JP4262176B2 - Capillary array, capillary electrophoresis apparatus, and method for manufacturing capillary array - Google Patents

Description

  The present invention relates to an electrophoresis apparatus for separating and analyzing samples such as DNA and proteins.

In a capillary electrophoresis apparatus, a capillary array composed of a bundle of many capillaries is used. The capillary is, for example, a quartz pipe having a jacket coated with a polyimide resin. One end of many capillaries is bundled and fixed, and is called a capillary head. In the capillary electrophoresis apparatus described in Japanese Patent Application Laid-Open No. 2001-324475, in the capillary head, the capillaries are cut out and closely inserted into the tube. Further, in the example disclosed in Japanese Patent Laid-Open No. 2002-39992, in a capillary head, a plurality of capillaries are bundled and bonded through a capillary receiver, and the ends of the capillary and the capillary receiver are aligned, Cut the parts at the same time to make them the same surface. The capillary head has a cylindrical shape to maintain a sealing property with the buffer solution container.
JP 2001-324475 A JP 2002-39992 A JP 2002-71642 A

  As in the capillary head disclosed in Japanese Patent Laid-Open No. 2002-39992, in a capillary head composed of a cylindrical capillary receiver, an adhesive, and a plurality of capillaries, the cylindrical capillary receiver and an adhesive, or a cylinder There is an interface between the cylindrical capillary receiver and a plurality of capillaries. Here, as a result of intensive studies by the inventor of the present application, it has been found that a slight gap is generated at this interface, and when the electrophoresis medium is filled in the capillary, the separation medium may leak from this gap. The inventor of the present application considers the following two causes for the occurrence of a gap at the interface.

First, in order to bring the capillary, the cylindrical capillary receiver, and the adhesive into contact with the buffer solution on the same surface, the capillary head is sandwiched from above and below in the diameter direction and stably fixed and cut with a diamond blade. . Here, if the cylindrical capillary receiver is strongly sandwiched in order to fix it firmly, the cylindrical capillary receiver is deformed, and a gap is generated between the inner wall and the adhesive of the cylindrical capillary receiver or the interface between the inner wall and the capillary. There is a fear.
If a low-viscosity adhesive such as a two-component mixed adhesive is used as the adhesive, the adhesive protrudes outside the cylindrical capillary receiver, and the adhesive inside the cylindrical capillary receiver can be sufficiently filled. However, there is a possibility that a gap is generated at the interface.

  Further, the inventor of the present application has found that there is a possibility that the electrophoretic analysis may fail if there are air pockets on the cut surface of the adhesive. In other words, when the capillary is filled with the electrophoresis medium, the air accumulated in the air pocket becomes a bubble, and the bubble is caught in the capillary. If bubbles are mixed in the capillary, there is a possibility that electrical conduction is interrupted and electrophoresis cannot be performed. Further, in an electrophoresis apparatus that irradiates laser light so as to propagate through a plurality of capillaries, the refractive index around the capillary, the electrophoresis medium, and the capillary is adjusted to reduce the divergence of the laser light propagating through the capillary. However, if bubbles exist in the capillary, this refractive index adjustment is not performed properly, and there is a possibility that a problem occurs in the propagation of laser light. For this reason, it is necessary to inspect the presence or absence of air pockets after the cutting operation.

  An object of the present invention relates to providing a capillary array having a capillary head that does not generate leaks and air pockets.

  The present invention relates to a resin material that fixes a plurality of capillaries together to form an outer peripheral surface of a capillary head. The present invention also relates to the fact that the capillary head that bundles the ends of a plurality of capillaries is substantially a single resin material.

  According to the present invention, the occurrence of leaks and air pockets can be prevented, and analysis accuracy can be improved and production costs can be reduced.

  The above and other novel features and benefits of the present invention will be described below with reference to the drawings. However, the drawings are for explanation only, and do not limit the scope of the present invention.

  FIG. 1 is a schematic diagram of an electrophoresis apparatus, and FIG. 2 is a schematic diagram of a capillary array. Hereinafter, an outline of the electrophoresis apparatus will be described.

  The electrophoresis apparatus includes a sample tray 3 that holds a plurality of sample containers 2, a capillary array 100 that includes a plurality of capillaries 1, an excitation optical system that irradiates each capillary with excitation light, and a detection optical that detects signal light from each capillary. The system includes a high-voltage power supply 15 and a signal processing device 21 that generate an electric field for electrophoresis of the sample.

  As shown in FIG. 2, the capillary array 100 includes a large number of capillaries 1, a load header 4, a detection unit 5, a capillary head 17, a separator 16, and a hollow electrode 20.

  The capillary 1 is generally a quartz pipe having an outer diameter of 0.1 to 0.3 mm and an inner diameter of 0.02 to 0.1 mm, and the jacket is coated with a resin such as polyimide. The capillary 1 holds therein a gel that is a separation medium for electrophoretic separation of the sample. The capillary array is configured by arranging several tens to 100 capillaries. In this embodiment, 96 capillaries are used in which a quartz pipe having an inner diameter of 0.050 mm and an outer diameter of 0.126 mm is coated with 0.012 mm of polyimide.

  The load header 4 holds one end of the capillary 1 and the hollow electrode 20 in a matrix corresponding to the arrangement of the sample container 2. Then, DNA is taken into the capillary by electrophoresis. The capillary head 17 on the opposite side of the load header 4 is formed by bundling 96 capillaries 1 into one, and its shape is made only with an adhesive, and is attached to a buffer solution container 14 containing the buffer solution 13 in a pressure-resistant and airtight manner. A large number of capillaries are aligned by the separator 16. The capillary array is a consumable item that is discarded when the separation performance deteriorates after electrophoresis for several months or several hundred times.

  The detection unit 5 arranges and fixes 96 capillaries 1 in parallel with an accuracy of several microns in height on the optical flat plane in the order of the sample number of the load header. When excitation light is irradiated from both sides, the fluorescent light 10, which is signal light depending on the sample separated by electrophoresis, is emitted.

  The sample tray 3 incorporates a plurality of sample containers 2 containing fluorescently labeled DNA samples. In this embodiment, 96 samples can be held simultaneously.

  The separation medium 13 is injected from the capillary head 17 at a high pressure, pressure is applied until the separation medium 13 comes out from the hollow electrode 20, and the inside of the capillary 1 is completely replaced with the separation medium 13.

  The high voltage power supply 15 applies a high voltage of about 15 kV to the buffer container 14 and the load header 4. At the time of sample introduction, a high voltage is applied to the current path including the buffer solution 13, the separation medium in the capillary 1, the sample in the sample container 2, and the hollow electrode 20. As a result, the sample in the sample container 2 is taken into the capillary 1. At the time of electrophoretic separation, a buffer solution container is disposed instead of the sample tray 3, and a high voltage is applied to the current path including the buffer solution 13, the separation medium in the capillary 1, the buffer solution, and the hollow electrode 20. Thereby, the sample introduced into the capillary 1 is separated by electrophoresis. While the DNA sample passes through the electrophoresis medium filled in the entire area of the capillary, the passage resistance varies depending on the size of the sample (also expressed as long or short), so the sample reaches the detection unit 5 in ascending order.

  The excitation optical system irradiates laser light emitted from the laser light source 6 from both sides of the detection unit 5 via the mirror 7, the beam splitter 8, and the condenser lens 9. Laser light, which is excitation light narrowed down to a thickness approximately equal to the inner diameter of the capillary, is irradiated from both sides of the capillaries arranged in a flat plate shape. The laser can be focused by the lens action of the capillaries, and all the capillaries can be irradiated with excitation light. By irradiating excitation light from both sides, each capillary can be excited to an equal intensity.

  The detection optical system is a sensor including a detection lens system 11 that detects fluorescence 10 that is signal light and a CCD camera 12. The CCD camera 12 detects fluorescence 10 that is signal light corresponding to four types of nucleoside bases of adenine, guanine, cytosine, and thymine.

  The signal processing device 21 analyzes the DNA sample from the detected fluorescence 10 and specifies the base sequence.

  FIG. 3 shows a capillary head of the capillary array in this example. FIG. 3A shows a planar configuration of the capillary head, and FIG. 3B shows a cross-sectional configuration of the capillary head. The capillary head of this example is composed only of the capillary 1 and the resin 27, and an interface is formed only between the capillary 1 and the resin 27. The resin 27 has a tip portion 27A having a small diameter and a capillary portion 27B having a large diameter. FIG. 4 shows a conventional capillary head, and a cylindrical capillary receiver 17 is attached to the tip of the capillary head. In this case, an interface is formed between the cylindrical capillary receiver 17 and the resin 27. In this case, as compared with the present example of FIG. 3, when there are about 50 capillaries, the interface that may be peeled off increases by about 1.5%. When there are about 100 capillaries, the interface that can be peeled off increases by about 10%. When the number of capillaries increases, the capillary 1 and the cylindrical capillary receiver 17 may approach or come into contact with each other. In this respect, there is a possibility of peeling.

  A method for manufacturing the capillary head of this example will be described with reference to FIG. First, a resin mold is formed by combining a resin mold upper 24, a resin mold 25, and a resin mold 26. The bundle of capillaries is inserted into the cavity 30 of the resin mold until the tip of the capillary contacts the lower mold 26 of the resin. Next, an adhesive resin 27 is filled into the cavity of the resin mold with the dispenser 23 or the like. The resin 27 may be, for example, a thermosetting adhesive. An example of such an adhesive is TB2210 manufactured by Three Bond. When the resin mold is removed after the resin 27 is cured, a member having a cylindrical resin attached to the tip of the capillary is completed. Finally, the capillary head is manufactured by cutting the tip BB.

  In this example, since the capillary head can be inspected with the naked eye, it does not occur that the inside of the capillary receiver is not sufficiently filled with the adhesive as in the example of FIG.

  With reference to FIG. 6, the adhesive vacuum defoaming step before curing when the resin 27 is a thermosetting adhesive will be described. When a thermosetting adhesive is used as the resin 27, since the curing time is relatively short, bubbles may remain inside the resin. Therefore, in this example, the vacuum deaerator 28 is used to prevent the generation of such bubbles. As shown in FIG. 5, after inserting a bundle of capillaries into a cavity of a resin mold and filling a thermosetting adhesive resin 27, it is placed in a vacuum deaerator 28. When the pressure in the vacuum deaerator 28 is lowered, the bubbles in the adhesive are removed. Accordingly, it is possible to eliminate air pockets generated when the capillary head is cut.

  FIG. 7 shows another example of the capillary head. In this example, the capillary head 17 extends to the detection unit 5. According to this example, the capillary between the capillary head 17 and the detection unit 5 can be protected, and breakage thereof can be avoided.

  In the example described in Patent Document 3, the capillaries are sandwiched by another member, for example, an aluminum array holder and a holder lid, in a region where a plurality of capillaries come into contact or close together during electrophoresis. Thereby, the heat generated in the capillary is dissipated, and the movement speed of the DNA in the gel is made constant. However, in this example, since the heat extraction member is already provided in the capillary array, it is possible to omit the troublesome work of sandwiching the capillary array in preparation for electrophoresis. Of course, the capillary head and the detection unit may be formed of the same member.

  FIG. 8 shows still another example of the capillary head. In the case of this example, the cross-sectional shape of the capillary head 17 is a quadrangular shape, and a plurality of capillaries are arranged at predetermined positions by the positioning member 29. In the example of FIG. 8, capillaries are arranged side by side on both surfaces of a plate-like positioning member 29. The positioning member 29 may be resin or glass.

  In this way, the capillary is fixed to the positioning member 29, temporarily fixed with a curing jig or the like, and then inserted into a mold as shown in FIG. 5, and an adhesive resin is loaded into the cavity. Once the resin is cured, the mold can be removed. Thus, a capillary head having a quadrangular cross section is manufactured. There is no problem even if the positioning member 29 remains in the capillary head 17 as it is. As shown in FIG. 8, the positioning member 29 is dimensioned so as not to be exposed from the detection unit side. Thereby, the occurrence of a leak at the interface between the positioning member 29 and the resin is avoided.

  If the capillary head is cylindrical and has a constant diameter, the number of capillaries must be reduced as the capillary diameter increases. However, in the example of FIG. 8, the diameter of the capillary can be increased.

It is a figure which shows the outline of an electrophoresis apparatus. It is a figure which shows the outline of a capillary array. It is a figure which shows a capillary head. It is a figure which shows the conventional capillary head. It is a figure which shows the manufacturing method (filling process) of a capillary head. It is a figure which shows the manufacturing method (vacuum defoaming process) of a capillary head. It is a figure which shows the other example of a capillary head. It is a figure which shows the further another example of a capillary head.

Explanation of symbols

  DESCRIPTION OF SYMBOLS 1 ... Capillary, 2 ... Sample container, 3 ... Sample tray, 4 ... Load header, 5 ... Detection part, 6 ... Laser light source, 7 ... Mirror, 8 ... Beam splitter, 9 ... Condensing lens, 10 ... Fluorescence, 11 ... Detection lens system, 12 ... CCD camera, 13 ... buffer solution (separation medium), 14 ... buffer solution container, 15 ... high voltage power source, 16 ... separator, 17 ... capillary head, 20 ... hollow electrode, 21 ... signal processing device, 22 ... cylindrical capillary receiver, 23 ... dispenser, 24 ... resin mold, 25 ... resin mold, 26 ... resin mold, 27 ... resin (adhesive), 28 ... vacuum defoamer, 29 ... positioning member, 30 ... cavity

Claims (10)

Ends of a plurality of capillaries having a capillary head which is and fixed are bonded together by a resin material, the resin material from a single resin material for forming the outer peripheral surface of and the capillary head filled between the capillary A capillary array characterized by comprising:   2. The capillary array according to claim 1, wherein the resin material covers the capillaries so that the plurality of capillaries are not exposed from the outer peripheral surface of the capillary head. 2. The capillary array according to claim 1, wherein the resin material is a thermosetting adhesive. Furthermore, it has a detection unit for arranging and fixing the plurality of capillaries in parallel, and when the detection unit is irradiated with excitation light, it emits fluorescence, which is signal light depending on the sample separated from the inside of the capillary. Configured as
Capillary array according to claim 1, wherein said resin material, characterized in that it extends to near the detecting section.   2. The capillary array according to claim 1, further comprising a plate-like positioning member, wherein the capillaries are arranged side by side on both sides of the positioning member. A capillary array including a plurality of capillaries, a high-voltage device that generates an electric field for electrophoresis of a sample in the capillary, a detection optical system that irradiates the capillary with excitation light and detects signal light from the capillary, In the capillary head of the capillary array, the ends of the plurality of capillaries are bonded and fixed to each other by a resin material , and the resin material fills the space between the capillaries and forms the outer peripheral surface of the capillary head A capillary electrophoresis apparatus comprising a single resin material. The capillary electrophoresis apparatus according to claim 6, wherein the resin material is a thermosetting adhesive. Insert a bundle of capillaries into the mold cavity,
Fill the mold cavity with resin ,
Curing the resin,
Remove the above mold,
A method for manufacturing a capillary array, in which a tip of a capillary to which a resin taken out from the mold is attached is cut so that an end face of the capillary is exposed from the cut surface. 9. The method for manufacturing a capillary array according to claim 8 , wherein the resin is vacuum degassed in a vacuum atmosphere after filling the cavity of the mold . 9. The method for manufacturing a capillary array according to claim 8, wherein the resin material is a thermosetting adhesive.

Images