JP6098471B2 - Capillary electrophoresis device - Google Patents

Description

  The present invention relates to a capillary electrophoresis apparatus for introducing a sample into a capillary tube for electrophoresis.

  Capillary electrophoresis (CE) is a method for rapidly separating and analyzing a very small amount of biological sample with high efficiency and high resolution. In addition to DNA fragment analysis and Sanger method sequence analysis, It is an indispensable technology in a wide range of application fields related to molecular structure analysis, antibody drugs, and low molecular ion components. As an apparatus for performing analysis using such capillary electrophoresis, various capillary electrophoresis apparatuses have been proposed (for example, see Patent Document 1 below).

  FIG. 9 is a schematic view showing a configuration example of a conventional capillary electrophoresis apparatus. The capillary electrophoresis apparatus includes a plurality of (for example, eight) capillary tubes 101, a detector 102, a temperature control unit 103, a polymer bottle 104, a polymer filling pump 105, a check valve 106, a buffer valve 107, and an anode side. A reservoir 108, a cathode side reservoir 109, a positive electrode 110, a negative electrode 111, a high voltage power source 112, a negative electrode 113 for sample injection, a syringe 114, a waste liquid bottle 115, an autosampler 116, a control unit 117, and the like are provided.

  One end of each capillary tube 101 constitutes a sample injection end 101a for injecting a sample. The other end of each capillary tube 101 is bound to form a binding end 101b. The detector 102 is provided on the binding end 101b side with respect to the plurality of capillary tubes 101, and extends from the sample injection end 101a to a detection window (not shown) formed on the capillary tube 101 in the detector 102. The length (separation effective length) is, for example, 50 cm.

  The plurality of capillary tubes 101 are accommodated in the temperature adjustment unit 103 in a curved state except for the vicinity of the sample injection end 101a and the vicinity of the binding end 101b. Thereby, during the analysis, the sample can be electrophoresed in the capillary tube 101 while the capillary tube 101 is heated.

  The temperature in the temperature control unit 103 is an important parameter with respect to resolution, migration speed, control of the denaturation state of single-stranded DNA, and the like. Therefore, the temperature control unit 103 accurately controls the temperature within a range from room temperature to about 70 ° C. using an air circulation oven or a system in which the capillary tube 101 is brought into contact with a thermally controlled surface. It is configured to be able to.

  The polymer bottle 104 contains a separated polymer. One end of a polymer suction pipe 121 is connected to the suction port of the polymer filling pump 105, and the other end of the polymer suction pipe 121 is inserted into the polymer bottle 104. A check valve 106 is interposed in the polymer suction pipe 121 so that the separated polymer can be prevented from flowing backward from the polymer filling pump 105 side to the polymer bottle 104 side.

  One end of the polymer supply pipe 122 is connected to the discharge port of the polymer filling pump 105, and the other end of the polymer supply pipe 122 faces the binding end 101 b of the capillary pipe 101. One end of the anode side connecting pipe 123 faces the binding end 101b of the capillary tube 101, and the other end of the anode side connecting pipe 123 is inserted into the anode side reservoir 108 in which the buffer solution is stored. Has been. A buffer valve 107 is interposed in the anode side connecting pipe 123. A positive electrode 110 is disposed in the anode side reservoir 108 while being immersed in a buffer solution.

  In this capillary electrophoresis apparatus, prior to analysis, each capillary tube 101, polymer supply tube 122, and anode side connection tube 123 are filled with a separation polymer. Specifically, first, the polymer filling pump 105 is driven to suck the separated polymer from the polymer bottle 104. At this time, by opening the buffer valve 107, the separated polymer is filled over the entire length of the polymer suction pipe 121, the polymer supply pipe 122, and the anode side connection pipe 123.

  The separated polymer once sucked by the polymer filling pump 105 does not flow back into the polymer bottle 104 by the check valve 106 and is hardly filled in each capillary tube 101 due to the back pressure. As a result, the inhaled separated polymer is discharged into the anode side reservoir 108.

  Next, the polymer filling pump 105 is driven again, whereby the separated polymer is sucked from the polymer bottle 104. At this time, by closing the buffer valve 107, the inside of each capillary tube 101 is replaced with a new separation polymer. The separated polymer discharged from the sample injection end 101a of each capillary tube 101 flows into the cathode side reservoir 109 in which the buffer solution is stored. In the cathode side reservoir 109, the negative electrode 111 is disposed so as to be immersed in the buffer solution.

  In general, the separation polymer includes a high viscosity polymer matrix and a DNA denaturant. Therefore, it is necessary to periodically replace the polymer after filling the polymer with a sealing member (not shown) trapped so that they do not stick or deposit in the polymer filling pump 105. Such an operation can be performed manually using a syringe 114 and a waste liquid bottle 115 respectively connected to the polymer filling pump 105.

  When a sample is injected into each capillary tube 101, the sample injection end 101 a of the capillary tube 101 is inserted into each of the plurality of sample containers 130 by operating the autosampler 116. A sample injection negative electrode 113 is provided at the sample injection end 101 a of each capillary tube 101. With the sample injection negative electrode 113 of each capillary tube 101 immersed in the sample in the sample container 130, a high voltage power source 112 is connected between the sample injection negative electrode 113 and the positive electrode 110 in the anode side reservoir 108. A sample can be injected into each capillary tube 101 by using and applying a voltage.

  The control unit 117 controls the voltage applied by the high-voltage power supply 112 at the time of sample injection, the time during which the voltage is applied, and the like. When the sample injection into each capillary tube 101 is completed, the sample injection end 101a of each capillary tube 101 is immersed in the buffer solution in the cathode side reservoir 109 by operating the autosampler 116. In this state, electrophoretic separation of the sample can be started by applying a voltage between the negative electrode 111 in the cathode side reservoir 109 and the positive electrode 110 in the anode side reservoir 108 using the high voltage power source 112. it can.

  The separated DNA fragments pass through the detector 102 in the order of the chain length, and are discharged into the buffer solution in the anode side reservoir 108. The detector 102 includes, for example, an excitation optical system that irradiates each capillary tube 101 with excitation light with a constant and uniform intensity, and a spectroscopic optical system that separates fluorescence collected from each capillary tube 101 side. . As the excitation light source, a laser diode (LD) or an LD excitation solid state laser can be used. Further, as the spectroscopic means in the spectroscopic optical system, a reflection type or transmission type diffraction grating, a prism, or the like can be used. As the light receiving element, a back-illuminated CCD area image sensor, an area scan CMOS image sensor, or the like can be used.

Japanese Patent No. 3417143

  In recent years, the next-generation sequencer has performed comprehensive analysis of the entire genome for the purpose of searching for target gene mutations of new molecular target drugs, searching for disease-related gene markers, and safety evaluation for clinical application of iPS cells. (NGS) is active. In connection with this, it is thought that the sequence by the Sanger method using a capillary electrophoresis apparatus will be divided as a means for complementing NGS and a routine examination of individual genotypes.

  For example, known genetic mutation analysis related to cancer and genetic diseases, or phylogenetic analysis of microorganisms and pathogens is required to analyze a relatively short DNA base length more quickly and with high throughput. Further, in the routine inspection field, simple operability and maintenance are required in addition to processing capability.

  In such a flow, in the conventional capillary electrophoresis apparatus as described above, for example, an effective separation length is 50 cm, and electrophoresis is performed under standard conditions using a commercially available POP7 (manufactured by Applied Biosystems) as a separation polymer. 850 base sequencing can be performed in 125 minutes on average. In the high-speed mode, 500 base sequences can be decoded in about 40 minutes. However, even in such a case, the upper limit is to analyze about 280 samples per day using eight capillary tubes, and the capillary electricity that enables analysis at higher speed and higher throughput. An electrophoresis apparatus is desired.

  The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a capillary electrophoresis apparatus that enables analysis at higher speed and higher throughput.

A capillary electrophoresis apparatus according to the present invention includes a sample inhalation tube for inhaling a sample, a pump driven to inhale the sample into the sample inhalation tube, and a linearly arranged in the horizontal direction. A capillary tube into which the sample sucked into the tube is introduced, a cathode side end of the capillary tube, the sample suction tube, and the pump are connected to each other, and a cathode side block in which an internal space is formed at the junction thereof; It is provided with.

According to such a configuration, the sample can be sucked into the sample suction tube using the pump, and the sample can be introduced into the capillary tube linearly arranged in the horizontal direction. Thus, since the sample is sucked through the sample suction tube, it is not necessary to bend the capillary tube and immerse the end portion in the sample. As a result, it is not necessary to lengthen the capillary tube in order to compensate for the resolution that is impaired by bending the capillary tube, and the capillary tube can be shortened, enabling faster and higher throughput analysis. can do. Further, since it is not necessary to immerse the electrode for each sample container at the time of electrical injection, the electrode is not contaminated with the sample, and there is no fear that the sample remains between the electrode and the capillary tube.
Further, the sample sucked into the sample suction tube using the pump can be drawn into the internal space of the cathode side block, and the sample can be introduced from the internal space to the cathode side end of the capillary tube. As a result, the sample can be reliably and stably introduced into the capillary tube through the cathode side block.

  A plurality of the capillary tubes and a plurality of sample suction tubes corresponding to the capillary tubes are connected to the cathode side block, and a plurality of the internal spaces corresponding to the capillary tubes are formed. Good.

  According to such a configuration, the samples are sucked into the plurality of sample suction tubes at once, and the samples are drawn into the separate internal spaces in the cathode side block, and then the plurality of capillary tubes corresponding to the respective internal spaces. A sample can be introduced into the cathode side end. Thereby, since it is possible to introduce a sample into a plurality of short capillary tubes at a time for electrophoresis, it is possible to perform analysis at higher speed and higher throughput.

  The capillary electrophoresis apparatus may further include a cathode-side reservoir having a buffer solution storage unit capable of storing a buffer solution, and a negative electrode immersed in the buffer solution in the cathode-side reservoir.

  According to such a configuration, after the sample is sucked into the internal space of the cathode side block via the sample suction tube, the sample suction tube is immersed in the buffer solution in the cathode side reservoir, and the negative electrode in the cathode side reservoir is obtained. The sample can be easily introduced into the end of the capillary tube on the cathode side simply by applying a voltage.

  The capillary electrophoresis apparatus has a buffer liquid storage section that can store a buffer liquid, and the buffer liquid storage section communicates with the internal space of the cathode side block; And a negative electrode immersed in the buffer solution. In this case, when the sample is sucked into the internal space of the cathode side block via the sample suction tube, the buffer solution in the cathode side reservoir is also sucked, and then the negative electrode in the cathode side reservoir is charged with voltage. May be applied to the capillary tube from the cathode side end.

  According to such a configuration, when the sample is sucked into the internal space of the cathode side block via the sample suction tube, the buffer liquid in the cathode side reservoir can also be sucked. If a voltage is subsequently applied to the negative electrode in the cathode reservoir, the sample can be introduced into the capillary tube from the cathode end, and the time for introducing the sample can be shortened. In addition, analysis at high throughput can be made possible. In addition, if the channel width of the sample suction channel is sufficiently smaller than the channel width of the buffer communication channel, the entire sample at the end of the capillary can be introduced, so that the quantitativeness can be improved.

  The capillary electrophoresis apparatus can store a buffer solution in a buffer solution storage unit, and has an anode side reservoir block in which an internal space communicating with the anode side end of the capillary tube is formed, and the anode side A polymer filling mechanism that pressurizes and fills the separated polymer from the end on the anode side into the capillary tube through the internal space of the reservoir block may be further provided.

  According to such a configuration, it is possible to fill the capillary tube with the separation polymer from the anode side end portion through the internal space of the anode side reservoir block. After filling the separation polymer, the buffer solution in the anode reservoir block and the separation polymer are in contact with each other, so that the electrophoresis can be performed as it is.

  The polymer filling mechanism may include a polymer filling needle inserted into the anode reservoir block. In this case, a connection port into which the tip of the polymer filling needle can be inserted and sealed may be formed at a boundary portion between the buffer liquid storage portion and the internal space in the anode side reservoir block.

  According to such a configuration, the tip of the polymer filling needle is inserted into the connection port of the anode side reservoir block, and the capillary filling tube is separated from the tip of the polymer filling needle through the internal space of the anode side reservoir block. The polymer can be filled. Thereby, the separated polymer can be easily injected under pressure into the capillary tube without performing the work of removing bubbles.

  The capillary electrophoresis apparatus may further include a cleaning water supply mechanism that supplies cleaning water from the anode side end to the capillary tube via an internal space of the anode side reservoir block.

  According to such a configuration, the inside of the capillary tube can be easily cleaned simply by supplying cleaning water from the anode side end to the capillary tube through the internal space of the anode side reservoir block. Furthermore, the internal space of the anode-side reservoir block and the buffer liquid storage portion communicating with the internal space can be cleaned with cleaning water, and after cleaning, the anode-side end of the capillary tube is in contact with the cleaning water. Since it can be made into a state, it can prevent that the said anode side edge part dries. Further, by performing such a process automatically, the maintainability can be improved.

  The washing water supply mechanism may include a washing water supply needle whose tip can be inserted into the connection port.

  According to such a configuration, the tip of the cleaning water supply needle is inserted into the connection port of the anode side reservoir block, and the capillary tube is inserted from the tip of the cleaning water supply needle through the internal space of the anode side reservoir block. Wash water can be supplied. As a result, the washing water can be easily injected under pressure into the capillary tube.

  The capillary electrophoresis apparatus may further include a temperature adjustment unit that is provided in a straight line along a direction in which the capillary tube extends, and that can adjust the temperature by accommodating the capillary tube therein. In this case, the capillary tube may be taken out of the temperature control unit by moving the temperature control unit in a direction intersecting the direction in which the capillary tube extends.

  According to such a configuration, the capillary tube arranged in a straight line in the horizontal direction is arranged so that almost the entire region excluding the fittings at both ends of the capillary tube is removed by the temperature control unit provided in a straight line along the same direction. The temperature can be adjusted uniformly. In addition, since the capillary tube can be easily taken out of the temperature control unit simply by moving the temperature control unit in a direction intersecting the direction in which the capillary tube extends, the maintainability can be improved.

  According to the present invention, it is not necessary to lengthen the capillary tube in order to compensate for the resolution that is impaired by bending the capillary tube, and the capillary tube can be formed short, so that analysis can be performed at higher speed and higher throughput. Can be made possible. Further, since the capillary tube is not directly inserted into the sample container, the electrode is not contaminated with the sample. Since the temperature of the entire region is uniformly controlled except for the connecting portions at both ends of the capillary tube, the maximum separation performance can be obtained even with a short capillary tube. Furthermore, since the inside of the capillary tube can be easily washed with water after the analysis is completed, it is excellent in operability and maintainability.

It is the perspective view which showed the structural example of the capillary electrophoresis apparatus which concerns on one Embodiment of this invention. It is the schematic of the capillary electrophoresis apparatus of FIG. It is the top view which showed the structural example of the capillary assembly. It is sectional drawing which showed the aspect at the time of attaching / detaching the polymer filling needle with respect to the connection port of an anode side reservoir block, and has shown the state by which the polymer filling needle was connected to the connection port. It is sectional drawing which showed the aspect at the time of attaching / detaching the polymer filling needle with respect to the connection port of an anode side reservoir block, and has shown the state by which the connection port was open | released. It is the perspective view which showed the aspect at the time of introduce | transducing a sample into each capillary tube. It is the figure which showed the structural example of the detection part. It is the schematic which showed the structural example of the capillary electrophoresis apparatus which concerns on another embodiment of this invention. FIG. 8 is a perspective view showing an aspect when a sample is introduced into each capillary tube in the capillary electrophoresis apparatus of FIG. 7. It is the schematic which showed the structural example of the conventional capillary electrophoresis apparatus.

  FIG. 1 is a perspective view showing a configuration example of a capillary electrophoresis apparatus according to an embodiment of the present invention. FIG. 2 is a schematic diagram of the capillary electrophoresis apparatus of FIG.

  This capillary electrophoresis apparatus includes a plurality of (for example, eight) capillary tubes 1, a cathode side block 2, an anode side reservoir block 3, a sample suction tube 4, a pump 5, a cathode side reservoir 6, a negative electrode 7, a positive electrode. Electrode 8, polymer filling needle 9, polymer cartridge 10, washing water supply needle 11, rinse port 12, washing water tank 13, washing water pump 14, drive mechanism 15, electromagnetic switching valve 16, opening / closing valve 17, drain 18, An autosampler 19 and a temperature control unit 20 are provided.

  The plurality of capillary tubes 1 are arranged in a straight line in the horizontal direction in a bundled state. One end portion (cathode side end portion) of each capillary tube 1 is connected to the cathode side block 2, and the other end portion (anode side end portion) is connected to the anode side reservoir block 3. The same number of sample suction tubes 4 as the capillary tubes 1 are provided, and one end of each is connected to the cathode side block 2. The pump 5 is also connected to the cathode side block 2 via a pipe 51.

  Each sample inhalation tube 4 is for inhaling a sample. By driving a pump 5 connected to the sample inhalation tube 4 via a cathode side block 2 and a pipe 51, each sample inhalation tube 4 The sample can be inhaled. The sample sucked into each sample suction tube 4 is introduced into each capillary tube 1 from the cathode side end of each capillary tube 1 connected via the cathode side block 2.

  The cathode-side reservoir 6 has a buffer solution reservoir 61 that can store a buffer solution, and is arranged in a state where the negative electrode 7 is immersed in the buffer solution in the buffer solution reservoir 61. The anode-side reservoir block 3 has a buffer liquid storage part 31 that can store a buffer liquid, and is arranged in a state in which the positive electrode 8 is immersed in the buffer liquid in the buffer liquid storage part 31. ing. Both the cathode side reservoir 6 and the anode side reservoir block 3 are formed of an insulating material such as a resin.

  The polymer filling needle 9 is connected to a polymer cartridge 10. With the polymer filling needle 9 inserted into the anode reservoir block 3, the separated polymer in the polymer cartridge 10 is supplied to the polymer filling needle 9, so that the tip of the polymer filling needle 9 is moved to the anode side. The separation polymer is filled into the capillary tube 1 through the reservoir block 3. The polymer filling needle 9 and the polymer cartridge 10 constitute a polymer filling mechanism for pressure-filling the capillary tube 1 with the separated polymer from the anode side end.

  The washing water supply needle 11 is connected to the pump 5 via a pipe 52. The rinse water stored in the washing water tank 13 is supplied to the rinse port 12 by the washing water pump 14. By driving the pump 5 with the cleaning water supply needle 11 inserted into the rinse port 12, the cleaning water can be sucked into the cleaning water supply needle 11 and the pipe 52.

  Thereafter, the pump 5 is driven in a state where the cleaning water supply needle 11 is inserted into the anode-side reservoir block 3, so that the cleaning water supply needle 11 and the cleaning water in the pipe 52 are supplied to the cleaning water supply. The capillary 11 is supplied from the tip of the needle 11 through the anode reservoir block 3. The cleaning water supply needle 11, the rinse port 12, the cleaning water tank 13, and the cleaning water pump 14 constitute a cleaning water supply mechanism that supplies cleaning water to the capillary tube 1 from the anode side end.

  The drive mechanism 15 is driven when the polymer filling needle 9 or the washing water supply needle 11 is moved. With this drive mechanism 15, either the polymer filling needle 9 or the cleaning water supply needle 11 can be inserted into the anode reservoir block 3 or inserted into the rinse port 12.

  The pipe 51 and the pipe 52 merge at the electromagnetic switching valve 16 and are connected to the pump 5 through a common pipe 53. By switching the electromagnetic switching valve 16, the pump 5 is switched between the state in which the pump 5 communicates with the cathode side block 2 through the pipe 51 and the state in which the pump 5 communicates with the cleaning water supply needle 11 through the pipe 52. Can do. The opening / closing valve 17 is provided in the pipe 53.

  The drain 18 is for accumulating waste liquid, and is provided side by side with the cathode side reservoir 6 in this example. The cathode side reservoir 6 and the drain 18 are held by the autosampler 19 together with the plurality of sample containers 30. Therefore, the sample suction tube 4 can be inserted into any of the sample container 30, the cathode side reservoir 6, and the drain 18 by moving the autosampler 19 in the horizontal direction and the vertical direction.

  The temperature control unit 20 is provided in a straight line along the direction in which the capillary tube 1 extends. In this example, the temperature control unit 20 is formed in a U-shaped cross section with one side open, and is opened by moving the temperature control unit 20 in a horizontal direction perpendicular to the direction in which the capillary tube 1 extends. The capillary tube 1 can be accommodated inside the temperature control unit 20 or the capillary tube 1 can be taken out of the temperature control unit 20 via the one side surface.

  However, the temperature control unit 20 is not limited to a U-shaped cross section, and may have another shape as long as the capillary tube 1 can be accommodated therein. The direction in which the temperature adjustment unit 20 is moved is not limited to the horizontal direction orthogonal to the direction in which the capillary tube 1 extends, and may be another direction that intersects the direction in which the capillary tube 1 extends.

  Hereinafter, the specific structure and operation of the capillary electrophoresis apparatus according to the present embodiment will be described in detail with reference to FIGS. 1 and 2 as well as other drawings as appropriate. The operation of the capillary electrophoresis apparatus is controlled by, for example, a control unit (not shown) including a CPU (Central Processing Unit), and the operation described below is executed by the CPU executing a program. Can be performed automatically.

<Attachment, temperature control and cleaning of capillary assembly>
FIG. 3 is a plan view showing a configuration example of the capillary assembly. In the present embodiment, a plurality of (for example, eight) capillary tubes 1 are combined at the anode side end portion to constitute a capillary assembly. Each capillary tube 1 is arranged in a straight line so as to extend in parallel with a predetermined distance from each other.

  The plurality of capillary tubes 1 are attached to a mail nut 1a made of PEEK, for example, with their anode side ends bound together. The mail nut 1 a constitutes an anode side attachment portion for attaching the anode side end portion of the capillary tube 1 to the anode side reservoir block 3. As shown in FIG. 2, an attachment hole 3a for attaching the mail nut 1a is formed on the side surface of the anode reservoir block 3, and the attachment can be done by screwing the mail nut 1a into the attachment hole 3a.

  For example, PEEK fittings 1b are attached to the cathode side ends of the plurality of capillary tubes 1. The fittings 1b attached to the cathode side end portions of the respective capillary tubes 1 are connected to each other. In this example, the fittings 1b are configured as eight continuous fittings. The tip of each fitting 1b is formed by a conical tapered surface. As shown in FIG. 2, a plurality of attachment holes 2a for attaching the fittings 1b are formed on the side surface of the cathode side block 2, and can be attached by press fitting the fittings 1b into the attachment holes 2a. .

  The end on the anode side of each capillary tube 1 is fixed to the mail nut 1a so as to protrude slightly from the mail nut 1a. Similarly, the cathode side end of each capillary tube 1 is fixed to each fitting 1b in a state of slightly protruding from each fitting 1b. The protruding amount of the anode side end of each capillary tube 1 with respect to the mail nut 1a is preferably the same, and the protruding amount of the cathode side end of each capillary tube 1 with respect to each fitting 1b is preferably the same.

  A detection window 1c for allowing light to enter at the time of detection is provided on the anode side of the center of each capillary tube 1. The length (effective separation length) from the cathode side end of each capillary tube 1 to the detection window 1c is, for example, 10 cm or less. In this example, the inner diameter of each capillary tube 1 is set to 50 μm and the effective separation length is set to 85 mm. However, the shape of each capillary tube 1 is not limited to such a shape. Further, the number of capillary tubes 1 is not limited to eight, but may be seven or less, nine or more, and may be one instead of plural.

  As shown in FIG. 2, the anode-side reservoir block 3 is formed with an internal space 3 b that communicates between the buffer liquid storage portion 31 and the anode-side end of each capillary tube 1. The internal space 3 b is formed in an L-shaped cross section, and has one end communicating with the mounting hole 3 a and the other end (upper end) communicating with the bottom of the buffer liquid storage unit 31. A conical connection port 3c, for example, is formed at the bottom of the buffer liquid storage section 31 at the boundary between the buffer liquid storage section 31 and the internal space 3b. The tip of the polymer filling needle 9 and the tip of the washing water supply needle 11 can be inserted (press-fitted) into the connection port 3c.

  In the cathode side block 2, an internal space 2 b is formed at the cathode side end of each capillary tube 1, and at the junction of each sample suction tube 4 and pump 5 (pipe 51). A plurality of the internal spaces 2b are provided in association with each capillary tube 1 and are formed so as to extend in the vertical direction. The lower end portion communicates with each sample suction tube 4, and the upper end portion joins the piping. 51 communicates. The attachment hole 2a of each capillary tube 1 communicates in the middle of each internal space 2b extending in the vertical direction, and the cathode side end of each capillary tube 1 protruding from the tip of each fitting 1b attached to each attachment hole 2a. The portion is slightly overhanging in each internal space 2b (see FIG. 2).

  Each capillary tube 1 is temperature-controlled by the temperature adjustment unit 20 at a portion between the mail nut 1a and the fitting 1b. The temperature control unit 20 is provided with a heater (not shown), and the capillary tube 1 accommodated in the temperature control unit 20 can be temperature controlled by driving the heater.

  In this embodiment, the capillary tube 1 arranged in a straight line in the horizontal direction is removed from the male nut 1a and the fitting 1b at both ends of the capillary tube by the temperature control unit 20 provided in a straight line along the same direction. The temperature can be controlled uniformly over almost the entire area. Further, since the capillary tube 1 can be easily taken out of the temperature control unit 20 simply by moving the temperature control unit 20 in a direction intersecting the direction in which the capillary tube 1 extends, the maintainability is improved. Can do.

  Each capillary tube 1 is washed with water by the following procedure. First, the cleaning water pump 14 is driven, so that the cleaning water in the cleaning water tank 13 is supplied to the rinse port 12, and a part thereof overflows to the outside. Then, the cleaning water supply needle 11 is immersed in the cleaning water in the rinse port 12 by the drive mechanism 15, and the pump 5 is driven in a state where the piping 52 and the piping 53 are communicated by the electromagnetic switching valve 16. Is sucked into the pump 5 side.

  Thereafter, the electromagnetic switching valve 16 is closed and the pump 5 is driven in a state where the opening / closing valve 17 is opened, thereby draining the washing water to the outside. By repeating such an operation, bubbles in the flow path from the washing water supply needle 11 to the pump 5 can be completely removed.

  Next, the cleaning water supply needle 11 is press-fitted into the connection port 3 c of the anode reservoir block 3 by the drive mechanism 15 and the pump 5 is driven, whereby each capillary tube is passed through the internal space 3 b of the anode reservoir block 3. 1 is supplied with cleaning water from the anode side end. The washing water supplied into each capillary tube 1 is led from the cathode side end portion to each sample suction tube 4 via the cathode side block 2 and drained from each sample suction tube 4 into the drain 18.

<Polymer assembly filling polymer>
Each capillary tube 1 is filled with the separation polymer by the following procedure. First, the buffer solution is stored in the buffer solution storage unit 31 of the anode reservoir block 3 and the buffer solution storage unit 61 of the cathode reservoir 6 respectively. Then, the polymer filling needle 9 is inserted into the rinse port 12, and the separated polymer is supplied from the polymer cartridge 10 to the polymer filling needle 9. As a result, a predetermined amount of the separated polymer is discharged from the tip of the polymer filling needle 9.

  Thereafter, the polymer filling needle 9 is press-fitted into the connection port 3c of the anode side reservoir block 3 by the drive mechanism 15 and the separated polymer is supplied from the polymer cartridge 10 to the polymer filling needle 9, thereby the inside of the anode side reservoir block 3 being inside. Each capillary tube 1 is filled with the separation polymer from the end on the anode side through the space 3b. At this time, the separated polymer flowing out from the cathode side end portion of each capillary tube 1 is guided to each sample suction tube 4 through the cathode side block 2 and discharged from the sample suction tube 4 into the drain 18.

  After the separated polymer is filled in each capillary tube 1 for a predetermined time, the polymer filling needle 9 is pulled out from the connection port 3c of the anode side reservoir block 3 by the drive mechanism 15, and the connection port 3c is opened. Thereafter, the pump 5 is driven in a state where the pipe 51 and the pipe 53 are communicated with each other by the electromagnetic switching valve 48, whereby the cleaning water in the drain 18 is sucked into the pump 5 side.

  Thereby, the separation polymer accumulated in the internal space 2b of the cathode side block 2 can be removed. However, the configuration is not limited to such a configuration, and when the separation polymer is filled in each capillary tube 1, the pump 5 is driven at the same time and the cleaning water in the drain 18 is sucked into the pump 5 side. There may be.

  4A and 4B are cross-sectional views showing a mode when the polymer filling needle 9 is attached to and detached from the connection port 3c of the anode side reservoir block 3, and FIG. 4A shows the polymer filling needle 9 at the connection port 3c. FIG. 4B shows a state in which the connection port 3c is opened.

  As shown in FIG. 4A, when the polymer filling needle 9 is connected to the connection port 3c, the tapered tip end portion of the polymer filling needle 9 is press-fitted into the tapered connection port 3c and the internal space. It will be in the state which faced 3b. By supplying the separation polymer from the polymer filling needle 9 in this state, the separation polymer can be supplied to the internal space 3 b without flowing into the buffer liquid storage portion 31.

  Thereafter, as shown in FIG. 4B, when the polymer filling needle 9 is removed from the connection port 3c of the anode reservoir block 3 and the connection port 3c is opened, as shown by hatching in the drawing, the internal space 3b In this state, the separated polymer remains.

  In the present embodiment, the capillary tube 1 can be filled with the separation polymer from the anode side end via the internal space 3 b of the anode side reservoir block 3. After filling the separation polymer, the buffer solution in the anode reservoir block 3 and the separation polymer are in contact with each other, so that the electrophoresis can be performed as it is.

  In the present embodiment, the tip of the polymer filling needle 9 is inserted into the connection port 3 c of the anode reservoir block 3, and from the tip of the polymer filling needle 9 through the internal space 3 b of the anode reservoir block 3. The capillary tube 1 can be filled with the separation polymer. Thereby, the separation polymer can be easily injected under pressure into the capillary tube 1 without performing the operation of removing bubbles.

<Sample introduction to capillary assembly>
A sample is introduced into each capillary tube 1 by the following procedure. First, the piping 51 and the piping 53 are communicated by the electromagnetic switching valve 16, the drain 18 is moved downward by the autosampler 19, and the sample suction pipe 4 is separated from the drain 18. By driving the pump 5 in this state, an air gap (for example, 5 μL) is sucked into the internal space 2 b of the cathode side block 2.

  FIG. 5 is a perspective view showing an aspect when a sample is introduced into each capillary tube 1. After inhalation of the air gap, each sample suction tube 4 is immersed in the sample in the sample container 30 by the autosampler 19, and the pump 5 is driven in this state, whereby the cathode side block 2 is passed through each sample suction tube 4. A predetermined amount of sample is sucked into the internal space 2b. At this time, for example, by inhaling the sample for 5 seconds at 1 μL / sec, 5 μL of the sample can be inhaled into the internal space 2 b of the cathode side block 2.

  Then, each sample suction tube 4 is immersed in the buffer solution in the cathode-side reservoir 6 by the autosampler 19, and a voltage is applied between the negative electrode 7 and the positive electrode 8, so that each capillary tube 1 is discharged from the internal space 2 b. A sample is introduced into the cathode side end of the sample. At this time, as a sample introduction voltage, for example, by applying a voltage between the negative electrode 7 and the positive electrode 8 only for 10 seconds at an electric field strength of 230 V / cm, the sample is satisfactorily applied to each capillary tube 1 from the cathode side end. Can be introduced.

  After the introduction of the sample, the applied voltage between the negative electrode 7 and the positive electrode 8 is stopped, and at the same time, the cathode-side reservoir 6 is moved downward by the autosampler 19 to separate the sample suction tube 4 from the cathode-side reservoir 6. By driving the pump 5 in this state, an air gap (for example, 10 μL) is sucked into the internal space 2 b of the cathode side block 2.

  Next, each sample suction tube 4 is immersed in the buffer solution in the cathode-side reservoir 6 by the autosampler 19 and the pump 5 is driven to suck the buffer solution by a predetermined amount. At this time, for example, by sucking the buffer solution at 1 μL / sec for 10 seconds, 10 μL of the buffer solution can be sucked into the internal space 2 b of the cathode side block 2.

  In the present embodiment, a sample can be sucked into the sample suction pipe 4 using the pump 5, and the sample can be introduced into the capillary tube 1 arranged linearly in the horizontal direction. Since the sample is sucked through the sample suction tube 4 in this way, it is not necessary to bend the capillary tube 1 and immerse the end portion in the sample. As a result, it is not necessary to lengthen the capillary tube 1 in order to compensate for the resolution that is impaired by bending the capillary tube 1, and the capillary tube 1 can be formed short, so that analysis can be performed at higher speed and higher throughput. Can be made possible.

  For example, when each capillary tube 1 has an inner diameter of 50 μm and an effective separation length of 85 mm, the capillary electrophoresis apparatus according to this embodiment can decode a length of 300 bases in about 10 minutes. When eight capillary tubes 1 are used in a 15-minute cycle, it is possible to analyze 32 samples in one hour and 768 samples in 24 hours. The conventional capillary electrophoresis apparatus shown in FIG. Compared with the upper limit of about 280 samples), analysis at a high throughput is possible.

  Furthermore, in this embodiment, the sample sucked into the sample suction tube 4 using the pump 5 is drawn into the internal space 2b of the cathode side block 2, and the sample is introduced from the internal space 2b to the cathode side end of the capillary tube 1. can do. Thereby, the sample can be reliably and stably introduced into the capillary tube 1 through the cathode side block 2.

  In particular, since a plurality of internal spaces 2 b corresponding to each capillary tube 1 are formed in the cathode side block 2, samples are sucked into the plurality of sample suction tubes 4 at a time, and these samples are taken into the cathode side block 2. After being drawn into the separate internal spaces 2b, the sample can be introduced into the cathode side ends of the plurality of capillary tubes 1 corresponding to the internal spaces 2b. Thereby, since it is possible to introduce a sample into a plurality of short capillary tubes 1 at a time and perform electrophoresis, it is possible to perform analysis at higher speed and higher throughput.

  In the present embodiment, the sample is sucked into the internal space 2 b of the cathode side block 2 through the sample suction tube 4, and then the sample suction tube 4 is immersed in the buffer solution in the cathode side reservoir 6. The sample can be easily introduced into the cathode side end of the capillary tube 1 simply by applying a voltage to the negative electrode 7 in the tube 6.

  The anode side end of the capillary tube 1 is always in contact with the buffer liquid stored in the buffer liquid storage part 31 of the anode side reservoir block 3. Therefore, when introducing a sample into the capillary tube 1 or performing electrophoresis, it is only necessary to apply a voltage to the positive electrode 8 immersed in the buffer solution in the anode side reservoir block 3. There is no need to separately perform an operation for immersing the anode side end of the electrode in the buffer solution.

<Electrophoresis, detection>
The sample introduced into each capillary tube 1 is subjected to electrophoretic separation by applying a voltage between the negative electrode 7 and the positive electrode 8. At this time, as a separation voltage, for example, by applying a voltage between the negative electrode 7 and the positive electrode 8 at an electric field strength of 230 V / cm, the sample in each capillary tube 1 can be favorably electrophoresed. The DNA fragments separated by electrophoresis are detected by the detection unit 21 with fluorescence.

  FIG. 6 is a diagram illustrating a configuration example of the detection unit 21. In this example, laser light having a wavelength of 505 nm is used as excitation light, and uniform excitation light is irradiated from the detection window 1c to each capillary tube 1 through the Powell lens 21a. The light (fluorescence) from each capillary tube 1 is converted into parallel light by the collimator lens 21b, passes through the filter 21c, and then enters the condenser lens 21d.

  The light condensed by the condenser lens 21d passes through the slit formed in the slit plate 21e, is reflected by the mirror 21f, and then enters the diffraction grating 21g. The diffraction grating 21g is, for example, a reflection type toroidal diffraction grating, and the light incident on the diffraction grating 21g is dispersed as wavelength dispersion and position dispersion of each capillary tube 1 and is received by the light receiving unit 21h. The light receiving unit 21h can be configured by, for example, an area scan CMOS image sensor. A signal (fluorescence signal) output from the light receiving unit 21h is sent to a personal computer for data analysis.

<End of analysis, washing>
After the analysis is completed, the cathode side block 2 and each capillary tube 1 are automatically cleaned by the following procedure. First, in a state where the piping 51 and the piping 53 are in communication with each other by the electromagnetic switching valve 16, each sample suction tube 4 is immersed in the cleaning water in the drain 18 by the autosampler 19, and the pump 5 is driven. As a result, the cleaning water in the drain 18 is sent to the pump 5 side through each sample suction tube 4 and the internal space 2 b of the cathode side block 2, and is drained to the outside through the open / close valve 17. .

  Thereafter, the polymer filling needle 9 is press-fitted into the connection port 3c of the anode side reservoir block 3 by the drive mechanism 15 and the separated polymer is supplied from the polymer cartridge 10 to the polymer filling needle 9, thereby the inside of the anode side reservoir block 3 being Each capillary tube 1 is filled with the separation polymer from the end on the anode side through the space 3b. At this time, the separated polymer flowing out from the cathode side end of each capillary tube 1 is sent to the pump 5 side through the internal space 2b together with the wash water sucked from each sample suction tube 4, and opened and closed. The liquid is discharged to the outside through the valve 17.

  After the separated polymers are filled into the capillary tubes 1 for a predetermined time, the polymer filling needle 9 is removed from the connection port 3c of the anode side reservoir block 3 by the driving mechanism 15, and the process proceeds to the next sample introduction process. When the series of analysis programs is finished, the pump 5 is immersed in the cleaning water supply needle 11 in the cleaning water in the rinse port 12 by the drive mechanism 15 and the piping 52 and the piping 53 are communicated by the electromagnetic switching valve 16. Is driven to suck the cleaning water into the pump 5 side.

  Thereafter, the drive mechanism 15 presses the cleaning water supply needle 11 into the connection port 3 c of the anode reservoir block 3 and drives the pump 5 to drive each capillary tube through the internal space 3 b of the anode reservoir block 3. 1 is supplied with cleaning water from the anode side end. The washing water supplied into each capillary tube 1 is led from the cathode side end portion to each sample suction tube 4 via the cathode side block 2 and drained from each sample suction tube 4 into the drain 18.

  The buffer liquid in the buffer liquid reservoir 31 of the anode side reservoir block 3 is sucked by the cleaning water supply needle 11 and then drained into the rinse port 12, and the outer surface of the cleaning water supply needle 11 is also rinse port 12. Washed in. The polymer filling needle 9 is also washed in the rinse port 12.

  In the present embodiment, the inside of the capillary tube 1 can be easily cleaned simply by supplying cleaning water from the anode side end to the capillary tube 1 via the internal space 3 b of the anode side reservoir block 3. Furthermore, the internal space 3b of the anode side reservoir block 3 and the buffer liquid storage part 31 communicating with the internal space 3b can also be washed with washing water. After washing, the anode side end of the capillary tube 1 is cleaned. Since it can be kept in contact with the washing water, the anode side end can be prevented from drying. Further, by performing such a process automatically, the maintainability can be improved.

  In the present embodiment, the tip of the cleaning water supply needle 11 is inserted into the connection port 3 c of the anode reservoir block 3, and the internal space 3 b of the anode reservoir block 3 passes through the tip of the cleaning water supply needle 11. Through this, the washing water can be supplied to the capillary tube 1. Thereby, it is possible to easily inject the washing water into the capillary tube 1 under pressure.

  FIG. 7 is a schematic view showing a configuration example of a capillary electrophoresis apparatus according to another embodiment of the present invention. FIG. 8 is a perspective view showing a mode when a sample is introduced into each capillary tube 1 in the capillary electrophoresis apparatus of FIG. In this embodiment, only the configuration of the cathode side reservoir 6 and the cathode side block 2 is different from the above embodiment, and the other configurations are the same as those in the above embodiment. Detailed description will be omitted.

  The cathode-side reservoir 6 in the present embodiment is not configured to be held by the autosampler 19 but is configured to be connected to the cathode-side block 2. Specifically, the buffer liquid storage portion 61 of the cathode side reservoir 6 communicates with each internal space 2 b of the cathode side block 2 through the same number of connection tubes 62 as the capillary tube 1. Each connection tube 62 is connected to the cathode side block 2 on the extension line of each capillary tube 1 with each internal space 2b interposed therebetween.

  In this case, when the sample is sucked into the internal space 2 b of the cathode side block 2 through the sample suction tube 4, after the buffer liquid in the buffer liquid storage part 61 of the cathode side reservoir 6 is also sucked, the cathode side A voltage is applied to the negative electrode 7 in the reservoir 6. As a result, the sample can be introduced into each capillary tube 1 from the cathode side end portion, and the time for introducing the sample can be shortened, so that analysis at high speed and high throughput can be made possible. .

  As an application field of the present invention, only the region of the genome where the target gene or gene mutation should be analyzed is amplified by PCR (Polymerase Chain Reaction) method, and the genotype is analyzed by reading a relatively short base sequence with high accuracy. A field called SBT (Sequencing Based Typing) can be mentioned. Examples of individual medical treatments for examining cancer gene mutations targeted for molecular targeted drugs and making treatment plans are K-ras genes (exon2, codon12 and 13) for colon cancer, BRAF gene (exon15 V600E), lung cancer EGFR gene (exon 18, 19, 21), c-kit gene (exon 9, 11) of gastrointestinal stromal tumor. All of these can be inspected by the decoding length of the capillary electrophoresis apparatus according to the present invention.

DESCRIPTION OF SYMBOLS 1 Capillary tube 2 Cathode side block 2b Internal space 3 Anode side reservoir block 3b Internal space 3c Connection port 4 Sample suction pipe 5 Pump 6 Cathode side reservoir 7 Negative electrode 8 Positive electrode 9 Polymer filling needle 10 Polymer cartridge 11 Washing water supply Needle 12 Rinse port 13 Washing water tank 14 Washing water pump 15 Drive mechanism 16 Electromagnetic switching valve 17 Open / close valve 18 Drain 19 Autosampler 20 Temperature control unit 21 Detection unit 30 Sample container 31 Buffer liquid storage unit 48 Electromagnetic switching valve 51 to 53 Piping 61 Buffer solution reservoir 62 Connection pipe

Claims (9)

A sample inhalation tube for inhaling the sample;
A pump driven to inhale the sample into the sample inhalation tube;
A capillary tube that is linearly arranged in the horizontal direction and into which the sample sucked into the sample suction tube is introduced ;
A capillary electrophoresis apparatus comprising: a cathode side end of the capillary tube; the sample suction tube; and the pump are connected to each other, and a cathode side block in which an internal space is formed at a junction thereof. A plurality of the capillary tubes and a plurality of the sample suction tubes corresponding to the capillary tubes are connected to the cathode side block, and a plurality of the internal spaces corresponding to the capillary tubes are formed. The capillary electrophoresis apparatus according to claim 1 . A cathode-side reservoir having a buffer solution reservoir capable of storing a buffer solution;
A negative electrode immersed in a buffer solution in the cathode-side reservoir,
After the sample is sucked into the internal space of the cathode side block through the sample suction tube, the negative electrode in the cathode side reservoir is immersed in the buffer solution in the cathode side reservoir after the sample is sucked into the buffer solution in the cathode side reservoir. capillary electrophoresis apparatus according when a voltage is applied, to claim 1 or 2, characterized in that the sample from the cathode side end portion is introduced into the capillary tube. A cathode-side reservoir having a buffer solution reservoir capable of storing a buffer solution, the buffer solution reservoir communicating with the internal space of the cathode block;
A negative electrode immersed in a buffer solution in the cathode-side reservoir,
When a sample is inhaled into the internal space of the cathode side block via the sample inhalation tube, the buffer solution in the cathode side reservoir is also inhaled, and then a voltage is applied to the negative electrode in the cathode side reservoir. the Rukoto, capillary electrophoresis apparatus according to claim 1 or 2, characterized in that the sample is introduced from the cathode side end portion in the capillary tube. An anode side reservoir block in which an internal space communicating with the anode side end of the capillary tube is formed, the buffer solution can be stored in the buffer solution storage unit;
Through the interior space of the anode-side reservoir block, to any one of claims 1 to 4, characterized in that the separated polymer further comprising a polymer filler mechanism is pressure filled from the anode side end portion to the capillary tube The capillary electrophoresis apparatus described. The polymer filling mechanism includes a polymer filling needle inserted into the anode side reservoir block,
The connection port in which a tip of the polymer filling needle can be inserted and sealed is formed at a boundary portion between the buffer liquid storage portion and the internal space in the anode side reservoir block. 5. A capillary electrophoresis apparatus according to 5. Through the interior space of the anode-side reservoir block, capillary electrophoresis according to claim 5 or 6, further comprising a cleaning water supply mechanism for supplying cleaning water from the anode side end portion to the capillary tube apparatus. The capillary electrophoresis apparatus according to claim 7 , wherein the washing water supply mechanism includes a washing water supply needle whose tip can be inserted into the connection port. A linear temperature adjustment unit is provided along the direction in which the capillary tube extends, and further includes a temperature adjustment unit that can accommodate and adjust the temperature of the capillary tube therein.
By moving the temperature control unit in a direction intersecting the direction in which the capillary tube extends, claim 1-8, characterized in that it is possible to take out the capillary tube to the outside of the temperature control unit Capillary electrophoresis apparatus according to claim 1.

Images