JP3548134B2 - Separation and separation equipment - Google Patents

Description

[0001]
[Industrial applications]
The present invention relates to an apparatus for separating and separating biological substances such as DNA and protein.
[0002]
[Prior art]
Gel or electrophoresis was used to separate DNA or protein. For the separation of the separated components, a method of cutting out a gel portion containing a target substance and extracting DNA therefrom is used. Recently, Applied Biosystems Inc. An apparatus that transports a sample eluted from a gel from another using a liquid stream and separates the sample using a method similar to a fraction collector of a liquid chromatograph is beginning to be marketed.
[0003]
[Problems to be solved by the invention]
However, they are suitable for fractionating relatively large amounts of samples, but are not suitable for fractionating small amounts of samples. In other words, since the sample is diluted with a large amount of solution and sampled, it requires time and effort for concentration, and the distance from the gel elution to the sampling section is long, causing problems such as loss due to adsorption or contamination due to desorption of the adsorbate. There was an easy difficulty.
[0004]
[Means for Solving the Problems]
In order to solve the above problems, in the present invention, a sample fractionation cell is inserted in the middle of the migration path. The bottom of the sample fractionation cell is opened so as not to block the electrophoresis current path. In the sample fractionation cell, an array is composed of a plurality of cells so as to fractionate and separate a separated sample, and can be moved in a direction crossing the migration path. The separation timing of the separated sample is determined based on a signal from a detector that monitors the passage of the sample, or based on the migration time.
[0005]
According to another solution of the invention, the separated sample, for example the sample eluted from the gel separation part, is sampled by spraying it with a trace amount of solvent into a sample fractionation cell.
[0006]
[Action]
Insert a cell array into the electrophoresis path, position it one cell at a time in the electrophoresis path, and move it across the electrophoresis path to separate fractionated samples in real time. it can. Since the cell array for fractionation is directly connected to the separation section, it is possible to prevent the loss due to adsorption and the decrease in separation ability.
[0007]
In addition, the sample eluted from the gel is flowed in a sheath flow around the gel capillary tube, introduced into the nebulizer, and atomized to prevent adsorption loss on the tube wall and sample without diluting too much. it can.
[0008]
【Example】
[Example 1]
FIG. 1 shows an example of a fractionating device according to the present invention. The DNA fragment sample to be fractionated is injected into the upper portion 11 of the capillary gel in the upper buffer tank containing the ordinary buffer solution 6. The DNA fragment is fluorescently labeled by adding a fluorescently labeled oligomer or the like to the end by ligation. A voltage is applied between the electrodes 9 and 10, and the DNA sample is electrophoresed. The electrophoresis section is made of a capillary tube, the inside of which is filled with agarose or polyacrylamide gel. An inner diameter of 0.2 to 1 mm can be selected and used according to the purpose. When a capillary tube filled with a polyacrylamide gel having an inner diameter of 0.2 mm and a length of 15 cm is used, a voltage of about 500 V to 1 kV is applied between the electrodes, and a capillary tube filled with an agarose gel having an inner diameter of 1 mm and a length of 10 cm is used. At this time, a voltage of about 100 V is applied between the electrodes. The sample is separated according to its size in the gel, and its state can be monitored using the fluorescence detector 14.
[0009]
The separated sample enters the fractionation cell 3 through the pores 13 of the upper cell holding plate 4. The diameter of the fractionation cell is 1 to 5 mm, about 5 times the size of a gel tube, and the volume is 40 to 100 μl. The DNA fragment that has flowed into the fractionation cell 3 spreads by diffusion, and at the same time, the electric field it receives decreases by the area ratio (1/25), and the DNA fragment stays in this region for a long time. The passage of the DNA fragment is detected by the fluorescence monitor, and the fraction cell cartridge 2 is moved by the motor 15 with a delay in consideration of the migration time from the detection section to the fraction section. The fractionation cell is sandwiched between the upper cell holding plate 4 and the lower cell holding plate 5, whereby the DNA fragment is held in the fractionation cell. The shape of the fractionation cell can take any shape such as a linear array or a disk array. Each fraction cell has pores of 0.1 mm diameter at the lower part, and pores 12 are also formed in the cell holding plate 5 supporting the fraction cell cartridge, thereby forming a migration current path. The fractionation cell is made of plastic, for example, polypropylene, and the upper and lower cell holding plates 4, 5 are made of Teflon, polyimide resin or the like.
[0010]
Although the flow of DNA is almost negligible only by providing pores at the bottom of the fractionation cell, a gel or molecular sieve membrane separation filter may be provided at the bottom instead of the pores to prevent DNA flow. In particular, if a gel or a molecular sieve membrane fractionation filter is further provided after providing pores, it becomes possible to concentrate and trap DNA in a minute region of the gel or molecular sieve membrane fractionation filter. It is.
[0011]
The movement of the fractionation cell cartridge may be performed based on the signal of the fluorescence monitor, or may be performed at regular time intervals for fractionation. These operations are controlled by the controller 16. Since the diameter of the fractionation cell is usually 2 mm, about 50 cells can be arranged. When more cells are needed, the cells can be arranged two-dimensionally and divided while moving in the front, rear, left and right directions.
[0012]
Although not shown in detail in the figure, the fractionation cell cartridge 2 can be removed from the tanks containing the buffer solutions 7 and 8 while keeping the airtightness of each fractionation cell. After the fractionation is completed, the fraction is taken out, and a small amount of a DNA probe or the like can be directly injected into the fractionation cell 3 of the fractionation cell cartridge 2 for analysis.
[Example 2]
FIG. 2 is a block diagram of another embodiment of the fractionating apparatus according to the present invention. In this embodiment, a sheath flow is used together, and the configuration of the sorting section is almost the same as that of the first embodiment. In the first embodiment, the electrode 10 is provided in the lower buffer tank. However, in the present embodiment, the electrode 10 may be provided in the sheath flow liquid tank 17 as shown.
[0013]
The DNA fragments eluted from the gel electrophoresis separation unit 1 pass through the pores 13 of the upper cell holding plate 4 and enter the fractionation cell 3 together with the sheath flow liquid (the composition is a buffer solution for electrophoresis). Although the fractionation cell is previously filled with the buffer solution, it is replaced with the sheath flow solution containing the sample. The flow rate of the sheath flow liquid can be controlled by the pressure applied from the pressurizer to the sheath flow liquid tank 17, and is usually about 10 μl / min. The volume of the fractionation cell is 40-100 μl. Although the sheath flow liquid passes through the fractionation cell and escapes to the buffer liquid tank 7 side, the pores 12 serving as outlet holes are shifted from the pores 13 serving as inflow holes as shown in FIG. However, the DNA fragment stays in the fractionation cell. Furthermore, when the sheath flow liquid flows into the fractionation cell, the inside of the cell is in a turbulent state, so that the DNA fragments are less likely to flow out of the cell and more effective fractionation can be performed. In this embodiment, the fluorescence detector 14 can be attached to the gel-free portion immediately before the fractionation cell, which is convenient for real-time fractionation. The pores 12 and 13 have a diameter of 0.1 to 0.2 mm to increase the linear velocity of the sheath flow liquid. As in the first embodiment, a molecular sieve membrane or the like may be attached to the outlet side pore of the fractionation cell, and the lower part may be sucked.
[Example 3]
FIG. 3 is a configuration diagram of another embodiment of the present invention. The DNA fragment separated by the gel electrophoresis separation unit 1 in which a capillary tube having an inner diameter of 0.2 to 1 mm is filled with agarose or polyacrylamide gel is supplied to a sheath flow liquid tank 17 (composition: electrophoresis). Buffer solution). The DNA fragment is sprayed together with the sheath flow liquid by the atomizer 18 and collected in the fractionation cell 20. The fractionation cell 20 has a diameter of 1 to 5 mm and a volume of 40 to 100 μl as in the above-described embodiment, and can be made of plastic such as polypropylene, but it is not necessary to provide pores at the bottom. Further, in this example, it is not necessary to put a buffer solution in the fractionation cell array in advance, and a high-concentration DNA fragment can be fractionated.
[0014]
As the spraying method, the sheath flow is introduced into a nickel tube with an inner diameter of 0.1 mm and the tip is heated and sprayed, the electric field is sprayed by applying an electric field to the tip, or the volume of the tube using a piezoelectric element such as an ink jet printer. There is a method of jetting while reducing the pressure, but any method can be used. Here, an ink jet method is adopted, and a sheath flow eluted with DNA fragments is guided at a flow rate of 10 μl / min to a thin tube having a diameter of 0.1 mm to which a piezoelectric element is attached, and an electric signal of several hundred kHz is supplied to the piezoelectric element for several tens to several times. A DNA fragment was fractionated into the fractionation cell array 20 by applying a voltage of 100 volts.
[0015]
Although not shown in FIG. 3, a detector for monitoring the passage of the DNA fragment is provided in the sample flow channel, and the fractionation cell cartridge is controlled by the controller based on a monitor signal from the detector, as in the above-described embodiment. It goes without saying that the motor 15 to be driven may be controlled.
Further, the separation unit of the present embodiment is not limited to a unit using electrophoresis, and may be a liquid chromatograph for separating a small amount of sample. In that case, the sheath flow is not necessary, and the eluate from the liquid chromatograph may be introduced into the atomizer 18 as it is.
[0016]
【The invention's effect】
According to the present invention, the separation unit and the sampling unit are directly connected, so that the system is compact and loss of the sample due to adsorption or the like can be reduced. Further, a small amount of a sample can be fractionated in a high concentration state without being diluted with a large amount of a solution. Then, since the sample is directly collected in the fractionation cell, the next process such as analysis can be easily performed by injecting a small amount of reagent into the fractionation cell.
[Brief description of the drawings]
FIG. 1 is a configuration diagram of a first embodiment of a separation / fractionation apparatus according to the present invention.
FIG. 2 is a configuration diagram of a second embodiment of the separation / fractionation apparatus of the present invention.
FIG. 3 is a configuration diagram of a third embodiment of the separation / fractionation apparatus of the present invention.
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 1 Gel electrophoresis separation part 2 Fractionation cell cartridge 3 Fractionation cell 4 Upper cell holding plate 5 Lower cell holding plate 6 Upper buffer solution 7 Lower buffer solution 8 Buffer solution 9,10 Electrode 11 Sample holding unit 12 Hole 13 Fraction cell entrance pore 14 Photodetector 15 Motor drive unit 16 Controller 17 Sheath flow liquid tank 18 Atomizer 19 Holding plate 20 Open type fractionation cell

Claims (6)

A separation unit for separating the sample,
A fractionation unit having a plurality of fractionation cells for storing the separated sample,
Means for moving the separated sample to the sorting section,
A detection unit that detects the sample that passes through the separation unit,
Moving means for moving the fractionation cell in a direction transverse to the direction of separation,
Comprising a fractionation cell holding plate provided between the separation unit and the fractionation unit,
The separation / fractionation apparatus, wherein the fractionation cell holding plate includes an inflow hole having a diameter smaller than an inner diameter of the separation unit and a diameter of the fractionation cell. The separation and separation device according to claim 1, wherein the separation unit is a capillary gel electrophoresis unit, and has an inner diameter smaller than a diameter of the fractionation cell. An electrophoresis section for separating the sample by electrophoresis,
A sheath flow cell provided so as to surround the sample elution end of the electrophoresis unit,
A fractionation unit having a plurality of fractionation cells for storing the separated sample,
Means for moving the separated sample to the sorting section,
A detection unit that detects the sample passing through the sheath flow cell,
Moving means for moving the fractionation cell in a direction transverse to the direction of the electrophoresis,
A fractionation cell holding plate provided between the sheath flow cell and the fractionation unit,
The separation / fractionation apparatus, wherein the fraction cell holding plate includes an inflow hole having an inner diameter at an outlet end of the sheath flow cell and a diameter smaller than the diameter of the fraction cell. 4. The separation according to claim 3, wherein the sorting unit has an outflow hole at a bottom, and the outflow hole is provided at a position shifted from the inflow hole in the direction of the electrophoresis. 5. Sorting device. The separation / fractionation apparatus according to claim 3, wherein the sheath flow cell has an inner diameter at an outlet end smaller than a diameter of the fractionation cell. The separation / fractionation apparatus according to claim 1 or 3, wherein the fractionation cell has one of a pore and a molecular sieve membrane at the bottom.

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