously described, the cuts are provided to make the leading edges thinner. The shape of the cuts is not particularly limited as long as the sample separation medium 131, the conductive medium 133, the sample separation section 110, and the conductive section 120, are tapered down toward their leading edges. For example, in (c) of
In one embodiment, as shown in
The film 700 is not particularly limited as long as it has a low protein adsorption property, strength, and narrow through holes. For example, the film 700 can be a hydrophilic PVDF (Polyvinylidene difluoride) film, a hydrophilic PTFE (Polytetra fluoro ethylene) film, a PES (Polyether sulphone) film, or the like film. The film 700 may be attached to the sample separation section 110 with use of an adhesive tape or an adhesive. Alternatively, the sample separation section 110 and the film 700 may be held together with use of a clip(s) or the like so that the film 700 can be attached to the sample separation section 110. In order to make the film 700 impregnated with the sample separation medium 131, the film 700 should be attached to the leading edge 112 of the sample separation section 110 before the sample separation section 110 is filled with the sample separation medium 131. For example, assume that polyacrylamide gel is used as the sample separation medium 131. In this case, an acrylamide solution before gel polymerization should be poured from the connection section 111 of the sample separation section 110 with the film 700 attached thereto, after which the gel polymerization is caused to develop. The presence of a clearance between the sample separation medium 131 and the sample adsorption member 132 may cause outflow of the sample from the clearance. Further, the clearance may cause an unstable passage of electric current, which may in turn cause irregular transfer of the sample. With attachment of the film 700 to the sample separation section 110 and impregnation of the film 700 with the sample separation medium 131, it becomes possible that even the leading edge 112 of the sample separation section 110 is easily filled with the sample separation medium 131. This makes it possible to bring the sample adsorption member 132 into close contact with the sample separation medium 131 without causing a clearance. Thus, it is possible to prevent outflow of the sample from the clearance and to secure a stably energized state.
In another embodiment, as shown in
The film 800 is not particularly limited as long as it has strength and narrow through holes. For example, the film 800 can be a PVDF film, a hydrophilic PVDF film, a PTFE film, a hydrophilic PTFE film, a PES film, or the like film. The film 800 may be attached to the conductive section 120 with use of an adhesive tape or an adhesive. Alternatively, the conductive section 120 and the film 800 may be held together with use of a clip(s) or the like so that the film 800 can be attached to the conductive section 120. In order to make the film 800 impregnated with the conductive medium 133, the film 800 should be attached to the leading edge 122 of the conductive section 120 before the conductive section 120 is filled with the conductive medium 133. For example, assume that polyacrylamide gel is used as the conductive medium 133. In this case, an acrylamide solution before gel polymerization should be poured from the connection section 121 of the conductive section 120 with the film 800 attached thereto, after which the gel polymerization is caused to develop. The presence of a clearance between the conductive medium 133 and the sample adsorption member 132 may cause an unstable passage of electric current, which may in turn cause irregular transfer of the sample. With attachment of the film 800 to the conductive section 120 and impregnation of the film 800 with the conductive medium 133, it becomes possible that even the leading edge 122 of the conductive section 120 is easily filled with the conductive medium 133. This makes it possible to bring the sample adsorption member 132 into close contact with the conductive medium 133 without causing a clearance. Thus, it is possible to secure a stably energized state.
The conductive section 120 is moved by a movable arm (pressing means) 144 and pressed against the sample adsorption member 132. This brings the sample adsorption member 132 into close contact with both the sample separation medium 131 and the conductive medium 133, which secures energization.
The sample separation medium 131 may be installed in the sample separation section 110 in such a state that the sample separation medium 131 contains a sample to be separated. Alternatively, the sample may be added to the sample separation medium 131 after the sample separation medium 131 is installed in the sample separation section 110. The sample is not particularly limited, but the sample can be a prepared product derived from a biological material (e.g. biological body, body fluid, cell strain, tissue culture, or tissue section), a commercially available reagent, or the like. The sample is more preferably polypeptide or polynucleotide.
At the time of adding the sample, for example, in two-dimensional electrophoresis, a gel strip 130 having undergone isoelectric focusing electrophoresis in the first dimension is connected to (brought into contact with) the sample separation medium 131 at a position corresponding to the connection section 111 of the sample separation section 110. The gel strip 130 may be a commercially available gel strip. Since a gel strip is generally thin and flexible, the gel strip is preferably fixed with use of a support plate 142 formed from acryl or the like material. The fixed gel strip 130 with the support plate 142 is moved by the movable arm 141 so as to be connected to the sample separation medium 131 inside the sample separation section 110. Note that the movable arm 141 may double as the movable arm 143. In this case, the movable arm 141 operates in the following manner. The movable arm 141 connects the gel strip 130 to the sample separation medium 131. After the sample gets into the sample separation medium 131, the movable arm 141 moves to catch an upper part of the sample adsorption member 132.
An electrophoretic separation system used in the first dimension may be incorporated into the appliance according to the present embodiment. In this case, it is possible to automate operations from the isoelectric focusing electrophoresis in the first dimension to the electrophoretic separation in the second dimension and transfer.
In a case where the electrophoresis in the first dimension is not carried out, a well (concavity) is formed at the time of filling the sample separation section 110 with the sample separation medium 131. After the sample is introduced into the well, the sample is immobilized with agarose gel or the like in order to prevent outflow of the sample into the first buffer solution tank 103 (the sample may be introduced together with the agarose gel so that the sample is solidified in the well). As in the case with a normal SDS-PAGE, the well (concavity) can be formed as follows. That is, a gel monomer solution (a liquid before changed to gel by polymerization) is poured into the sample separation section 110. Before gel monomer polymerization occurs, a comb (usually, a comb-like plate with teeth each about 5 mm long) is inserted. After gelation, the comb is removed to complete the well.
After the sample introduction, an electric current is passed between the cathode 101 and the anode 102, which makes it possible to carry out electrophoretic separation. The electric current is preferably 50 mA or lower, more preferably in the range from 20 mA to 30 mA. When the electric current is in the above range, it is possible to curb heat generation while electrophoresis is carried out at a sufficient speed. If the electric current is much higher, the electrophoresis becomes faster. However, a higher electric current causes heat generation, which may affect the gel, the sample, a resolution of electrophoretic separation, and others. Note that in order to prevent heat generation, a cooling system using a peltier device or the like may be provided in the appliance according to the present embodiment.
When the sample reaches the leading edge 112 of the sample separation section 110, the sample adsorption member 132 is lifted up by the movable arm 143 so that the sample can be continuously adsorbed (transferred) to the sample adsorption member 132. Whether the sample reaches the leading edge 112 of the sample separation section 110 may be determined with a previously-stained marker introduced into the sample or may be determined by monitoring of a voltage value.
As a colored sample, a sample into which, for example, BPB (Bromphenol Blue) is introduced can be preferably used. The BPB is used as a frontline usually indicating a leading edge of electrophoresis in a commercially available molecular-weight marker, a SDS-PAGE kit, or the like.
For monitoring of a voltage value, for example, a well-known, commonly used voltage monitor (voltage detecting means) is used that monitors a voltage between the cathode 101 and the anode 102. When the sample reaches the leading edge 112 of the sample separation section 110, electroconductivity decreases at the position where the sample separation medium 131 is connected to the sample adsorption member 132. This increases a value of resistance between the electrodes and therefore sharply increases a voltage value. Monitoring such a voltage value enables detection of emission of the sample.
By incorporating a program (sample position detecting means) for monitoring a voltage value into the appliance according to the present embodiment, it is possible to automatically detect emission of the sample and control the movable arm 143 so that the movable arm 143 lifts up the sample adsorption member 132. In this manner, it is possible to control lifting of the sample adsorption member 132 by a current value or a voltage value. In the same manner, it is possible to control a speed of lifting which is carried out after the initiation of adsorption. This makes it possible to use the sample adsorption member 132 without waste (to minimize an area where the sample is not adsorbed in the sample adsorption member 132), and it is possible to downsize the appliance. The speed of lifting should be such a speed that enables adsorption of the sample with sufficient resolution, and the speed of lifting can be appropriately determined by a person skilled in the art.
After completion of the sample adsorption, the sample adsorption member 132 is collected by the movable arm 143 and is subjected to staining, immunoreaction, etc. Thereafter, a separation and transfer pattern of the sample is detected by a fluorescence detector or the like. A system for fluorescence detection may be also incorporated into the appliance according to the present embodiment in order to automate all the operations, i.e. electrophoresis, transfer, and detection.
As shown in
Further, a movable arm (pressing means) 244 is equipped with: a first buffer solution tank 203 for holding a first buffer solution; and a sample separation section 210 that holds a sample separation medium 231. In the first buffer solution tank 203, a cathode (first electrode) 201 is placed. The sample separation medium 231 and the first buffer solution tank 203 are connected to each other via a connection section 212 of the sample separation section 210. The sample separation section 210 has a shape that allows it to stand upright. The sample separation section 210 is provided with a leading edge 211 that exposes the sample separation medium 231, at the lower part of the sample separation section 210 which lower part is at an opposed location to the connection section 212.
The sample adsorption member 232 is pulled out of the third buffer solution tank 205 by a movable arm (sample adsorption member moving means) 243 and passes through a guide roll 245 so as to be in a horizontal position. Then, the sample adsorption member 232 comes into contact with the sample separation medium 231 and the conductive medium 233 in such a state so as to be positioned between the sample separation medium 231 and the conductive medium 233. A movable arm 244 moves the sample separation section 210 downward. This brings the sample adsorption member 232 into close contact with both the sample separation medium 231 and the conductive medium 233, which secures energization. With this arrangement, separation of the sample introduced into the sample separation medium 231 and transfer of the sample to the sample adsorption member are carried out.
As in the case with the sample separation/adsorption appliance 100 according to First Embodiment, introduction of the sample into the sample separation/adsorption appliance 200 can be achieved by various kinds of methods. As shown in
As described above, with an arrangement of the sample separation/adsorption appliance 200 according to the present embodiment, i.e. the arrangement in which the sample adsorption member is caused to move horizontally, it is also possible to obtain the effect of the present invention.
The sample separation/adsorption appliance 100 according to First Embodiment as shown in
The sample separation section 110 (70 mm in width, 70 mm in length, and 5 mm in thickness) was filled with 10% polyacrylamide gel (60 mm in width, 30 mm in length, and 1 mm in thickness) including Tris-HCl buffer of pH 6.8, as the sample separation medium 131. In order to improve resolution, the sample separation section 110 was formed so that the tapered leading edge 112 has a shape tapered down at a tilt angle of 45°. Further, the sample emission port of 100 μm in thickness was provided in the center of an end face of the leading edge 112, so that the sample separation medium 131 was exposed to the outside. On the other end of the sample separation section 110, the first buffer solution tank 103 was provided so as to be full of a buffer solution. As the buffer solution, a commercially available MOPS buffer (Invitrogen) of pH 7.3 was used. Into the first buffer solution tank 103, the cathode 101 formed from platinum wire was inserted.
Further, the sample adsorption member 132 was inserted into the third buffer solution tank 105, which was provided on the stage 140. The third buffer solution tank 105 was kept full of a buffer solution in which commercially available NuPAGE transfer buffer (Invitrogen) of pH 7.2 was mixed with 20% methanol. That is, the sample adsorption member 132 was in a state of being immersed into the buffer solution. Further, the upper part of the sample adsorption member 132 was fixed to the movable arm 143. As the sample adsorption member 132, Immobiron PSQ (Millipore), which is a commercially available PVDF film, was used after being subjected to pretreatment for hydrophilicity with methanol.
The conductive section 120 (70 mm in width, 50 mm in length, and 5 mm in thickness) was provided in such a manner that it can slide over the stage 140 in a horizontal direction (toward the sample separation section 131) to make the sample adsorption member 132 positioned between the sample separation section 110 and the conductive section 120. The conductive section 120 was formed from glass and filled with 10% polyacrylamide gel (60 mm in width, 15 mm in length, and 1 mm in thickness) including Tris-HCl buffer of pH 6.8. The leading edge 122 of the conductive section 120 facing the sample separation section 110 was formed to have a shape that tapered down, which causes lines of electric force to be dense. This improved resolution of sample adsorption. On the other end of the conductive section 120, the second buffer solution tank 104 was provided so as to be full of a buffer solution. As in the case with the foregoing third buffer solution tank 105, the buffer solution used was a buffer solution in which commercially available NuPAGE transfer buffer (Invitrogen) of pH 7.2 was mixed with 20% methanol. Into the second buffer solution tank 104, the anode 102 formed from platinum wire was inserted.
The conductive section 120 was moved by the movable arm 144, so that the sample adsorption member 132 came into close contact with the sample separation medium 131 provided in the sample separation section 110 and the conductive section 120 provided in the conductive medium 133.
As the sample, SeeBlue Plus2 Pre-stained Standard (Invitrogen) commercially available as a molecular-weight marker was used. At the time of filling the sample separation section 110 with the sample separation medium 131, a well (concavity of 4 mm by 6 mm by 1 mm) was previously formed in the sample separation medium 131. After the sample was introduced into the well, the sample was immobilized with agarose gel in order to prevent outflow of the sample into the first buffer solution tank 103.
After introduction of the sample, a voltage was applied between the cathode 101 and the anode 102, and electrophoretic separation was carried out for 1 hour under conditions of a constant current of 20 mA. The previously-described sample, SeeBlue Plus2 Pre-stained Standard (Invitrogen), is a colored protein. Electrophoretic separation of the sample was therefore confirmed by visual observation.
Thereafter, the sample emitted from the sample separation section 110 was continuously adsorbed (transferred) to sample adsorption member 132, and the sample adsorption member 132 was collected by the movable arm 143.
The present invention is not limited to the aforementioned embodiments and is susceptible of various changes within the scope of the accompanying claims. Also, an embodiment obtained by suitable combinations of technical means disclosed in the different embodiments are also included within the technical scope of the present invention.
Further, all of the academic literatures and patent literatures listed herein are incorporated by reference herein.
Specific embodiments or examples implemented in the description of the embodiments only show technical features of the present invention and are not intended to limit the scope of the invention. Variations can be effected within the spirit of the present invention and the scope of the following claims.
The present invention can be used in the field of analysis of biological samples, chemical samples, and the like and in the field of manufacture of appliances for the analysis.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2008-171272 | Jun 2008 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP2009/059992 | 6/1/2009 | WO | 00 | 12/23/2010 |
| Publishing Document | Publishing Date | Country | Kind |
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| WO2010/001675 | 1/7/2010 | WO | A |
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| 20110094887 A1 | Apr 2011 | US |
