JP4423810B2 - Electrophoresis device - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
In the field of life science, the present invention relates to an apparatus for sorting a predetermined fragment portion of a sample of a polymer substance such as DNA or protein separated by electrophoresis.
[0002]
[Prior art]
For example, in the field of genetic engineering, it is necessary to perform gel electrophoresis between a standard sample and a target sample, compare the migration patterns of both samples, verify the difference, and determine the molecular sequence of the difference. For this purpose, it is necessary to sort out a portion of the migration pattern of the target sample that is different from the migration pattern of the standard sample.
[0003]
Conventionally, the gel of the fragment to be separated from the pattern of the target sample subjected to gel electrophoresis is directly cut out manually with a scalpel, or the developed migration pattern is transferred to a nylon membrane and then the nylon membrane is sandwiched between scissors. Sorting by cutting with a scalpel.
[0004]
[Problems to be solved by the invention]
However, conventionally, the electrophoresis pattern is optically recognized by another recognition device, and a predetermined portion is cut out based on the information, but the electrophoresis pattern recognition and the cutting work are independent work by a device, Even if the portion to be cut out is determined by the electrophoresis pattern recognition device, it is necessary to reconfirm the position when cutting out. Moreover, the manual cutting operation using a knife or the like is complicated.
[0005]
Recently, instead of gel electrophoresis, an electrophoresis chip formed by joining two substrates has been proposed as a form that can be expected to speed up analysis and reduce the size of the apparatus (DJ Harrison). et al./Anal. Chem. 1993, 283, 361-366). This electrophoresis chip consists of a base material made of a pair of transparent plate-like inorganic materials, and capillary grooves for electrophoresis that cross each other on the surface of one base material by semiconductor photolithography technology and etching technology or micromachining technology. The other base material is provided with through holes at positions corresponding to the ends of the grooves as an anode reservoir, a cathode reservoir, a sample reservoir, and a waist reservoir.
If this electrophoresis chip is used, it is possible that a very small amount of sample can be handled and the electrophoresis sample can be easily separated.
Therefore, an object of the present invention is to provide a novel sorting apparatus using an electrophoresis chip.
[0006]
[Means for Solving the Problems]
In order to solve the above problems, the present invention provides at least one analysis channel in a plate-shaped member, a sample and waste liquid reservoir connected to both ends of the analysis channel, electrodes accommodated in both reservoirs, and the analysis In an electrophoresis apparatus comprising a detector disposed on the downstream side of a flow path, a plurality of branch flow paths branched from the analysis flow path downstream from the detector position of the analysis flow path, and at the end thereof The electrophoresis apparatus is characterized in that a reservoir containing an electrode is provided, and each separated sample is selectively separated into the reservoirs of the plurality of branch channels for each target component .
[0007]
Here, for example, various glass, quartz or Si substrates are used as the plate-like member, and the thickness thereof is preferably about 0.2 to 1 mm, for example. Grooves (flow paths and reservoirs) are formed in the plate member by, for example, photofabrication technology. Photofabrication technology refers to a technology that creates a replica by transferring a photomask pattern. Generally, a photosensitive material called photoresist or resist is applied to the surface of a substrate through a metal mask, and the pattern is transferred by light. To do. Then, the transferred planar pattern is processed into a certain three-dimensional shape by etching or the like.
As the photoresist (or resist) to be used, for example, OFPR5000 manufactured by Tokyo Ohka Co., Ltd., Microposit S1400 manufactured by Shipley Far East Co., Ltd., OMR83-100cp can be used, but not limited thereto, and can withstand a subsequent etching process. If it is a thing, it will not specifically limit. Moreover, the thickness needs to be able to withstand a later etching process, and a thickness of 1 to 2 μm is common.
[0008]
For the transfer of the mask pattern, as in the case of a general integrated circuit, contact exposure for bringing a photomask into close contact with a substrate coated with a resist or projection exposure using a stepper (reduced projection exposure apparatus) is performed. Moreover, holographic exposure may be used. In addition, as a light source used in the exposure, for example, g-line (436 nm) of an ultrahigh pressure mercury lamp can be used, and the exposure condition depends on the thickness of the resist material and the resist. When the mask pattern is transferred and the metal is exposed, the metal mask is patterned to expose the substrate surface. The patterning of the metal mask is performed with aqua regia, for example, when gold is used as the metal.
[0009]
The etching method includes wet etching when various types of glass and quartz are etched. The etchant is not particularly limited as long as it is a solution in which various types of glass and quartz are etched. For example, a hydrofluoric acid type solution is generally used. Moreover, wet etching (anisotropic etching) is mentioned as a method for etching the Si substrate. The etchant used for anisotropic etching is not particularly limited as long as it is an etchant used in this field, such as a KOH aqueous solution, TMAH (tetramethylammonium hydride), or hydrazine.
[0010]
The plate-like member may be used singly or may be used by being bonded with the groove inside. In the case of a single sheet, the flow path is an open system. When the plate members are bonded together, for example, a tapered through hole is formed in one plate member. Here, the method of forming the through hole in the glass or quartz substrate is not particularly limited, but ultrasonic processing is generally used. The size of the through hole is not particularly limited. For example, the opening diameter is preferably about 0.1 to several mm.
Lamination of the plate-like members is performed with the grooves facing inward. The means for joining (joining) the two plate-like members is not particularly limited, but in the case of the present invention, it is desirable to directly join the plate-like members without using an adhesive because of the microanalyzer. For joining the glasses, a means for fusing the two glasses by heating to about 600 to 900 ° C. in a vacuum or in a nitrogen substitution atmosphere is desirable. In addition, for quartz bonding, for example, a means for heating in the same manner as described above after sputtering glass on at least one substrate bonding surface is desirable. Further, when glass and silicon are bonded, for example, an anodic bonding method may be used in which heating is performed at about 400 ° C. and a negative voltage of about −1 kV is applied to the glass side for bonding.
[0011]
The number of analysis channels may be one or more. The branch channel preferably has the same inner diameter as the analysis channel, and the branch channel preferably has a structure in which a plurality of branch channels branch from one analysis channel.
Reservoirs are provided at both ends of the analysis flow path and at the end of the branch flow path. Electrophoresis buffers and electrodes are placed in the reservoirs, and the flow paths can be electrically connected. As the electrophoresis buffer, for example, Tris EDTA, phosphate buffer or the like can be used, but is not limited thereto.
[0012]
A detector is disposed downstream of the analysis flow path. As the detector, a light detector, a light detector, a photodetector, an electrochemical detector, or the like can be used. When a photodetector is used, aluminum or the like may be deposited on the inner surface of the light incident / exit groove by, for example, sputtering. The light from the light source is incident on the light incident groove, and the light passes through the migration groove and is reflected by the light exit groove to reach the detection means. As the light source, an ultraviolet / visible light source, for example, a He—Cd semiconductor laser, a deuterium lamp, or a tungsten lamp is used. As the detector, for example, a photomultiplier tube or a silicon photodiode is used.
The switching of the plurality of branch channels can be performed by switching energization to the reservoirs of the branch channels according to the signal from the detector, for example.
[0013]
DETAILED DESCRIPTION OF THE INVENTION
The electrophoresis apparatus of the present invention will be described with reference to the drawings. FIG. 1 is a plan view showing an embodiment. 1 is a single flat plate made of, for example, glass or silicon, and the flat plate 1 has dimensions of, for example, 10 to 30 mm and a width of 30 to 120 mm. On the surface of the flat plate 1, a sample reservoir 2, a waste liquid reservoir 3, and sorting cells d1 to d4 each formed of a cylindrical recess are formed.
Each reservoir contains a buffer as an electroosmotic flow medium. For example, the sample reservoir 2 and the waste liquid reservoir 3 have a diameter of 1 to 5 mm, a depth of 1 to 3 mm, and sorting cells d1 to d4. Is 0.5 to 3 mm in diameter and 1 to 3 mm in depth.
[0014]
The sample reservoir 2 and the waste liquid reservoir 3 communicate with each other through an electrophoresis path (analysis channel) 4 formed on the surface of the flat plate 1. The migration path 4 has, for example, a width of 50 to 100 μm and a depth of 50 to 100 μm. Further, four branch flow paths (sorting paths) f1 to f4 are branched on the downstream side of the migration path 4, and the sorting paths f1 to f4 communicate with the sorting cells d1 to d4. The sorting paths f1 to f4 have, for example, a width of 30 to 100 μm and a depth of 30 to 100 μm.
[0015]
A detector 5 is disposed on the migration path 4 at a position between the sample reservoir 2 and the sorting paths f1 to f4. The detector 5 includes, for example, a light source and a photodetector, and a signal from the detector 5 is sent to a control unit (not shown).
Further, electrodes e1 to e6 are immersed in each of the reservoirs 2 and 3 and the sorting cells d1 to d4, and voltage application to these electrodes is also controlled by a control unit (not shown).
[0016]
In the above configuration, for example, electrophoresis and fractionation of a negatively charged sample are performed as follows.
First, each of the reservoirs 2 and 3 and the sorting cells d1 to d4 are packed with an electrophoresis buffer such as Tris EDTA (TE) buffer, and the separation path such as linear polyacrylamide is packed into the electrophoresis path 4.
Next, as shown in FIG. 2A, the electrode e1 of the sample reservoir 2 is negatively charged and the electrode e2 of the waste liquid reservoir 3 is positively charged, so that the sample S in the sample reservoir 2 is moved toward the waste liquid reservoir 3. Start electrophoresis. After the detector 5 in the middle of the migration path 4 confirms the passage of the sample S, the electrode e2 of the waste liquid reservoir 3 is turned off, and for example, the electrode of the sorting cell d1 is positively charged. As shown in FIG. 2B, the sample S moves toward the sorting cell d1 and can be sorted.
Similarly, by sequentially switching the charge of the sorting cells d2 to d4, the sample can be sorted into each cell.
[0017]
In addition, this invention is not limited to the said structure, For example, it is also possible to fractionate and remove the unnecessary component in which electrophoresis speed differs from a target component. Further, when a target band component is separated from a plurality of bands, if the number of bands is known, the flow path is switched from the number of bands passing through the detector, and the target component can be separated. Further, when the number of bands is unknown, the target component can be selectively separated by simultaneously running a marker substance that sandwiches the target component.
[0018]
【The invention's effect】
According to the present invention, it is possible to automatically collect a target product and remove contaminants having different migration speeds by simply switching the charge.
[Brief description of the drawings]
FIG. 1 shows an example of an electrophoresis apparatus according to the present invention. FIG. 2 shows an example of sample collection.
1: Flat plate 2: Sample reservoir 3: Waste liquid reservoir 4: Migration path (analysis flow path)
5: Detectors d1 to d4: Sorting cells f1 to f4: Sorting path (branch flow path)

Claims (2)

At least one analysis channel in a plate-like member, and the sample and waste reservoir to be connected to both ends of the analysis channels, the electrodes which are accommodated in said two reservoir, detector disposed downstream of the analysis channel A plurality of branched flow channels branched from the analysis flow channel downstream from the detector position of the analysis flow channel, and a reservoir containing electrodes at the end thereof , An electrophoresis apparatus, wherein each sample is selectively dispensed into a reservoir of the plurality of branch channels for each target component . 2. The electrophoretic device according to claim 1, further comprising a control unit that switches energization to the reservoir of the branch channel in accordance with a signal from the detector.

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