JP2008309539A - Two-dimensional electrophoretic apparatus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a two-dimensional electrophoretic apparatus easy to automate. <P>SOLUTION: The two-dimensional electrophoretic apparatus is constituted so as to perform electrophoresis by introducing a migration liquid, which is prepared by mixing a wide region buffer solution using an ampholyte, either one of a water soluble straight chain polymer and a water-soluble straight chain polymer containing a branch structure and a sample, into a unidimensional separation flow channel and a two-dimensional electrophoretic flow channel composed of a plurality of two-dimensional separation flow channels crossing the unidimensional separation flow channel to communicate with it. By using this two-dimensional electrophoretic apparatus, two-dimensional electrophoresis can be performed immediately after the completion of the unidimensional electrophoresis. Since conventionally required operation for transferring a support containing the sample after the unidimensional electrophoresis to the surface of the two-dimensional electrophoresis is dispensed with, the automation of a two-dimensional electrophoretic apparatus becomes easy. Further, the electrophoretic tank is formed into a microchip structure to further increase the speed of electrophoresis. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

The present invention relates to a two-dimensional electrophoresis apparatus for performing protein separation analysis and an electrophoresis method using the same.

Gel electrophoresis using polyacrylamide gel as a support for electrophoresis is widely used as a means for separating and analyzing proteins, peptides, neurotransmitters, hormones, nucleic acids and the like in the fields of life science, medicine, environment and food. In particular, polyacrylamide gel two-dimensional electrophoresis, which separates proteins in two different separation modes, isoelectric point and molecular weight, is known as an analytical method with excellent resolution.

Explaining one example of the conventional method described above, two-dimensional electrophoresis is a combination of first-dimension isoelectric focusing using tube gel and second-dimension molecular weight separation electrophoresis using slab gel. In the electrophoresis, after the first-dimensional separation was completed, the gel was taken out from the tube, and it was necessary to perform the second-dimensional electrophoresis after adhering it to the slab gel for the second-dimensional separation. .
O'Farrell, J. Biol. Chem., 250, 4007-4021, 1975

In polyacrylamide gel two-dimensional electrophoresis, preparation of polyacrylamide gel, which is a support for electrophoresis, is complicated, and quality standardization is difficult. In recent years, the gel size has been reduced and the electrophoresis time has been shortened. However, a simpler and more practical electrophoresis method and electrophoresis apparatus are desired.

The main problem to be solved by the present invention is to provide a two-dimensional electrophoresis apparatus excellent in speeding up and separation performance of two-dimensional electrophoresis. Furthermore, the entire system including peripheral devices must be miniaturized and automated.

The solution according to the present invention will be described below.
The invention described in claims 1 and 2 constitutes the basis of the present invention, and in the case of two-dimensional electrophoresis, a single first-dimensional separation channel that does not have a sample-only introduction channel, and a plurality of crossing channels. The purpose is to perform two-dimensional electrophoresis by providing parallel separation channels for the second dimension. Furthermore, an electrophoresis solution in which one or more of a water-soluble linear polymer or a water-soluble linear polymer containing a branched structure and a wide-area buffer solution using an amphoteric electrolyte are mixed in the entire region of the flow path, or a sample is further added thereto. Two-dimensional electrophoresis is performed by introducing a mixed electrophoresis solution.

Hereinafter, the above-described invention will be described in detail with reference to FIG. 1A is a perspective view of the two-dimensional electrophoresis tank 100, and FIG. 1B is a partial cross-sectional view taken along the line PP in FIG. 1A. A second-dimensional electrophoresis separation channel 120 constituted by a plurality of channels 121 and 122 intersects and is integrated with the first-dimensional electrophoresis separation channel 110. In the intersection, the flow paths 110, 121, and 122 communicate with each other, and fluid can freely pass between the flow paths. As shown in the figure, the electrophoresis solution 130 is introduced into the entire area of the first-dimensional separation channel and the second-dimensional separation channel. As described above, this electrophoresis solution is a mixture of at least one of a water-soluble linear polymer or a water-soluble linear polymer containing a branched structure and a wide-area buffer solution using an amphoteric electrolyte, or a mixture thereof. Furthermore, the sample is mixed.

The plurality of flow paths for second-dimensional electrophoretic separation are physically independent from each other except at the intersection with the flow path for first-dimensional electrophoretic separation. For this reason, even when the first-dimensional electrophoresis is performed by applying a voltage to both ends of the first-dimensional electrophoresis separation channel with an appropriate electrode and electrode liquid, the two-dimensionally crossed and integrally connected Less susceptible to the presence of multiple flow paths for eye electrophoresis separation.

For voltage application in the first-dimensional electrophoresis, for example, a pair of first-dimensional electrophoresis electrode systems 510 illustrated in FIG. 5A may be used. In the first-dimensional electrophoresis electrode system 510, the capillary 513 is filled with the electrode solution 511, and a constant amount is held by capillary action. It is also possible to mix a sample (protein, etc.) in advance in the electrode solution. An electrode wire 512 is provided in the electrode liquid. Now, if each of the electrode liquid surfaces 515 of the pair of first-dimensional electrophoretic electrode systems is brought into contact with both ends of the first-dimensional electrophoretic separation channel, the electrode liquid and the first-dimensional electrophoretic liquid are brought into contact with each other. Therefore, the first-dimensional electrophoresis can be performed by applying a predetermined voltage to the electrode lines. At this time, the plurality of second-dimensional electrophoretic separation channels crossing and integrally connecting the first-dimensional electrophoretic separation channels are arranged at both ends to make it difficult to influence the first-dimensional electrophoresis. Is open.

In order to apply a voltage to the second-dimensional electrophoresis separation channel 120, for example, a pair of second-dimensional electrophoresis electrode systems 520 shown in FIG. 5B may be used. The configuration of the electrode system for the second dimension electrophoresis is similar to that for the first dimension, and the electrode liquid container 523 is filled with the electrode liquid 521, and a constant amount is held by capillary action. An electrode wire 522 is provided in the electrode liquid.

According to the present invention, the second-dimensional electrophoresis can be started immediately after the completion of the first-dimensional electrophoresis. After completion of the first-dimensional electrophoresis required by the conventional method, the complicated operation of transferring the electrophoresis support containing the sample onto the support for the second-dimensional electrophoresis is not required, and the electrophoresis time is shortened. At the same time, since the process is simplified, it is easy to automate the apparatus. The water-soluble linear polymer (or the water-soluble linear polymer containing a branched structure) mixed in the electrophoresis solution suppresses the diffusion of the separation spot or improves the separation ability by the molecular sieve effect.

The point of the invention shown in claim 2 is that the sample is further mixed in the electrophoresis solution shown in claim 1. This eliminates the need to introduce a sample when starting the first-dimensional electrophoresis, and simplifies the electrophoresis operation and process.

Further, the point of the invention shown in claim 3 is that the flow path is constituted by a capillary. Furthermore, in the invention shown in claim 4, electrode tanks are provided at both ends of the capillary for separation of the first dimension electrophoretic separation shown in claim 3, and also at both ends of the plurality of capillaries for electrophoresis of the second dimension. Each capillary is provided with a common electrode tank, and the inner surface of the electrode tank for the second dimensional electrophoresis is subjected to water repellent treatment. By applying the above water-repellent treatment, the inner surface of the electrode tank is wetted from the end of each capillary for 2D electrophoretic separation when the electrophoresis solution is introduced to the entire area of the 1D and 2D electrophoresis capillaries. However, it is possible to prevent the electrophoresis solution from leaking.

Furthermore, the point of the invention shown in claim 5 is that the first-dimensional and second-dimensional electrophoretic separation flow paths are constituted by groove-shaped flow paths provided on the substrate. Furthermore, in the invention shown in claim 6, as in the invention of claim 4, electrode tanks are provided at both ends of the groove-shaped channel. A common electrode tank is provided at each end of the plurality of groove-shaped channels for separation of the second-dimensional electrophoresis, and the inner surface of the electrode tank for the second-dimensional electrophoresis is further provided. It is to give water repellent treatment.

According to the present invention, the time required for two-dimensional electrophoresis is shortened based on the simplification of the process, and the apparatus can be easily downsized and automated. Furthermore, an electrophoretic separation image with excellent resolution can be obtained by using an electrophoretic solution in which a wide-area buffer using an ampholyte and a water-soluble linear polymer (or a water-soluble linear polymer containing a branched structure) are mixed. It is done.

The best mode for carrying out the present invention will be described below with reference to the drawings. FIG. 2 shows a two-dimensional structure in which the first-dimensional and second-dimensional electrophoresis separation channels are constituted by capillaries, and electrode tanks 251 and 252 are provided at both ends of the first-dimensional and second-dimensional electrophoresis separation capillaries. An electrophoresis apparatus 200 is shown. Into each capillary, an electrophoretic solution having a specification of A or B shown below is introduced.
A specification electrophoresis solution: Electrophoresis solution in which either a wide-area buffer solution using an ampholyte and one or more of a water-soluble linear polymer or a water-soluble linear polymer containing a branched structure are mixed B specification electrophoresis solution: the above A specification electrophoresis Electrophoresis solution in which the sample is mixed with the solution in advance

Examples of the water-soluble linear polymer or the water-soluble linear polymer containing a branched structure include linear polyacrylamide, cellulose, methyl cellulose, hydroxymethyl cellulose, dextran, and polyethylene glycol.

FIG. 6 shows an electrode system for applying a voltage to the electrophoresis solution for the first-dimensional electrophoresis and the electrophoresis solution for the second-dimensional electrophoresis. FIG. 6A shows an electrode system 610 for first-dimensional electrophoresis, in which an electrode solution and an electrode wire are set in a capillary vessel. FIG. 6B shows an electrode system 620 for second-dimensional electrophoresis, in which an electrode solution and an electrode wire are set in an electrode solution container.

FIG. 3 shows an example of a microchip in which the electrophoresis channel is formed by a groove-like channel. FIG. 4 shows an example in which an electrode tank is provided at each end of the first-dimensional and second-dimensional electrophoresis channel provided in the microchip. The electrode system shown in FIG. 6 can also be applied when applying a voltage to the electrophoresis solution introduced into the flow path shown in FIGS.

FIG. 2 shows a two-dimensional electrophoresis apparatus according to the first embodiment of the present invention. FIG. 2A is a perspective view of the two-dimensional electrophoresis apparatus 200 of the present embodiment, which is characterized in that the first-dimensional / second-dimensional electrophoresis separation channel is constituted by a capillary. FIG. 2B is a partial cross-sectional view taken along the line S-S in FIG. The first-dimension electrophoretic separation capillary 210 and the plurality of second-dimensional electrophoretic separation capillaries 221 and 222 intersect and are integrated. Further, as shown in FIG. 2B, the first-dimensional electrophoresis separation capillary and the second-dimensional electrophoresis separation capillary communicate with each other at the intersection. Furthermore, a first-dimensional electrophoresis electrode tank 251 is provided at both ends of the first-dimensional electrophoresis separation capillary.

Furthermore, a two-dimensional electrophoretic electrode tank 252 is also provided at both ends of the plurality of second-dimensional electrophoretic separation capillaries. Further, as shown in the drawing, a water repellent coating layer 255 is provided on the inner surface of the electrode tank 252 for the second dimensional electrophoresis, and the electrophoretic solution 230 is introduced to the entire area of the first and second dimensional electrophoresis capillaries. Occasionally, the electrophoresis solution is prevented from leaking out while wetting the inner surface of the electrode tank from the end of each capillary for second-dimensional electrophoresis separation.

An example of separating proteins using the above two-dimensional electrophoresis apparatus will be described below. First, the electrophoresis solution 230 is introduced from the first-dimensional electrophoresis electrode tank to the entire area of the first- and second-dimensional electrophoresis separation capillaries. At this time, the amount of the introduced solution is adjusted so that the electrophoresis solution does not flow into the second-dimensional electrophoresis electrode tank. As the electrophoresis solution, the aforementioned A-specific electrophoresis solution was used. In this example, the A-specific electrophoretic solution is a commercially available amphoteric electrolyte (wide-area buffer solution using an amphoteric electrolyte, pH 3.5 to pH 10: pH 3.5 to pH 5.0 = 1: 1), 0.1%, linear polyacrylamide. An aqueous solution prepared in a solution (3% T, 0% C) was obtained.

Next, the first-dimensional electrophoresis will be described. In order to apply a voltage to the electrophoretic solution in the first-dimensional electrophoresis separation capillary, a first-dimensional electrophoresis electrode system 610 shown in FIG. 6A is used. In the capillary 613, an electrode solution 611 and an electrode wire 612 are provided. The capillary has a structure capable of sucking and holding a predetermined amount of a predetermined electrode solution from another container by capillary action. In this example, 0.01 M phosphoric acid solution (for anode) and 0.04 M sodium hydroxide solution (for cathode) are used as electrode solutions, and a sample (protein) is mixed in the anolyte in advance. After bringing the pair of electrode systems 610 into contact with the electrophoretic solution 230 in the electrode tank 251 for the first-dimensional electrophoresis, voltage is applied to perform the first-dimensional isoelectric focusing.

Immediately after the completion of the first-dimensional electrophoresis, the process proceeds to the second-dimensional electrophoresis. For this purpose, a voltage is applied to the electrophoresis solution for second dimension separation using the electrode system 620 for second dimension electrophoresis shown in FIG. 6B. The electrode system for the second dimensional electrophoresis has a structure substantially similar to that of the first dimension, and an electrode liquid 621 and an electrode wire 622 are provided in the electrode liquid container 623. The electrode solution container has a structure capable of sucking and holding a predetermined amount of a predetermined electrode solution from another container by capillary action. In this example, 0.05 M Tris (hydroxys aminomethane) -0.38 M glycine buffer was used as the anode / cathode electrode solution.

After setting the pair of electrode systems for the second-dimensional electrophoresis on the upper part of the electrode tank for the second-dimensional electrophoresis, measure the liquid junction between the electrophoresis solution for the second-dimensional electrophoresis and the electrode liquid, and apply a voltage. Second-dimensional electrophoresis (zone or molecular weight electrophoresis) is performed. Trace analysis samples can be used with pretreatment that enables high-sensitivity detection such as fluorescent labeling, and the fluorescence generated by irradiating the electrophoresis channel with excitation light is captured by a CCD camera in the computer. Observation, image analysis, and display. The excitation light source is selected according to the type of phosphor used.

A second embodiment of the present invention is shown in FIG. FIG. 3 shows a microchip 300 for two-dimensional electrophoresis in which the first-dimensional / second-dimensional separation channel is constituted by a fine groove-like channel (hereinafter referred to as a microchannel). 3A is a perspective view of the microchip, and FIG. 3B is a partial cross-sectional view taken along a line TT in FIG. The first-dimensional separation microchannel 310 and the plurality of second-dimensional separation microchannels 321 and 322 are integrated so as to intersect. 3A and 3B, the first-dimensional electrophoretic separation microchannel and the second-dimensional electrophoretic separation microchannel are in communication with each other at the intersection.

In the microchip described above, a microchannel is formed on the substrate 340 by applying a semiconductor microfabrication technique. The substrate material is silicon. The microchannel has a channel width of 100 μm, a depth of 100 μm, and a length of 20 mm. The plurality of micro-channels for second-dimensional electrophoresis are composed of 100 micro-channels while keeping the interval between adjacent channels (the thickness of the partition walls of the parallel channels) at about 20 μm. An oxide film (SiO ₂ film) having a thickness of about 1 μm is formed on the inner surface of the first and second dimension microchannels.

In the microchannel, the electrophoresis solution 330 is introduced over the entire area. As the electrophoresis solution, the aforementioned B-specific electrophoresis solution was used. In this example, a B-type electrophoretic solution was prepared by using a commercially available amphoteric electrolyte (wide-area buffer solution using an amphoteric electrolyte, pH 3.5 to pH 10: pH 3.5 to pH 5.0 = 1: 1), 0.1%, linear polyacrylamide. A solution (3% T, 0% C) and two kinds of samples such as laver-derived fluorescent protein phycoerythrin were prepared to be 0.7 μg / μl.

In order to apply a voltage to the electrophoretic solution 330 in the microchannel, the above-described electrode system shown in FIG. 6 is used. The electrode solution to be used is the same as that shown in Example 1. However, since the sample is mixed in advance in the electrophoresis solution, the sample is not mixed again in the electrode solution.

First, in the first-dimensional separation, a pair of electrode systems for first-dimensional electrophoresis 610 are brought into contact with the region of the first-dimensional electrophoresis electrode liquid contact zone 351 on the microchip, so that the liquid of the electrode liquid and the electrophoretic liquid is obtained. After entanglement, voltage is applied to perform first-dimensional electrophoresis (isoelectric focusing).

As described above, when the first-dimensional electrophoresis is completed, the process immediately proceeds to the second-dimensional electrophoresis. A pair of 2D electrophoretic electrode system 620 is brought into contact with the 2D electrophoretic electrode liquid contact zone 352 on the microchip, and the voltage between the electrophoretic liquid and the electrode liquid is measured. Second-dimensional electrophoresis (zone or molecular weight electrophoresis) is performed. Since the two-dimensional electrophoresis process and the observation method of the electrophoresis image at the end are the same as those in the first embodiment, description thereof is omitted.

  A third embodiment of the present invention is shown in FIG. FIG. 4 shows that the separation channel for the first and second dimension electrophoresis is composed of microchannels as in the second embodiment, and is provided at both ends of the first and second dimension electrophoresis microchannels. The microchip 400 for two-dimensional electrophoresis is provided with an electrode tank. 4A is a plan view of the microchip, and FIG. 4B is a cross-sectional view taken along a line YY in FIG. 4A.

The structure of the first and second dimension electrophoresis microchannels is substantially the same except that the first dimension electrophoresis electrode tank 451 and the second dimension electrophoresis electrode tank 452 are provided at both ends. Since it is similar to Example 2, detailed description is omitted. For the same purpose as described in the first embodiment, as shown in the figure, a water repellent coating layer 455 is provided on the entire inner surface of the electrode tank 452 for second-dimensional electrophoresis. Here, in the plan view shown in FIG. 4A, the drawing of the water-repellent coating layer applied to the bottom surface of the electrode tank 452 for the second-dimensional electrophoresis is omitted. Further, the electrophoresis solution and the electrophoresis method used in the present embodiment are very similar to the contents described in the first and second embodiments, and thus detailed description thereof is omitted.

In the microchips shown in Examples 2 and 3, no cover is provided on the microchannel, but a cover plate may be provided as necessary. The cover plate at this time must be transparent to illumination light for observing the electrophoretic image and excitation light for causing the fluorescent label to emit light, and a plate-like cover made of glass or synthetic resin is required. Is preferred. Furthermore, the substrate material of the microchip is not particularly limited to silicon as long as it can take an insulating substrate or a structure in which the flow path can be electrically insulated from the substrate. A material made of glass or synthetic resin can be irradiated with illumination light or excitation light from the back surface of the substrate, and is therefore suitable as a substrate material for a microchip. The substrate material must not emit fluorescence when irradiated with light.

As an application example of the present invention, it can be applied to a wide range of fields such as medical care, food, environmental analysis, or quality control thereof.

The perspective view and partial sectional view of the two-dimensional electrophoresis tank according to the present invention The perspective view and partial sectional view of the two-dimensional electrophoresis apparatus shown in Example 1 Perspective view and cross-sectional view of a microchip for two-dimensional electrophoresis shown in Example 2 The perspective view and sectional drawing of the microchip for two-dimensional electrophoresis shown in Example 3 The perspective view of the electrode system for electrophoresis used for explanation of the present invention The perspective view of the electrode system for electrophoresis used in each example

Explanation of symbols

100-2D electrophoresis tank
110-Separation flow path for 1D electrophoresis
120-Separation flow path for 2D electrophoresis
121, 122-Single flow path for separation of 2D electrophoresis
130, 230, 330, 430-electrophoresis solution
200--two-dimensional electrophoresis device
210--Capillary for separation of first dimension electrophoresis
221 and 222-Capillaries for separation of the second dimension electrophoresis
251 and 451-Electrode bath for first dimension electrophoresis
252 and 452-Electrode bath for second-dimensional electrophoresis
255, 455-water repellent coating layer
300, 400-Microchip for two-dimensional electrophoresis
310, 410-Microchannel for separation of 1st dimension electrophoresis
320, 420-Microchannel for separation of 2D electrophoresis
321, 322, 421, 422-Single microchannel for separation of 2D electrophoretic separation
340, 440--Board
351-Electrolyte contact zone for 1D electrophoresis
352-Electrolyte contact zone for 2D electrophoretic electrophoresis
510, 610-1D electrophoretic electrode system
511, 521, 611, 621-Electrode solution
512, 522, 612, 622-electrode wire
513, 613-Capillary
520, 620-2D electrophoretic electrode system
523, 623-Electrolyte container
515, 525--Electrode liquid level

Claims (8)

A plurality of flow paths that are separation flow paths for the second dimensional electrophoresis intersect and are integrated with the separation flow path for the first dimensional electrophoresis, and the first dimensional electrophoresis and the second dimensional electrophoresis are intersected at the intersection. Electrophoresis channels are in communication with each other, and the plurality of separation channels for the second-dimensional electrophoresis are independent from each other except for the intersections, and are electrically connected to each other. In the electrophoresis tank for two-dimensional electrophoresis which is configured to be insulated,
A water-soluble linear polymer containing a wide-area buffer solution using an amphoteric electrolyte and a water-soluble linear polymer or a branched structure in the separation channel for the first-dimensional electrophoresis and the separation channel for the second-dimensional electrophoresis A two-dimensional electrophoresis apparatus, wherein an electrophoretic solution in which any one of the above is mixed is introduced. In the electrophoresis solution according to claim 1,
The two-dimensional electrophoresis apparatus according to claim 1, wherein the sample is further mixed to obtain an electrophoresis solution. 3. The two-dimensional electrophoresis apparatus according to claim 1, wherein the separation channel for the first-dimensional electrophoresis and the separation channel for the second-dimensional electrophoresis are configured by capillaries. 4. . Electrode chambers are provided at both ends of the separation capillaries for the first dimension electrophoresis, and common electrode chambers are provided for both capillaries at both ends of the plurality of separation capillaries for the second dimension electrophoresis. The two-dimensional electrophoresis apparatus according to claim 3, wherein a water repellent treatment is applied to an inner surface of the eye electrophoresis electrode tank. 3. The separation channel for the first-dimensional electrophoresis and the separation channel for the second-dimensional electrophoresis are configured by groove-shaped channels provided on the substrate. 2. The two-dimensional electrophoresis apparatus described in 1. Electrode tanks are provided at both ends of the separation channel for separation of the first-dimensional electrophoresis provided on the substrate,
In addition, a common electrode tank is provided at each end of the plurality of second-dimensional electrophoresis separation groove-shaped channels for each of the plurality of second-dimensional electrophoresis separation groove-shaped channels. 6. The two-dimensional electrophoresis apparatus according to claim 5, wherein a water repellent treatment is applied to the inner surface of a common electrode tank for each of the grooves. A two-dimensional electrophoresis method using the two-dimensional electrophoresis apparatus according to any one of claims 1 to 6. The two-dimensional electrophoresis method according to claim 7, wherein the first-dimensional electrophoresis is isoelectric point separation, and the second-dimensional electrophoresis is zone or molecular weight separation.

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