JP2013195240A - Capillary assembly - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a capillary assembly in which a separation speed of a sample is fast and an analysis time is short.SOLUTION: An anode reservoir 2 and a cathode reservoir 3 each including a storage tank which stores liquid are connected to opposite respective ends of a linear capillary 4 whose surface is exposed. Both of the reservoirs are adhesively fixed by a frame 14 with a predetermined distance provided between the centers of the reservoirs. The anode reservoir 2, the cathode reservoir 3, and the capillary 4 are installed in such a manner as to be tightly attached with a temperature control stage in an electrophoresis apparatus, and thus the temperature of the whole of a capillary assembly 1 is controlled.

Description

  The present invention relates to a capillary electrophoresis apparatus, and more particularly to an electrophoresis separation device.

  In the case of analyzing a very small amount of protein or nucleic acid, an electrophoresis apparatus has been conventionally used, and a typical example is a capillary electrophoresis apparatus.

  A capillary electrophoresis apparatus is filled with a separation medium in a glass capillary having an inner diameter of 100 μm or less (hereinafter also simply referred to as a capillary), a sample is introduced into one end, and both ends are brought into contact with a buffer solution. Then, a high voltage is applied between both ends to separate the analyte in the capillary. Further, the temperature of the capillary or the entire apparatus is adjusted in order to increase the migration speed of the sample in electrophoresis and shorten the analysis time.

  FIG. 4 shows an outline of a conventional capillary electrophoresis apparatus 41. The capillary 44 is installed on the temperature adjustment stage 48 of the capillary electrophoresis apparatus 41, and has a curved portion 45 in order to fix one end to the anode reservoir 42 and to make the other capillary release end 44a downward in the vertical direction. The anode reservoir 42 has an injection port 43 for filling the capillary 44 with a separation medium. The injection port 43 is also an injection port for a buffer solution or a cleaning solution, and also serves as an insertion port for an electrode 46 (plus electrode).

  A triaxial stage 52 is arranged vertically below the capillary release end 44a, and the triaxial stage is formed from the separation medium container 50, the sample container 51, the buffer liquid container 53, and the cleaning liquid container 54 mounted on the triaxial stage 52. After selecting necessary containers by moving the X and Y directions of 52, the triaxial stage 52 is moved vertically upward (Z direction) to contact the capillary release end 44a. The electrode 47 (minus electrode) is immersed at the same time when the capillary open end 44a comes into contact with the buffer solution. A detector 49 that detects an analysis object analyzed by electrophoresis is installed in proximity to the capillary 44 in the vicinity of the anode reservoir 42.

  FIG. 5 shows an outline of the configuration of a microchannel chip disclosed in Patent Document 1 as a conventional example. (A) is a perspective view, (b) is an exploded perspective view, and (c) is a cross-sectional view at the AA position of (a). The microchannel chip disclosed in Patent Document 1 is devised as an improvement on the conventional example shown in FIG. The microchannel chip 61 includes a pair of transparent plate-like inorganic materials (for example, glass, quartz, silicon, etc.) or base materials 62 and 63 and a capillary 64 made of resin.

  A groove 67 for arranging the capillary 64 is formed on the lower surface of the upper substrate 62 by, for example, a semiconductor photolithography technique, an etching technique, a micromachining technique, a normal machining technique, or a laser processing technique. The base material 62 is formed with through holes constituting reservoirs 65 and 66 at both end positions of the groove 67. The groove 67 is formed such that the width and depth dimensions are several tens of μm larger than the outer diameter of the capillary 64, for example, 500 μm.

  In the microchannel chip 61, the capillary 64 is fixed in the groove 67 in a state where both ends of the capillary 64 are located in the reservoirs 65 and 66, and both the base materials 62 and 63 are as shown in (a) and (c). Used in a state where they are joined together. The joining of the two base materials 62 and 63 and the fixing of the capillary 64 to the groove 67 are performed by application of an adhesive or water glass, fusion by heating, or the like.

Next, operations when performing electrophoresis using the microchannel chip 61 will be described.
1) A separation medium such as a polymer is filled into the capillary 64 from one of the reservoirs 65 or 66 by, for example, pumping using a syringe.

  2) After injecting the sample from one of the reservoirs 66, the reservoirs 65 and 66 contain the buffer solution. Thereafter, both electrodes are inserted into the buffer solution, a high voltage is applied between the reservoirs 65 and 66 through the buffer solution, and the sample is separated in the capillary 64 by electrophoresis.

  3) Next, for example, a detection mechanism (not shown) such as an optical detection mechanism or an electrochemical detection mechanism using ultraviolet absorption or fluorescence detection is disposed at a predetermined position (detection site) on the reservoir 65 side of the capillary 64. The separated samples are detected sequentially.

JP 2002-207031 A

  In the conventional example shown in FIG. 4, since the sample is introduced from the capillary 44 on the cathode side, the capillary 44 is opened downward in the vertical direction. Therefore, a heater having a temperature adjustment function for keeping the temperature of the entire capillary 44 at a high temperature cannot be attached, and only the capillary portion excluding the capillary release end 44a can be adjusted in temperature. Therefore, the presence of the portion where the temperature cannot be adjusted (capillary open end 44a) becomes an obstacle to increasing the migration speed of the sample and shortening the analysis time. Therefore, it is required to eliminate the capillary open end 44a.

  Further, in order to adjust the temperature of the entire capillary 44, it is possible to make the temperature of the entire capillary uniform by adjusting the temperature of the entire apparatus, but this is a large scale and further prevents the effects of sample evaporation and the like. In addition, it is necessary to provide an inexpensive temperature adjustment mechanism.

  In order to solve the above-mentioned problem, Patent Document 1 discloses a method for eliminating the capillary open end, which is a problem of the conventional example shown in FIG. 4, but this method also has the following problems.

  It is necessary to form a groove 67 for disposing the capillary 64 on the lower surface of the base material 62 by a semiconductor photolithography technique, an etching technique, a micromachining technique, a normal machining technique, or a laser processing technique. Expensive.

  The joining of both base materials 62 and 63 and the fixing of the capillary 64 to the groove 67 are performed by application of an adhesive, water glass or the like, or fusion by heating, but the capillary 64 and the groove 67 and the lower base material 63 are used. Since it is difficult to remove a liquid such as a sample or a buffer solution in the gap between the capillary 64 and the groove 67, it is necessary to seal the gap between the capillary 64 and the groove 67 and the substrate. However, when the amount of the adhesive or water glass is small, cavities and gaps are generated, and when the amount is too large, it leaks into the reservoir or closes the capillary hole. Therefore, the bonding and sealing work is difficult and requires skill.

  When the material of the substrate 63 interposed between the capillary and the heater is resin, the thermal conductivity is poor, and it takes time to heat the capillary to a predetermined temperature, and the start of analysis is delayed. In addition, when a cavity or a gap is generated at the joint between both the base materials 62 and 63, the temperature of the capillary 64 may become non-uniform.

  In order to solve the above problems, the present invention includes a capillary filled with a separation medium, two reservoirs having reservoirs for storing liquid at both ends of the capillary with exposed surfaces, and the two reservoirs It is provided with a frame for holding.

  In the present invention, it is preferable that the material of the two reservoirs is a resin or an inorganic material.

  In the present invention, it is preferable that the capillary is fixed to the two reservoirs by adhesion or ferrule.

  In the present invention, the frame may include holding means for holding a distance between the two reservoirs constant.

  The present invention may further include a temperature adjustment stage for adjusting the temperatures of the capillary and the two reservoirs.

  By connecting the two ends of a capillary with a bare surface to two reservoirs, it is possible to eliminate the capillary open end where temperature cannot be adjusted, and it is possible to adjust the temperature of the capillary and the two reservoirs at both ends simultaneously.

  Since the capillary assembly of the present invention is exposed, the groove processing for embedding the capillary is unnecessary, and the processing cost can be reduced. Further, the connection between the capillary and the reservoir is an easy operation that does not require any skill because it is a connection by bonding or ferrule at the connection point.

  Furthermore, since there are no inclusions that hinder heat transfer by bringing the capillary directly into contact with the temperature adjustment stage in the electrophoresis apparatus, the temperature of the capillary can be reliably adjusted in the shortest time.

1 shows an outline of the configuration of a capillary assembly that is an embodiment of the present invention. (A) is a front view and (b) is a top view. The outline | summary of a structure of the capillary assembly of the modification in this invention is shown. An outline of the configuration of a capillary assembly mounted on a temperature adjustment stage is shown as another modification of the present invention. (A) is a front view, and (b) is a top view with the frame 14 omitted. An outline of a conventional capillary electrophoresis apparatus is shown. An outline of a configuration of a microchannel chip disclosed in Patent Document 1 is shown as a conventional example. (A) is a perspective view, (b) is an exploded perspective view, and (c) is a cross-sectional view at the AA position of (a).

  FIG. 1 shows an outline of the configuration of a capillary assembly 1 according to an embodiment of the present invention. 1A is a front view, and FIG. 1B is a top view. The capillary assembly 1 includes a capillary 4, an anode reservoir 2 and a cathode reservoir 3 connected to both ends of the capillary 4 whose surface is exposed, and a frame 14.

  The connecting portions of the anode reservoir 2 and the cathode reservoir 3 and the capillary 4 are bonded by an adhesive 11. The capillary 4 includes a glass capillary 9 and a coating 10 on the outer periphery of the glass capillary 9, and a part of the coating 10 is removed to form a detection unit 4a for detecting a sample. The glass capillary 9 has an outer diameter of 0.5 mm or less and an inner diameter of 0.1 mm or less, for example, an outer diameter of 0.36 mm and an inner diameter of 0.05 mm. The material of the covering 10 is, for example, polyimide.

  The anode reservoir 2 has a separation medium filling hole 7 communicating with the capillary 4 and a storage tank 5 for storing a buffer solution or a cleaning solution. The cathode reservoir 3 has a sample well 8 communicating with the capillary 4 and a storage tank 6 for storing a buffer solution or a cleaning solution. The anode reservoir 2 and the cathode reservoir 3 are made of resin or inorganic material.

Next, an operation when performing electrophoresis using the capillary assembly 1 will be described.
1) The capillary 4 is filled with a separation medium such as a polymer through the separation medium filling hole 7 of the anode reservoir 2 by pressure feeding using, for example, a syringe (not shown). After filling, excess separation medium is removed.

  2) Next, after injecting the sample into the sample well 8 of the cathode reservoir 3 using, for example, a sample syringe (not shown), the buffer solution is stored in the reservoirs 5 and 6 of the anode reservoir 2 and the cathode reservoir 3. To do. Thereafter, both electrodes (not shown) are inserted into the buffer solution, a high voltage (about 10 kV) is applied between both reservoirs via the buffer solution, and the sample is separated by electrophoresis in the capillary 4.

3) Next, the separated samples are sequentially detected by an optical detector (not shown) by, for example, ultraviolet absorption or fluorescence detection.
4) After the analysis by sample detection is completed, the buffer solution is discharged, and then the cleaning solution is injected, cleaned, and discharged using, for example, a syringe (not shown).
An autosampler, an XY stage (not shown), or the like is used for the operations associated with the above-described electrophoretic analysis.

  The anode reservoir 2 and the cathode reservoir 3 generate a voltage difference of about 10 kV between both ends (anode and cathode) through the capillary tube during electrophoresis. The creepage distance necessary to maintain the insulation of the cable is secured.

  In order to set the linear distance of the capillary 4 from the cathode reservoir 3 to the detection unit 4a, that is, the separation length to a standard separation length of 85 mm, the distance between the centers of the anode reservoir 2 having an outer diameter of 50 mm and the cathode reservoir 3 is set to 135 mm. The capillary assembly 1 having a standard separation length of 85 mm is used for a DNA decoding length of about 300 bases. In order to keep the distance between the centers of the anode reservoir 2 and the cathode reservoir 3 at 135 mm, the two reservoirs are bonded and fixed to the frame 14 having the through holes 14 a and 14 b into which the two reservoirs are inserted at the position of the distance between the centers of 135 mm. .

  FIG. 2 shows an outline of the configuration of a capillary assembly 21 according to a modified example of the present invention. The capillary assembly 21 includes a capillary 24, an anode reservoir 22 and a cathode reservoir 23 connected to both ends of the capillary 24 having a bare surface, and a frame 15, and the connecting portion is coupled by a ferrule 32 and a screw 31. . The anode reservoir 22 and the cathode reservoir 23 are bonded and fixed at a predetermined center distance by the frame 15.

  As the screw 31 is screwed in, the ferrule 32 is pushed deeper, the contact surface of the taper portion 22a of the anode reservoir 22 and the taper portion 32a of the ferrule 32 are strongly pressed to contact each other, and the tip of the taper portion 32a shrinks to the inner diameter side. By closely contacting with the capillary 24, the capillary 24 and the anode reservoir 22 are hermetically fixed. Similarly, the capillary 24 and the cathode reservoir 23 are hermetically fixed.

  The capillary 24 includes a glass capillary 29 and a coating 30 on the outer periphery of the glass capillary 29, and a portion of the coating 30 is removed to form a detection unit 24a for detecting an analyte. The anode reservoir 22 has a separation medium filling hole 27 communicating with the capillary 24 and a storage tank 25 for storing a buffer solution or a cleaning solution. The cathode reservoir 23 has a sample well 28 that communicates with the capillary, and a storage tank 26 that stores a buffer solution or a cleaning solution. The anode reservoir 22 and the cathode reservoir 23 are made of resin or inorganic material.

  FIG. 3 shows an outline of the configuration of the capillary assembly 1 mounted on the temperature adjustment stage 17 as another modification of the present invention. FIG. 3A is a front view, and FIG. 3B is a top view with the frame 14 omitted. Show. The temperature adjustment stage 17 has holes 17a and 17b into which the anode reservoir 2 and the cathode reservoir 3 are inserted. The depths of the holes 17a and 17b are set so that the capillary 4 comes into contact with the upper surface of the temperature adjustment stage 17 when the anode reservoir 2 and the cathode reservoir 3 are installed in the holes 17a and 17b. The entire capillary assembly 1 installed on the adjustment stage 17 is temperature-controlled uniformly.

  The temperature adjustment stage 17 that is a component of the electrophoresis apparatus includes a heater and a temperature sensor (not shown), and the temperature of the temperature adjustment stage 17 is set and controlled at about 60 ° C. Further, in order to detect the sample separated by electrophoresis in the capillary 4, for example, an optical detector 12 by ultraviolet absorption or fluorescence detection is installed on the temperature adjustment stage 17.

  In FIG. 1 to FIG. 5, the same reference numerals represent the same thing and have the same function. The capillaries are exaggerated for easy understanding.

1, 21, Capillary assembly 2, 22, 42 Anode reservoir 3, 23, Cathode reservoir 4, 24, 44, 64 Capillary 4a, 24a Detector 5, 6, 25, 26 Storage tank 7, 27 Separation medium filling hole 8 28, sample well 9, 29 glass capillary 10, 30 coating 11 adhesive 12, 49 detector 14, 15 frame 14a, 14b punch hole 17a, 17b hole 17, 48 temperature adjustment stage 22a, 32a taper part 31 screw 32 ferrule 41 Capillary electrophoresis apparatus 43 Inlet 44a Capillary open end 45 Curved portion 46, 47 Electrode 50 Separation medium container 51 Sample container 52 Triaxial stage 53 Buffer liquid container 54 Washing liquid container 61 Microchannel chip 62, 63 Base material 65, 66 Reservoir 67 Groove

Claims (5)

  It comprises a capillary filled with a separation medium inside, two reservoirs having storage tanks for storing liquid at both ends of the capillary with exposed surfaces, and a frame for holding the two reservoirs. Capillary assembly.   The capillary assembly according to claim 1, wherein the material of the two reservoirs is a resin or an inorganic material.   The capillary assembly according to claim 1 or 2, wherein the capillary is fixed to the two reservoirs by adhesion or ferrule.   The capillary assembly according to any one of claims 1 to 3, wherein the frame includes holding means for holding a distance between the two reservoirs constant.   The capillary assembly according to any one of claims 1 to 4, further comprising a temperature adjustment stage that adjusts temperatures of the capillary and the two reservoirs.

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