JP2007304117A - Device for electrophoresis, electrophoretic equipment, electrophoretic method, and specimen detection method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a device for electrophoresis, electrophoretic equipment, an electrophoretic method and a specimen detection method, which are particularly suitable for a capillary array sheet. <P>SOLUTION: The present invention relates to an electrophoretic device in the form of a casing having inside sealed spaces isolated by an electrophoretic carrier, wherein the device comprises at least one liquid injection/discharge opening, communicable with the outside, on the outer wall of the sealed spaces, and an electrophoretic method and a specimen detection method using the device. Additionally, the present invention relates to an electrophoresis apparatus having a structure for sandwiching the electrophoretic carrier between a pair of electrodes, and having a space capable of holding liquid between the sandwiched electrophoretic carrier and the respective electrodes, and to an electrophoretic method using the same device. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to an electrophoresis device, an electrophoresis apparatus, an electrophoresis method, and a specimen detection method for detecting biologically relevant substances such as nucleic acids, proteins, polypeptides, and polysaccharides.

  In recent years, in order to diagnose diseases and elucidate the causes, biochips (DNA chips, etc.), which have been fixed on a large number of spots related to biologically related substances such as nucleic acids on a flat surface, are classified by type. It is starting to be used.

  As a biochip, a method of spotting and immobilizing a nucleic acid on a chemically or physically modified substrate [Science 270, 467-470 (1995)], a short chain by photolithography on a substrate such as silicon. There is known a method [US Pat. No. 5,445,934, US Pat. No. 5,774,305] for directly solid-phase synthesizing the above nucleic acid. Since the spotting method has a large variation in the amount of nucleic acid immobilized for each spot, the reproducibility for each chip is poor, and it is difficult to produce a uniform chip in large quantities. The photolithography method has little variation in the amount of nucleic acid immobilized for each spot and is excellent in reproducibility from chip to chip. However, the chip is expensive due to an expensive manufacturing apparatus and a multistage manufacturing process. In addition, since nucleic acid synthesis is performed on a substrate, it is difficult to synthesize long-chain nucleic acids.

  Therefore, recently, there is little variation in the amount of nucleic acid immobilized from spot to spot, excellent reproducibility from chip to chip, and easy to immobilize on a substrate, etc., regardless of the chain length of the nucleic acid. A biochip in which a substance probe is immobilized on a gel (see Japanese Patent Application Laid-Open Nos. 2000-270878 and 2000-60554) is attracting attention. In particular, the biochip disclosed in Japanese Patent Application Laid-Open No. 2000-270878 is a fiber assembly in which a plurality of biologically relevant substance-immobilized gel-holding hollow fibers each having a biologically relevant substance such as a different nucleic acid immobilized thereon are regularly arranged. It is a thin piece (hereinafter referred to as a capillary array sheet) obtained by cutting a (three-dimensional array) at a cross section intersecting with the fiber axis, and is highly expected from the market as a biochip capable of mass production at low cost.

  In a biochip in which a biological substance probe such as a nucleic acid is immobilized on a gel, a biological substance probe such as a nucleic acid in an electrolytic solution is migrated into the gel by electrophoresis. Biologically related substance specimens can be efficiently hybridized, and unnecessary specimens that have not been hybridized can be washed. The electrophoresis of DNA chips is described in Japanese Patent Application Laid-Open No. 2000-60554. However, this publication is an electrophoresis method using a chip provided with electrodes and cannot be applied to the capillary array sheet. In addition, it is not preferable to provide an electrode on a chip for electrophoresis because the chip is expensive.

  Capillary array sheets are characterized by being inexpensive and capable of mass production. In particular, an object of the present invention is to provide an electrophoresis device, an electrophoresis apparatus, an electrophoresis method, and a specimen detection method suitable for the capillary array sheet.

  In order to prevent contamination of the chip during storage and transportation, and to maintain quality, the chip is preferably stored in a housing. Furthermore, in a chip in which a biological substance probe is immobilized on the gel, The chips are preferably stored in a liquid so that they do not dry.

  In addition, it is desirable that the temperature can be controlled in order to efficiently perform hybridization between the sample and the probe immobilized on the gel and to wash unnecessary samples. Furthermore, when various types and sizes of nucleic acids are electrophoresed as specimens, the migration behavior of each nucleic acid is different, so if the state of nucleic acid migration can be monitored in real time, reliable hybridization reactions and unnecessary specimen washing can be performed. Can be removed.

  The present invention aims to solve these problems as well.

  As a result of intensive studies in order to solve the above-described problems, the present inventors have reduced the cost by placing an electrophoretic carrier such as a capillary array sheet in a casing capable of electrophoresis, hybridization, washing, and detection. A device for electrophoresis for detecting biologically relevant substances such as nucleic acids, proteins, polypeptides, polysaccharides and the like that can be mass-produced and stored well, an electrophoresis method using the same, and a specimen detection method are provided. Found to get.

  Furthermore, the present inventors have found that an electrophoresis apparatus and an electrophoresis method suitable for an electrophoresis carrier such as a capillary array sheet can be provided.

  That is, the present invention is a housing having a sealed space separated by an electrophoretic carrier inside, and has at least one liquid inlet and outlet that can communicate with the outside on the outer wall portion of the sealed space. A device for electrophoresis. Preferably, the present invention is a housing having two sealed spaces separated by an electrophoretic carrier inside, at least one liquid inlet that can communicate with the outside on each of the outer wall portions of the two sealed spaces, and An electrophoretic device having a discharge port. As the electrophoresis carrier, a capillary array sheet in which a polymer gel on which a probe is fixed is held in a hollow portion of the capillary is suitable. Moreover, a housing is comprised with a transparent material, for example.

  Furthermore, the present invention injects the specimen solution and the electrolyte solution from the liquid inlet and outlet respectively into the separated sealed space inside the casing of the electrophoresis device, and then separates from the liquid inlet and outlet. In this electrophoresis method, an electrode is inserted into the sealed space and a voltage is applied to migrate the analyte molecules onto the electrophoresis carrier. The analyte solution and the electrolyte solution can be injected through, for example, an injector, and the injector can be an electrode. Preferably, in the present invention, the specimen solution and the electrolyte solution are respectively injected from the liquid inlet and outlet into the two sealed spaces inside the casing of the electrophoresis device, and then the liquid inlet and outlet are discharged. In this electrophoresis method, an electrode is inserted into two sealed spaces separated from an outlet, and a voltage is applied to migrate an analyte molecule onto an electrophoresis carrier.

  Further, according to the present invention, a conductive liquid injector and a discharger are inserted into the separated sealed space inside the casing of the electrophoresis device from the liquid inlet and the outlet, and the cleaning liquid is inserted into the separated sealed space. In the electrophoresis method, unnecessary voltage molecules are migrated and removed from the surface and the inside of the electrophoresis carrier by applying a voltage between the liquid injector and the discharger while injecting and discharging the liquid. Preferably, in the present invention, a conductive liquid injector and a discharger are inserted into at least one liquid injection port and a discharge port, respectively, into two separated sealed spaces inside the casing of the electrophoresis device. Applying a voltage between the liquid injector and the discharger while injecting and discharging the cleaning solution into each of the two sealed spaces separated to migrate and remove unnecessary analyte molecules from the surface and inside of the electrophoresis carrier. An electrophoresis method characterized by

  Furthermore, the present invention provides a detection method characterized in that, in the above-described electrophoresis method, light is irradiated to the surface of the electrophoretic carrier at right angles to detect the analyte molecules present on or inside the carrier surface. The detection of the analyte molecule can be performed, for example, by detecting the fluorescence from the fluorescent molecule bonded to the analyte molecule.

  Furthermore, the present invention is an electrophoresis apparatus having a mechanism for sandwiching an electrophoresis carrier between a pair of electrodes, and having a space capable of holding a liquid between the sandwiched electrophoresis carrier and each electrode. As the electrophoresis carrier, a capillary array sheet in which a polymer gel on which a probe is fixed is held in a hollow portion of the capillary is suitable. The space capable of holding the liquid is formed by, for example, a concave portion of the electrode portion. Further, as a mechanism for sandwiching the biochip between a pair of electrodes, one of the pair of electrodes can be moved so as to face the other electrode. Examples of the electrode include an electrode having a liquid injection / discharge port and a light transmission window that communicate with at least one space capable of holding a liquid. Furthermore, the electrode can be temperature-controllable.

  Furthermore, the present invention is an electrophoresis method in which electrophoresis is performed while an analyte is optically detected by an electrophoresis apparatus having a light transmission window on an electrode.

  Capillary array sheets are housed in a case that can be electrophoresed, hybridized, washed, and detected to detect biologically relevant analytes such as nucleic acids, proteins, polypeptides, and polysaccharides that are inexpensive and can be mass-produced. An electrophoresis device, an electrophoresis apparatus, an electrophoresis method, and an analyte detection method are provided. In addition, chip contamination and gel drying during storage and transportation can be prevented. Furthermore, according to the present invention, it is possible to detect a biopolymer specimen such as a nucleic acid with high accuracy and efficiency using a capillary array sheet.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings by illustrating a capillary array sheet using hollow fibers as an electrophoretic carrier. However, the electrophoresis carrier is not limited to the capillary array sheet.

  FIG. 1 is a schematic view of the surface of a capillary array sheet. 11 is a polymer gel such as acrylamide and agarose on which probes (hereinafter referred to as probes) such as nucleic acids and proteins are immobilized, 12 is a hollow fiber holding the gel 11, and 13 is a hollow fiber 12 bonded thereto. Represents a matrix. In FIG. 1, a capillary array sheet, which is an electrophoresis carrier in which a gel in which a probe is immobilized in a hollow portion of a hollow fiber, is illustrated, is illustrated. However, the form of an electrophoresis carrier that holds a polymer gel that is an electrophoresis medium is shown here. It is not limited. Any gel may be used as long as it is filled with a gel with a probe fixed in the thickness direction of the electrophoresis carrier. As an electrophoretic carrier for holding a gel other than hollow fibers, for example, a plurality of stacked sheet-like members or block-like members as described in JP-A-2000-78998 are perforated with a laser or the like. Examples thereof include an electrophoretic carrier which is formed into a sheet after injecting a gel having a probe fixed therein.

  FIG. 2 is a schematic diagram of an electrophoresis device. It shows that the capillary array sheet 23 is sandwiched between the casing component 21 and the casing component 22 and has two spaces 24 and 25 separated by the capillary array sheet 23. Reference numeral 20 denotes an adhesive that fixes the casing component 21 and the casing component 22. Reference numerals 26, 27 and 28, 29 denote liquid inlets and outlets provided in the casing parts 21 and 22, which communicate with the internal spaces 24 and 25, respectively. The thickness of the capillary array sheet is in the range of several tens of microns to 1 mm. A size of several millimeters to several centimeters is usually used. The casing parts 21 and 22 can be produced in large quantities at a low cost if an injection-moldable plastic material is used. Moreover, if the casing components 21 and 22 are made of a transparent material such as glass, acrylic resin, or transparent electrode, the state of the capillary array sheet inside the casing can be observed. Furthermore, if the housing is a transparent material, the fluorescence-labeled specimen can be optically detected by irradiating the capillary array sheet with light. In the case where the housing is not a transparent material, a window may be appropriately provided and the portion may be made of a transparent material.

  In FIG. 2, a capillary array sheet is used instead of a kind of packing. Therefore, the two spaces 24 and 25 separated by the capillary array sheet 23 can be used as a liquid holding space. Further, if the capillary array sheet is used instead of packing, the capillary array sheet having an arbitrary thickness can be sandwiched between the casing parts 21 and 22 having the same shape. In the capillary array sheet, a relatively soft resin is used so that the matrix 13 in FIG. 1 can be easily sliced. Therefore, a method using the capillary array sheet instead of packing is a method suitable for the capillary array sheet.

  FIG. 3 is a schematic diagram of an electrophoresis device having a liquid inlet and outlet that can communicate with the internal space on the outer wall. 31, 32, 33 and 34 are liquid inlets and outlets which can communicate with the internal space, and communicate with the internal space separated by the capillary array sheet. The liquid inlet and outlet are closed with flexible rubber or film. Reference numeral 35 denotes a liquid injector / discharger. The liquid injection needle / discharge needle 36 is inserted into the liquid inlet / outlet to inject the sample solution, and inject / discharge the cleaning solution. In addition, a conductive liquid injection needle and discharge needle 36 subjected to gold plating or the like is used. When a voltage is applied to the needle, the analyte molecule can be migrated into the gel on which the biological substance-related probe of the capillary array sheet is immobilized. Further, if electrophoresis is performed while injecting and discharging the washing liquid in the same manner, dehybridization, washing and the like can be performed efficiently.

  FIG. 4 is a schematic diagram in the case of using a conductive liquid injector and discharger having a flat shape. 41 and 42 are liquid inlets and outlets that can communicate with the internal space, and 43 is a conductive liquid injector and outlet that has a flat shape. Reference numeral 44 denotes a liquid injection / discharge hole, through which liquid is injected and discharged. Examples of the material for the conductive liquid injector and discharger include graphite, platinum, gold, and metal plated with gold. Examples of the shape of the conductive liquid injector / discharger include those having a plurality of conductive needles in a comb shape, and flat and flat shapes. In order to migrate into the gel on which the probe is immobilized, a flat and wide one as shown in FIG. 4 is preferable.

  FIG. 5 is a schematic diagram illustrating a specimen detection method. If the specimen is fluorescently labeled, the specimen can be optically detected. As shown in FIG. 5, the light irradiation device 56 passes the casing part 51 to the capillary array sheet, and the direction perpendicular to the capillary array sheet surface. The fluorescence emitted from the specimen hybridized with the biological substance-related probe immobilized in the gel of the capillary array sheet can be detected by the light detection device 57 such as a CCD camera. Furthermore, if the matrix part of the capillary array sheet does not transmit light, fluorescence emitted from the specimen can be detected with a high S / N. Here, 54 and 55 are the conductive liquid injection needle and discharge needle shown in FIG. As shown in Fig. 5, if the needle is inserted into a position that does not interfere with light irradiation and fluorescence detection, the state of the sample can be monitored in real time while performing electrophoresis, washing, etc., so reliable and efficient hybridization is possible. In addition, unnecessary specimens can be washed, and specimens can be detected with high accuracy.

  In FIG. 6, the capillary array sheet 60 whose vertical direction is fixed by the packing material 61 is housed in a housing frame 62, the upper and lower sides are covered with a glass plate 64, and the inside of the packing material 61 to which the capillary array sheet 60 is attached is sealed. It is a schematic diagram of an electrophoresis device. The packing material 61 is preferably a flexible rubber material or the like that can seal a liquid and can be injected with a liquid injection and discharge needle. The housing frame 62 is provided with an insertion hole 63 so that a liquid injection and discharge needle can be inserted.

  As shown in FIG. 7, liquid injection / discharge is performed by inserting liquid injection and discharge needles 75 and 76 through an insertion hole 73 provided in the housing frame 72 and inserting the needle into the packing material 71, for example, liquid injection and discharge. While injecting the liquid from the needle 75, the liquid or the gas in the sealed space is discharged from the liquid injection and discharge needle 76. At this time, if the liquid is injected from the lower side and discharged from the upper side, the liquid can be filled without leaving bubbles in the sealed space.

  When the sealed space is filled with the sample solution, the sample naturally diffuses to the surface and the inside of the gel on which the biological substance-related probe of the capillary array sheet 70 is immobilized, and forms a hybrid with the probe. Here, if the sealed solution is filled with the sample solution, and the sample solution is further injected and discharged from the discharge needles 75 and 76 alternately, a liquid flow as indicated by the arrows in FIG. 7 occurs in the sealed space. The specimen can be circulated uniformly throughout the sealed space, and hybridization can be performed with little unevenness in place. Further, if a voltage is alternately applied to the liquid injection and discharge needles 75 and 76, an electric field as shown by an arrow in FIG. 7 is generated in the sealed space, and the specimen is uniformly migrated to the entire sealed space, and there is little unevenness in place. Hybridization is possible, and if it is performed simultaneously with circulation by a liquid flow, hybridization with even less unevenness in location is possible.

  Similarly, when the cleaning solution is alternately injected and discharged from the discharge needles 75 and 76, a liquid flow as shown by the arrows in FIG. 7 is generated in the sealed space, and the hybrid is introduced from the surface and inside of the electrophoresis carrier. Unnecessary specimens that have not been formed can be removed by liquid flow. Further, when a voltage is alternately applied to the liquid injection and discharge needles 75 and 76, an electric field as shown by an arrow in FIG. 7 is generated in the sealed space, and an unnecessary hybrid formed from the surface and inside of the electrophoretic carrier is unnecessary. The specimen can be removed by electrophoresis, and if it is performed simultaneously with the liquid flow, an unnecessary specimen can be removed more efficiently.

  FIG. 8 is a schematic diagram of the electrophoresis apparatus, and shows a state where a capillary array sheet is sandwiched between electrodes. The thickness of the capillary array sheet is in the range of several tens of microns to 1 mm. In addition, a size of several millimeters to several centimeters is usually used. Reference numeral 28 is a base of the apparatus, 21 and 22 are electrodes, 24 and 25 are insulators, 23 is a capillary array sheet, 26 is a manual moving stage, 27 is a moving base, and 29 is a power source. The electrode 21 is insulated by an insulator 24 and fixed to a base 28 of the apparatus, and the electrode 22 is insulated by an insulator 25 and fixed on a moving base 27 of a moving stage 26 fixed to the base 28 of the apparatus. Therefore, the electrode 22 can be moved together with the moving table 27 of the moving stage 26. The movable table 27 is moved to widen the gap between the electrodes 21 and 22, the capillary array sheet 23 is inserted between them, and then the movable table 27 is moved in the opposite direction to narrow the gap between the electrodes 21 and 22. 21 and 22 can sandwich the capillary array sheet 23 with an appropriate force. In addition, a capillary array sheet having an arbitrary thickness can be sandwiched between the electrodes in this way.

  In the sandwiching mechanism of FIG. 8, a capillary array sheet is used instead of a kind of packing. However, when the packing effect of the capillary array sheet is poor, a thin packing may be inserted and tightened between the capillary array sheet and the electrode. . In order to enhance the packing effect of the capillary array sheet, a relatively soft resin may be used for the matrix in FIG.

  Examples of the sandwiching mechanism include a method of holding and fixing the electrodes 21 and 22 using an insulating clamp other than that shown in FIG. 8, and a method of tightening with an insulating screw.

  FIG. 9 is a perspective view of the electrode. When the capillary array sheet is sandwiched between the electrodes, the surface indicated by the electrode 31 and the matrix portion of the capillary array sheet are brought into close contact with each other, and a space is formed between the electrode and the capillary array sheet. Is done. That is, since this electrode has a structure in which a concave portion is formed on the electrode surface, it is possible to perform electrophoresis by injecting an electrolytic solution and a specimen into this space and applying a voltage between the electrodes. When the electrode has no recess, insert the packing with an appropriate thickness between the capillary array sheet and the electrode as described above, and sandwich the capillary array sheet to use the packing thickness space as the liquid holding space. Can do.

  There are various electrode shapes, but the shape of 31 that is in close contact with the matrix portion of the capillary array sheet is optimized according to the size, thickness, and hardness of the capillary array sheet, so that the capillary array sheet can be obtained with an appropriate force. If the electrode is sandwiched between the electrodes, it is possible to form a space where no liquid leakage occurs between the electrode and the capillary array sheet. For example, in a capillary array sheet having a size of 20 × 20 mm, a thickness of 0.5 mm, and a matrix portion made of urethane resin, the portion 31 that is in close contact with the matrix portion of the capillary array sheet is flat and has a width of 1 to 2 mm. The degree is preferred. In this case, if the capillary array sheet is sandwiched with an appropriate force, a space can be formed between the electrode and the capillary array sheet, even if the capillary array sheet is somewhat uneven in thickness.

  Examples of the electrode material include graphite, platinum, gold, and metal plated with gold. A transparent electrode may be used. Furthermore, if an electric heater is embedded in the electrode, or the inside of the electrode is made hollow, a liquid at an arbitrary temperature is allowed to flow from the outside, and the temperature of the electrode is controlled, it is possible to efficiently perform hybridization or defragmentation under optimum temperature conditions. Hybridization, washing operations and the like can be performed.

FIG. 10 is a perspective view of an electrode composed of the conductor portion 41 and the insulating portion 42.
In electrophoresis, bubbles may be generated from electrodes by electrolysis. If the generated bubbles adhere to a spot on which a polymer probe such as nucleic acid is immobilized, the migration behavior of the specimen differs from the other spots due to the bubbles, and uniform hybridization or washing cannot be performed between the spots. The sample cannot be detected accurately. However, if the capillary array sheet is vertically sandwiched between the pair of electrodes shown in FIG. 10, a space opened upward is formed between the electrode and the capillary array sheet, and bubbles generated from the conductor portion of the electrode are formed in the capillary array sheet. It floats upward without adhering to the surface, and is further released into the atmosphere from the upper opening.

  Here, if a liquid injection / discharge port is provided in at least one position of the electrode, the specimen, the electrolyte solution, the electrolyte solution, the liquid injection / discharge port, even if the space formed between the electrode and the capillary array sheet is sealed The cleaning liquid can be injected and discharged. Furthermore, even when a space having an open top is formed between the electrode and the capillary array sheet, the specimen, the electrolytic solution, and the washing solution can be circulated, so that hybridization or washing can be performed efficiently.

  FIG. 11 is a view of the electrode as viewed from above. The electrode 51 is provided with a liquid inlet 52 and a liquid outlet 53. These liquid injection / discharge ports can inject and discharge the specimen, the electrolytic solution, and the cleaning liquid into the space formed between the electrode recess 54 and the capillary array sheet.

  FIG. 12 shows an example of an electrode capable of temperature control. The electrode 61 has a hollow inside, and the temperature of the electrode 61 can be controlled by circulating a liquid at an arbitrary temperature from the outside through the inlets 62 and 63 communicating with the hollow inside.

  FIG. 13 is a schematic view of a state in which the capillary array sheet is sandwiched between electrodes having windows as viewed from above. 71 is an electrode, and 74 and 75 windows are open. Reference numeral 72 is also an electrode, and is an example in which the entire portions 76 and 77 are windows. Examples of the window material include transparent materials such as glass and acrylic. 73 is a capillary array sheet, 78 and 79 are light irradiation devices, and 80 and 81 are light detection devices. In this figure, an example is shown in which windows are provided on both electrodes, but a window may be provided on only one of the electrodes to detect the analyte concentration in the liquid on one side of the capillary array sheet.

  In the capillary array sheet, a specific specimen is detected by binding to a probe fixed to a specific spot on the capillary array sheet. For example, when detecting one kind of specimen that can be combined with one of the 25 kinds of capillary array sheets having spots on which 25 different probes are immobilized, the capillary array sheet is sandwiched between a pair of electrodes, When the sample is uniformly migrated to a spot on the capillary array sheet by electrophoresis, 1/25 sample migrates to each spot, the sample and the probe are combined in one spot, and the sample and probe are combined in the other 24 spots. The probe passes through the spot without binding. Therefore, the polarity of the electrode is reversed, or at least one liquid injection / discharge port is provided in the electrode, and the sample and electrolyte separated by the capillary array sheet are injected and discharged without changing the polarity of the electrode. If the sample is circulated through the outlet and further subjected to electrophoresis, the amount of binding between the specimen and the probe can be increased, and the S / N of the detection can be increased.

  However, for example, when various types and sizes of nucleic acids are electrophoresed simultaneously as specimens, the migration behavior of each nucleic acid is different, so that all short-chain nucleic acids migrate in the gel. In the stage where only a part of the long-chain nucleic acid migrates in the gel, the polarity of the electrode is reversed, or the sample and the electrolyte are circulated, so that there is a possibility that the nucleic acid cannot be detected with high accuracy. Therefore, if the state of nucleic acid migration can be monitored in real time, reliable and efficient hybridization and unnecessary sample washing can be performed, and nucleic acid can be detected with high accuracy.

  Therefore, if the specimen is fluorescently labeled, the specimen can be detected optically, and the electrodes are passed through the windows 74 and 76 provided in the electrodes 71 and 72 by the light irradiation devices 78 and 79 as shown in FIG. The space between the electrodes 71 and 72 and the capillary array sheet 73 is irradiated with light, and the fluorescence emitted from the fluorescently labeled specimen existing in the space between the electrodes 71 and 72 and the capillary array sheet 73 is applied to the electrodes 71 and 72. If detection is performed by the photodetectors 80 and 81 through the provided windows 75 and 77, the migration behavior of the specimen can be monitored in real time. In addition, based on the monitored fluorescence intensity, control of inversion of applied voltage, change of applied voltage, circulation of specimen and electrolyte, etc. enables efficient and reliable hybridization and washing of unnecessary specimens. It is possible to detect a sample with high accuracy. It is also possible to monitor the migration behavior of the specimen in real time by guiding the specimen and the electrolytic solution to the outside with a transparent glass tube or the like from the liquid inlet / outlet provided on the electrode, and similarly irradiating light.

  In FIG. 13, the light irradiation devices 78 and 79 and the light detection devices 80 and 81 are arranged on a straight line, but the light irradiation devices 78 and 79 and the light detection devices 80 and 81 may be arranged at right angles. For example, referring to FIG. 13, the light detection devices 80 and 81 are arranged above or below, and the fluorescence emitted from the fluorescently labeled specimen is emitted in the space formed between the electrodes 71 and 72 and the biochip 73. A window may be provided below the upper opening or the electrodes 71 and 72, and detection may be performed from the window. Further, the windows 74, 75, 76, 77 provided in the electrodes 71, 72 and the light irradiation devices 78, 79 and the light detection devices 80, 81 may be connected by optical fibers.

  In FIG. 13, the light irradiation direction is irradiated in parallel with the surface of the capillary array sheet. As shown in FIG. 14, the electrode 91 is provided with a window 94 positioned perpendicular to the surface of the capillary array sheet. Through the window 94, the light irradiation device 96 irradiates the surface of the capillary array sheet 93 with light, and the fluorescence emitted from the fluorescently labeled specimen existing in the gel of the capillary array sheet 93 is applied to the opposing electrode 92. If the photodetection device 97 such as a CCD is detected through the provided window 95, it is possible to detect the gel drop of the capillary array gel, detection of bubble adhesion, and real-time monitoring of the migration behavior of the sample inside the gel of the capillary array sheet. is there. At this time, if the hollow fiber holding the matrix and the gel of the capillary array sheet does not transmit light, fluorescence can be detected with high S / N.

  This specification includes the contents described in the specification and / or drawings of Japanese Patent Application Nos. 2001-300107 and 2001-300108 which are the basis of the priority of the present application.

  EXAMPLES The present invention will be specifically described below with reference to examples, but the scope of the present invention is not limited to the scope of these examples.

[Example 1]
(1) Preparation of capillary array sheet:
Two perforated plates using two 0.1 mm thick perforated plates in which 5 × 5 and 25 holes of 0.32 mm in diameter are arranged in a lattice form at intervals of 0.42 mm in the center of the plate A hollow fiber made of polymethylmethacrylate (outer diameter 0.25 mm, inner diameter 0.18 mm, length 500 mm) was passed through all of the holes. Next, the two perforated plates were spread at intervals of 50 mm, and a polyurethane resin colored with carbon black was poured between the fibers, and embedded in a prismatic shape with a length of 50 mm and a square of 20 mm. A hollow fiber array having portions not fixed with resin at both ends was obtained. Twenty-five hollow fibers were arranged in a 20 mm square prismatic central 2.1 mm square.
Twenty-two hollow fibers in the obtained hollow fiber array were filled with only the gel precursor solution. The remaining three hollow fibers were filled with a gel precursor solution containing 40-base nucleic acid probe A and polymerized. Thereafter, a thin slice having a thickness of 0.5 mm was cut out in a direction perpendicular to the fiber axis with a microtome to obtain a capillary array sheet.

(2) Detection of specimen The obtained capillary array sheet was housed in an acrylic resin casing shown in FIG. At this time, as shown in FIG. 2, the capillary array sheet was sandwiched between the casing parts with an appropriate pressure, and the casing parts were bonded and fixed using an adhesive. Here, the periphery may be trimmed so that the capillary array sheet is sandwiched between the casing components. The space separated by the capillary array sheet sandwiched between the casing components was 10 mm in length, 10 mm in width, and 1 mm in height. For storage, sterilized water was filled from a liquid injection port and a discharge port using a liquid injection device and a discharge device in which a stainless steel hollow tube having an inner diameter of 0.3 mm shown in FIG. After filling with sterilized water, the liquid inlet and outlet liquids were closed with rubber lids.

  Next, in order to detect the specimen, two liquid injectors and dischargers were inserted into one of the spaces separated by the capillary array sheet into the liquid injection port and the discharge port. 40 bases complementary to 0.5 × TBE and probe A by another liquid injector and drainer while draining sterilized water filled by one liquid injector and drainer 120 fmol of the nucleic acid sample labeled with Cy5 dye and 120 fmol of the nucleic acid sample labeled with 40 base Cy3 dye that does not bind to probe A in a complementary manner.

  Similarly, two liquid injectors and dischargers were inserted into the liquid inlet and outlet in the space separated by the other capillary array sheet. While discharging the sterilized water filled by one liquid injector and discharger, only 0.5 × TBE was injected by another liquid injector and discharger. After the discharge and injection operations, the liquid injector and discharger on the side where the nucleic acid sample was injected was used as the negative electrode, the liquid injector and discharger on the side where only the electrolyte was injected was used as the positive electrode, and 0.5 V between the electrodes. Voltage was applied and electrophoresis was performed for 3 hours.

  After completion of the electrophoresis, the sterilized water was again filled with a liquid injector and a discharger, and the capillary array sheet was observed with a fluorescence microscope through an acrylic resin casing. As a result, fluorescence emitted by the Cy5 dye could be detected only at the three spots where the probe A of the capillary array sheet was immobilized. The fluorescence emitted by the Cy3 dye was not detected from any spot.

[Example 2]
The capillary array sheet obtained in Example 1 is housed in an acrylic resin casing to form a device. Similarly, an electrolyte and a fluorescently labeled nucleic acid sample are injected by a liquid injector and an ejector. Time electrophoresis was performed. Thereafter, with the voltage applied, cleaning was performed for 30 minutes while injecting and discharging 0.5 × TBE as a cleaning liquid with a liquid injector and discharger.
After completion of the washing, the sterilized water was again filled with a liquid injector and a discharger, and the capillary array sheet was observed with a fluorescence microscope through a case made of acrylic resin. As a result, fluorescence emitted by the Cy5 dye could be detected only at the three spots where the probe A of the capillary array sheet was immobilized. The fluorescence emitted by the Cy3 dye was not detected from any spot. In addition, the S / N of the fluorescence intensity emitted by the Cy5 dye detected only from the three spots where the probe A was immobilized was improved about 3 times as compared with that detected in Example 1.

Example 3
FIG. 8 shows a pair of electrodes as shown in FIG. 9 in which the capillary array sheet obtained in Example 1 is gold-plated with stainless steel having a concave dimension of 10 mm in width, 15 mm in height, and 1 mm in the depth of the recess. The sandwiching mechanism sandwiched vertically to form a space having an open top between the electrode and the capillary array sheet. At this time, the space between the electrode and the capillary array sheet was 10 mm wide, 1 mm high, and 15 mm deep when viewed from above.

  Nucleic acid sample labeled with 40 base Cy5 dye that binds complementarily to 0.5 × TBE, 120 μl and nucleic acid probe A as an electrolyte solution from the upper opening in the space between the electrode on the negative electrode side and the capillary array sheet a, 120 fmol of nucleic acid sample b labeled with 40 base Cy3 dye that does not bind complementarily to nucleic acid probe A, 120 fmol are injected, and an upper opening is formed in the space between the electrode on the positive electrode side and the capillary array sheet Then, only 0.5 × TBE, 120 μl was injected as an electrolytic solution, a voltage of 0.5 V was applied between the electrodes, and electrophoresis was performed for 3 hours.

  As a result of observation with a fluorescence microscope after completion of electrophoresis, it was possible to detect fluorescence emitted by the Cy5 dye only at three spots where the probe A of the capillary array sheet was immobilized. The fluorescence emitted by the Cy3 dye was not detected from any spot.

Example 4
Similarly to Example 3, the capillary array sheet obtained in Example 1 was gold-plated with stainless steel having a concave size of 10 mm, a height of 15 mm, and a recess depth of 1 mm and an acrylic resin window. A pair of electrodes is vertically sandwiched by the sandwiching mechanism shown in FIG. 8 to form an open space between the electrode and the capillary array sheet. In the space between the electrode on the negative electrode side and the capillary array sheet, Through the opening, 0.5 × TBE, 120 μl as an electrolyte, and nucleic acid sample a, 120 fmol labeled with 40-base Cy5 dye that binds complementarily to nucleic acid probe A are injected, the electrode on the positive electrode side and the capillary array sheet In the space between them, only 0.5 × TBE, 120 μl, is injected as an electrolytic solution from the upper opening, and a voltage of 0.5 V is applied between the electrodes, 13 (however, the detectors corresponding to 80 and 81 are arranged on the upper space side perpendicular to the irradiation light), the space between the electrode on the positive electrode side and the capillary array sheet, and on the negative electrode side Electrophoresis was performed while monitoring the fluorescence intensity emitted from the specimen present in the space between the electrode and the capillary array sheet. As the light irradiation device, a 633 nm laser was used, and the fluorescence intensity was detected from above by detecting the fluorescence emitted from the opening between the electrode and the capillary array sheet with an optical filter with a photo sensor. After 1.5 hours from the change in fluorescence intensity, the specimen that was present in the space between the electrode on the negative electrode side and the capillary array sheet is inside the gel of the capillary array sheet and between the electrode on the positive electrode side and the capillary array sheet. It was confirmed that it moved to the space. Therefore, the polarity of the electrode was further reversed and electrophoresis was performed. As a result, the moved specimen moved again to the space between the electrode and the capillary array sheet in the opposite direction in one hour.

  Here, as a result of taking out the capillary array sheet and observing with a fluorescence microscope, the fluorescence emitted by the Cy5 dye was detected only at the three spots where the nucleic acid probe A of the capillary array sheet was immobilized. It was almost twice the fluorescence intensity detected in Example 3.

  All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety.

It is a schematic diagram of the capillary array sheet surface. It is a schematic diagram of an electrophoresis device. It is a schematic diagram of an electrophoresis device having a liquid inlet and outlet that can communicate with an internal space on an outer wall. It is a schematic diagram in the case of using a conductive liquid injector and discharger having a flat shape. It is a schematic diagram showing the detection method of a test substance. It is a schematic diagram of an electrophoresis device. It is a schematic diagram showing the cross section of an electrophoresis device. It is a schematic diagram of an electrophoresis apparatus. It is a perspective view of an electrode. It is a perspective view of the electrode comprised from a conductor part and an insulation part. It is a schematic diagram of the electrode which has a liquid injection | pouring / discharge port. It is a schematic diagram of an electrode capable of temperature control. It is the schematic diagram which looked at the state which pinched | interposed the capillary array sheet | seat with the electrode which has a window from upper direction. It is the schematic diagram which looked at the state which pinched | interposed the capillary array sheet | seat with the electrode which has a window from upper direction.

Explanation of symbols

The meanings of the symbols in FIGS. 1 to 7 are as follows.
11... Gel 12 in which biological materials such as nucleic acid are immobilized 12... Hollow fiber 13 that holds gel... Matrix 20 that adheres hollow fiber ········ Adhesives 21 and 22 ··· Housing parts 23 ··· Capillary array sheets 24 and 25 ··· Sealed spaces 26 and 27 ··· Liquid inlet and outlets 28 and 29 ... Liquid inlet and outlet 31, 32 ... Liquid inlet and outlet 33, 34 ... Liquid inlet and outlet 35 ... Liquid injector and outlet 36 ... ... Conductive liquid injection and discharge needles 41, 42 ... Liquid injection and discharge holes 43 ... Conductive liquid injection and discharge 44 ... Liquid injection Discharge holes 51, 52... Housing parts 53... Capillary array sheets 54, 55 .. Conductive liquid injection and discharge needle 56... Light irradiation device 57... Photodetection device 60... Capillary array sheet 61. 62... Frame 63... Insertion hole 64... Glass plate 70... Capillary array sheet 71. .... Frame 73 ... Insertion hole 74 ... Glass plates 75, 76 ... Liquid injection needle and discharge needle

The meanings of the symbols in FIGS. 8 to 14 are as follows.
21, 22 ... Electrode 23 ... Capillary array sheet 24, 25 ... Insulator 26 ... Manual moving stage 27 ... Moving table 28 ... .. Electrophoresis device base 29... Power source 31... Electrode and biochip in close contact 41... Electrode conductor 42. Insulating part 51... Electrode 52... Liquid inlet 53... Liquid outlet 54. ... an inlet 63 communicated with the cavity inside the electrode ... an outlet 71, 72 communicated with the cavity inside the electrode 73 ... an electrode 73 having a window ... a capillary array sheet 74, 75... Window 76 provided on the electrode, 77... Window 78, 79. ... Photodetectors 91, 92 ... Electrodes 93 having windows ... Capillary array sheet 94 ... Windows provided on electrodes 95 ... Windows provided on electrodes 96 ··· Light irradiation device 97 ··· Photodetection device

Claims (10)

  An electrophoretic device having a sealed space separated by an electrophoretic carrier inside and having at least one liquid inlet and outlet that can communicate with the outside on an outer wall portion of the sealed space.   2. The electrophoresis device according to claim 1, wherein the electrophoresis carrier is a capillary array sheet in which a plurality of through holes are filled with a polymer gel containing a biological substance.   The electrophoresis device according to claim 1 or 2, wherein the casing is made of a transparent material.   A specimen solution and an electrolyte solution are injected from the liquid inlet and outlet of the electrophoresis device according to any one of claims 1 to 3, and then the liquid inlet and outlet are sealed into a sealed space. Electrophoresis method in which an electrode is inserted and a voltage is applied to migrate an analyte molecule onto an electrophoresis carrier.   5. The specimen detection method according to claim 4, wherein the specimen molecules are detected by irradiating light onto the surface of the electrophoresis carrier at right angles.   6. The specimen detection method according to claim 5, wherein the specimen molecule is detected by detecting fluorescence from a fluorescent molecule bonded to the specimen molecule.   An electrophoresis apparatus having a mechanism for sandwiching an electrophoresis carrier between a pair of electrodes, and having a space capable of holding a liquid between the sandwiched electrophoresis carrier and each electrode.   The electrophoresis apparatus according to claim 7, wherein the electrophoresis carrier is composed of a capillary array sheet in which a polymer gel having a probe fixed is held in a hollow portion of the capillary.   The electrophoresis apparatus according to claim 7 or 8, wherein the temperature of the electrode is controllable.   An electrophoretic device having a mechanism for sandwiching an electrophoretic carrier between a pair of electrodes, and having a space capable of holding a liquid between the electrophoretic carrier thus sandwiched and each electrode, the electrode having at least one light transmission window An electrophoresis method in which electrophoresis is performed while optically detecting a specimen by the electrophoresis apparatus.

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