Abstract:
The purpose of this invention has to do with being able to eliminate, using a small amount of an electrophoresis medium, air bubbles that get mixed in when loading an electrophoresis-medium container into a capillary electrophoresis device. This invention has to do with being able to simplify a positive-electrode-side channel in a capillary electrophoresis device by electrophoresing using only an electrophoresis medium on the positive-electrode side. This invention makes it possible to eliminate, easily and using a small amount of an electrophoresis medium, air bubbles that had become mixed in each time an electrophoresis-medium container was connected to the device. This invention also makes it easier to manage consumables and reduces the number thereof, making pre-electrophoresis preparation simple, and makes it possible to simplify and reduce the size of the device.

Description:
TECHNICAL FIELD 
       [0001]    The present invention relates to a technique for separating and analyzing the nucleic acid, protein, or the like through electrophoresis, and particularly to a capillary electrophoresis device. 
       BACKGROUND ART 
       [0002]    In recent years, a capillary electrophoresis device having a capillary filled with a phoresis medium such as polymer gel or polymer solution has been widely used. 
         [0003]    For example, the capillary electrophoresis device as disclosed in PTL 1 has conventionally been used. This device has features of having the higher heat dissipation property than the flat-type electrophoresis device and being capable of faster electrophoresis because the higher voltage can be applied to the sample. Other features are: the necessary amount of sample is small, filling with the separation medium can be automatically carried out, the sample injection can also be automatically carried out, and the like. Such a device is used in various separation and analysis measurements including the analysis of the nucleic acid and protein. 
         [0004]      FIG. 1  illustrates an example of the conventional capillary electrophoresis device. The capillary electrophoresis device includes a capillary  101 , a power source  102  that applies high voltage to both ends of the capillary  101 , an illumination system, which is not shown, including a laser light source and the like, a light-reception optical system, which is not shown, for detecting fluorescence, a thermostat tank  103  that controls the temperature of the capillary, a phoresis medium filling unit  104  that fills the capillary  101  with the phoresis medium, and a carrier, which is not shown, that carries a container containing the sample. 
         [0005]    An anode side of the capillary  101  is bonded to a flow channel of the phoresis medium filling unit  104 . The flow channel of the phoresis medium filling unit  104  is branched into two channels. One of the channels is bonded to a phoresis medium container  105  while the other is bonded to a buffer solution container A  106 . 
         [0006]    In the capillary electrophoresis device, the capillary  101  having an inner diameter of as small as 50 μm needs to be filled with the phoresis medium whose viscosity is several hundred times as high as that of water. In view of this, the phoresis medium filling unit  104  has a mechanism that can apply the pressure of several megapascals to one end of the channel for the phoresis medium. One example of this type of mechanism is a plunger pump  107 . In the case of  FIG. 1 , the plunger pump  107  is driven in a direction perpendicular to the paper surface. The driving of the plunger pump  107  changes the capacity of the channel to produce the pressure necessary for filling with the phoresis medium. 
         [0007]    In the analysis of the sample, high voltage is applied between opposite ends of the flow channel connected to the capillary  101  (between the buffer solution container A  106  and a buffer solution container B  109 ), thereby having a fluorescence-labelled sample such as DNA subjected to the electrophoresis in the phoresis medium of the capillary. Here, the charges used in the electrophoresis are mostly the charges in the buffer solution on the anode side. The sample differs in the phoresis speed depending on the molecular size and is detected in the detection unit  108 . 
         [0008]    Incidentally, the capillary electrophoresis device needs the exchange of the phoresis medium container  105  or the capillary  101 . In the exchange of these components, part of the flow channel is exposed to the air, in which case the air may be mixed into the flow channel. 
         [0009]    In the electrophoresis, voltage as high as several to several tens of kilovolts is applied between the opposite ends of the flow channel. Therefore, if there is an air bubble in the channel, the bubble may block the channel electrically. If the channel is electrically blocked, the high voltage difference is caused in the blocked portion, which results in the discharge. Depending on the magnitude of the discharge, the capillary electrophoresis device may be destroyed. 
         [0010]    In view of the above, it is necessary to remove the air bubble out of the flow channel before the start of the electrophoresis. 
         [0011]    For example, if there is an air bubble in the flow channel of the phoresis medium filling unit  104 , the connected flow channel between the phoresis medium filling unit  104  and the capillary  101  is closed and in this state, the phoresis medium is supplied to the buffer solution container A  106  in a manner that the medium returns at the branched path in the unit. Thus, the air bubble is removed from the flow channel section of the phoresis medium filling unit  104 . 
         [0012]    On the other hand, if there is an air bubble in the flow channel of the capillary  101 , the capillary  101  is filled with the phoresis medium whose amount is 1.5 times as large as the capacity of the capillary  101 . Here, the inner diameter of the capillary  101  is as small as 50 μm. Thus, the air bubble flows inside the capillary  101  together with the phoresis medium and is discharged from the other end of the capillary  101 . In other words, the air bubble can be removed from the inside of the capillary. 
         [0013]    PTL 2 discloses the mechanism for removing the air bubble from the flow channel of the phoresis medium filling unit  104  with a small amount of phoresis medium. Specifically, the structure is employed which forms the connected flow channel so that the phoresis medium flows from the bottom to the top in the connected portion between the phoresis medium filling unit  104  and the capillary  101 . 
       CITATION LIST 
     Patent Literatures 
       [0014]    PTL 1: Japanese Patent No. 2776208 
         [0015]    PTL 2: JP-A-2008-8621 
       SUMMARY OF INVENTION 
     Technical Problem 
       [0016]    In the case of the conventional device, since the phoresis medium filling unit  104  has the long flow channel, a large amount of phoresis medium is consumed in removing the air bubbles from the flow channel. 
         [0017]    In view of the above, an object of the present invention is to provide a capillary electrophoresis device with the phoresis medium filling unit  104  having the shorter flow channel so as to consume less phoresis medium in removing the air bubbles. 
       Solution to Problem 
       [0018]    In order to achieve the object, in the present invention, the electrophoresis is carried out with the charges necessary for the electrophoresis not from the buffer solution but from the phoresis medium, i.e., only with the electrophoresis medium in regard to the capillary anode end. 
       Advantageous Effects of Invention 
       [0019]    According to the present invention, the flow channel from the capillary connected portion to the container containing the buffer solution in the phoresis medium filling unit  104  can be omitted from the flow channel in the electrophoresis. This can suppress the consumption of the phoresis medium required for removing the air bubble out of the phoresis medium filling unit  104 . 
         [0020]    Furthermore, the buffer solution container  106  is no longer necessary, so that the number of consumption articles can be reduced, which can simplify the preparation before the analysis and the device. As a result, it becomes easier to operate the electrophoresis device. 
     
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         [0021]      FIG. 1  is a diagram illustrating a conventional example of a capillary electrophoresis device. 
           [0022]      FIG. 2  is a diagram schematically illustrating an entire structure of an electrophoresis device according to Example 1. 
           [0023]      FIG. 3  is an external diagram of a capillary array. 
           [0024]      FIG. 4A  is an external structure diagram of a phoresis medium container. 
           [0025]      FIG. 4B  is a sectional diagram of the phoresis medium container. 
           [0026]      FIG. 4C  is an external exploded structure diagram of the phoresis medium container. 
           [0027]      FIG. 4D  is a structure diagram of a component (lid) of the phoresis medium container. 
           [0028]      FIG. 4E  is a structure diagram of a component (middle lid) of the phoresis medium container. 
           [0029]      FIG. 4F  is a structure diagram of a component (rubber film) of the phoresis medium container. 
           [0030]      FIG. 4G  is a structure diagram of a component (main body portion) of the phoresis medium container. 
           [0031]      FIG. 5  is a diagram illustrating a structure of a resin flow channel block with the high electric insulating property used in Example 1. 
           [0032]      FIG. 6  is a diagram illustrating a process step of filling the capillary with the phoresis medium. 
           [0033]      FIG. 7A  is a diagram illustrating the flow channel in the resin flow channel block with the high electric insulating property according to a modified example. 
           [0034]      FIG. 7B  is a structure diagram in which a hollow pipe is used as an electrode according to a modified example. 
       
    
    
     DESCRIPTION OF EMBODIMENTS 
       [0035]    An embodiment of the present invention will be hereinafter described with reference to the drawings. Note that the device structure and the content of the process to be described below correspond to an example of the present invention and will not limit the content of the present invention. Embodiments can be combined with each other, or the embodiment can be combined with a known technique or replaced by a known technique to achieve another embodiment. 
         [0036]    A specific example of the device structure of the electrophoresis device suggested by the present inventor is hereinafter described. 
       Example 1 
     Summary of System 
       [0037]      FIG. 2  schematically illustrates the entire structure of an electrophoresis device according to Example 1. The electrophoresis device according to Example 1 includes a capillary array  202 , which is a single capillary  201  or a group of capillaries  201 , a laser light source  203  that irradiates a fluorescence-labelled sample in the capillary with a laser beam, a light-reception optical system  204  that detects the fluorescence emitted from the sample, a high-voltage application unit  205  that applies high voltage to the capillary, and a thermostat tank  206  that maintains the capillary at a constant temperature. 
         [0038]    The capillary array  202  is fixed to the thermostat tank  206 . Outside the thermostat tank  206  is provided a detection unit  207  that is used for testing the sample. In the drawing, the side provided with a buffer solution container  208  corresponds to the cathode end of the capillary array  202  and also to a sample suction end  209  through which the sample is injected. 
         [0039]    The sample suction end  209  is immersed in a buffer solution  210  in the buffer solution container  208  while the other (capillary head  302 ) is connected to a resin flow channel block  211  with the high electric insulating property. The resin flow channel block  211  is bonded to a hollow pipe  212  in addition to being bonded to the capillary array  202 . This hollow pipe  212  is connected to a phoresis medium container  214  containing a phoresis medium  213 . In the resin flow channel block  211 , an electrode  215  is also installed. 
       (Structure of Capillary Array) 
       [0040]      FIG. 3  is an external diagram of the capillary array  202 . Description is hereinafter made with reference to  FIG. 2  and  FIG. 3 . Each capillary  201  included in the capillary array  202  has an outer diameter of 0.1 to 0.7 mm and an inner diameter of 0.02 to 0.5 mm, and is coated with polyimide resin on the outside. The capillary  201  itself is a quartz pipe, and one capillary  201  or a plurality of (eight in this example) capillaries  202  is arranged to constitute the capillary array  202 . The capillary array  202  includes a load header  302  that takes the sample into the capillary  201  from the sample container containing a fluorescence-labelled DNA sample or the like by the electric operation, the detection unit  207  that arranges and fixes the capillaries  201  in the order of the sample number of the load header  302 , and a capillary head  301  binding and bonding the plural capillaries  201 . The sample suction end  209  projecting from the load header  302  is provided with a hollow electrode A  303  for applying the high voltage to the capillary  201 . The detection unit  301  includes an opening  304  through which the aligned and held capillary array  202  is irradiated with the laser beam from the side, and an opening  305  through which the light emitted from the capillary is extracted. 
         [0041]    In regard to the shape of the connected portion between the capillary head  301  of the capillary array  202  and the resin flow channel block  211 , a sleeve is attached to the round capillary head  301  binding the capillaries  201 , and the sleeve is deformed by fastening a setscrew, thereby filling the space. This enables the capillary head  301  to be fixed to the resin flow channel block  211 . 
       (Structure of Phoresis Medium Container) 
       [0042]      FIGS. 4(A) to 4(G)  illustrate the detailed structure of the phoresis medium container  214  used in the examples. FIG.  4 (A) is an external structure diagram of the phoresis medium container  214 ,  FIG. 4(B)  is a sectional structure diagram,  FIG. 4(C)  is an external exploded structure diagram, and  FIG. 4(D)  to  FIG. 4(G)  are external structure diagrams of the components. 
         [0043]    The phoresis medium container  214  includes a lid  401 , a middle lid  402 , a rubber film  403 , a main body portion  404 , and a plunger  405 . The rubber film  403  is fixed to the main body portion  404  with the middle lid  402  interposed therebetween when the lid  401  is rotated by a screw portion  406  provided for the lid  401 . On this occasion, the middle lid  402  is set so that a tapered portion A  407  of the rubber film  403  is not twisted by the rotation of the lid  401 . In this structure, as illustrated in  FIG. 00 , a protrusion  409  of the middle lid  402  is fitted to a groove  408  of the main body portion  404 , and when the lid  401  is fastened, the middle lid  402  transmits only the force in the vertical direction to the rubber film  403 . Further, the hollow pipe  212  is penetrated through a depressed portion  410  above the rubber film  403 . When the phoresis medium  214  is supplied by the plunger  405 , the tapered portion A  407  of the rubber film  403  is pressed by a tapered portion B  411  of the middle lid  402 , whereby the leakage from around the hollow pipe  212  is prevented during the penetration of the hollow pipe  212 . 
       (Structure of Resin Flow Channel Block  211 ) 
       [0044]      FIG. 5  illustrates the structure of the resin flow channel block  211  used in Example 1. The resin flow channel block  211  includes the hollow pipe  212  and the electrode  215 . 
         [0045]    Moreover, the flow channel in the resin flow channel block  211  has the smaller diameter than the air bubble generated in the flow channel so that when the capillary  201  is filled with the phoresis medium  213 , the air bubble in the flow channel in the resin flow channel block  211  can move for sure. In this example, the flow channel has an inner diameter of φ0.5 mm. 
       (Operation of Entire Device) 
       [0046]    Next, description is made of a series of process operations of the capillary electrophoresis device according to this example. The operation including the application of voltage for the electrophoresis in the capillary electrophoresis device to be described below is performed through a control unit (such as a computer), which is not shown. 
         [0047]      FIG. 6  illustrates a process step of filling the capillary array  202  with the phoresis medium  213 . 
         [0048]    First, the hollow pipe  212  is penetrated into the phoresis medium container  214 . After that, the plunger  405  of the phoresis medium container  214  is pressed to inject the phoresis medium  213  into the capillary  201 . On this occasion, the air bubbles mixed into the resin flow channel block  211  and the hollow pipe  212  go through the resin flow channel block  211  and moreover through the capillary  201  together with the phoresis medium  213  because the inner diameter of the capillary  201  is small, and then is discharged out of the sample suction end  209 . The amount of phoresis medium  213  injected into the capillary  201  is about 1.5 times as large as the inner capacity of the hollow pipe  212  and the rein flow channel block  211 +the inner capacity of the capillary array  202 . In the flow channel of the resin flow channel block  211  and the phoresis medium container  214 , the phoresis medium  213  with the charges necessary for one electrophoresis is left. In this example, the capillary array  202  has a length of 26 cm, 8 channels, and an inner diameter of φ50 μm. The amount of charges necessary for the electrophoresis is set to 87 mC from the experiments, and this amount is satisfied by approximately 60 μl of phoresis medium (POP-7™) manufactured by Life Technologies. When the phoresis medium  213  is filled, the sample suction end  209  is immersed in a waste tank (filled with pure water), which is not shown, carried by a carrier tray, which is not shown. 
         [0049]    After that, the sample suction end  209  is sank into the sample container, which is not shown, carried by the carrier tray, which is not shown, and then sank into the container containing pure water (for cleaning), which is not shown, and into the buffer solution container  208  in this order. After that, the electrophoresis is started in the state that the sample suction end  209  of the capillary array  202  is immersed in the buffer solution container  208 . 
         [0050]    As described above, the use of the electrophoresis device according to this example can easily remove the air bubbles, which are mixed in the setting of the phoresis medium container  214  and the capillary array  202 , with a small amount of phoresis medium  213  and can drastically reduce the running cost. Furthermore, the preparation for the electrophoresis can be facilitated as compared to the conventional device. 
       Example 2 
       [0051]    In the description above, the flow channel of the resin flow channel block  211  has the circular shape with the diameter smaller than that of the air bubble generated in the flow channel, so that the air bubble moves certainly and is not left in the flow channel. Even if the air bubble is mixed in the resin flow channel block  211 , a problem does not occur as long as the air bubble does not block the flow channel, i.e., the air bubble is not left in the place where the electrophoresis is interrupted. For example, the micro-channel may be provided for trapping the air bubble, which is well known as the flow channel for the micro-chemical chip like the flow channel illustrated in  FIG. 7A . In the micro-channel, the air bubble is easily formed on the smaller channel side due to the surface tension. Using this phenomenon, the air bubble mixed in the resin flow channel block  211  is moved toward the micro-channel, so that the wider flow channel can secure the bypass flow. Accordingly, the electrophoresis is not interrupted. 
         [0052]    In the above description, the resin flow channel block  211  includes the hollow pipe  212  and the electrode  215 . However, the hollow pipe may be used as the electrode and the electrode may be omitted as illustrated in  FIG. 7B . 
         [0053]    In the above description, the resin flow channel block  211  and the capillary head  301  are structured as separate parts. However, these parts may be an integrated component. 
       REFERENCE SIGNS LIST 
       [0000]    
       
           101  capillary 
           102  power source 
           103  thermostat tank 
           104  phoresis medium filling unit 
           105  phoresis medium container 
           106  buffer solution container A 
           107  plunger pump 
           108  detection unit 
           109  buffer solution container B 
           201  capillary 
           202  capillary array 
           203  laser light source 
           204  light-reception optical system 
           205  high-voltage application unit 
           206  thermostat tank 
           207  detection unit 
           208  buffer solution container 
           209  sample suction end 
           210  buffer solution 
           211  resin flow channel block 
           212  hollow pipe 
           213  phoresis medium 
           214  phoresis medium container 
           215  electrode 
           301  capillary head 
           302  load header 
           303  hollow electrode A 
           304  opening for delivering laser beam 
           305  opening for extracting emitted light 
           401  lid 
           402  middle lid 
           403  rubber film 
           404  main body portion 
           405  plunger 
           406  screw portion 
           407  tapered portion A 
           408  groove 
           409  protrusion 
           410  depressed portion 
           411  tapered portion B