JP4935750B2 - Bidirectional electrophoresis apparatus and bidirectional electrophoresis method - Google Patents

Description

  The present invention uses a microchip, a bidirectional electrophoresis apparatus capable of performing electrophoresis in a first direction and a second direction 180 degrees different from the first direction, and is used in the bidirectional electrophoresis apparatus. The present invention relates to a suitable microchip and a bidirectional electrophoresis method using the bidirectional electrophoresis apparatus.

  In capillary electrophoresis (capillary electrophoresis) in which electrophoresis is performed by applying a high voltage of about 10 kV to 30 kV using a capillary tube (capillary) having an inner diameter of 0.05 to 0.2 mm, the outer wall of the capillary is protected. A protective film such as a polyimide film for coating is applied. This is because the capillary of quartz glass or fused silica is physically and chemically stabilized (see Patent Document 1). However, when performing electrophoretic measurement, etc., optical means such as UV absorption is used as the detection means, so that the detection efficiency of the signal is reduced in the optical detection section, so the outer wall coating is made thin or removed. Thus, optical detection is performed, and the strength is lower than in other places. In order to compensate for this decrease in strength, the optical detection unit has been devised such as having a mechanism for complementing the strength of the capillary. In a capillary having this strength complementation mechanism of the optical detection unit only in one direction, there is a problem that the capillary cannot be used if a defect occurs on the injection end side of the capillary channel.

  Further, in the capillary electrophoresis method, there is a problem that only one direction of migration can be performed on the separation channel because the optical detection part of the capillary is on the opposite side of the sample injection. Capillary electrophoresis, in which only one-way electrophoresis can be performed on the separation flow path formed by the capillary, separation performance is caused by problems on the injection side such as adsorption of proteins to the inner wall of the capillary and physical damage. As a result, it is difficult to use the capillary repeatedly, and it is necessary to discard the capillary. As a result, there is a problem in that the cost of analysis by capillary electrophoresis increases.

In capillary electrophoresis, if the optical detection part is placed at the center of the capillary, bidirectional electrophoresis can be performed. However, in this case, there is a problem that the effective separation length is remarkably shortened, which is not realistic.
JP-A-7-151729

  In the present invention, electrophoresis can be performed from both ends of the separation channel in the first direction and in the second direction that is 180 degrees different from the first direction. When used in the first direction, proteins and the like are separated. If problems occur on the injection side, such as problems with adsorption to the inner wall of the flow path or physical damage, use from the second direction can increase the number of reuses and reduce analysis costs. Furthermore, even when trouble occurs only on one side of the separation channel during production, the bidirectional electrophoresis device that can be used from the opposite side and has a high production yield, a microchip suitable for use in this bidirectional electrophoresis device, and An object of the present invention is to provide a bidirectional electrophoresis method using this bidirectional electrophoresis apparatus.

  In order to achieve the above object, the first aspect of the present invention includes: (a) a holding substrate having in part an optical window penetrating from the upper surface to the lower surface; and (b) an optical window mounted on the upper surface and on the surface. A separation channel for electrophoresis that is aligned with each other, a microchip made of a transparent material having sample reservoirs connected to both ends of the separation channel, and (c) a voltage for electrophoresis in the separation channel The gist of the present invention is a bidirectional electrophoretic apparatus having a pair of electrodes for applying selenium and (d) an optical measurement system for measuring electrophoresis in a separation channel through an optical window. The bidirectional electrophoresis apparatus according to the first aspect of the present invention includes a second direction in which the direction of the microchip is 180 degrees different from the first direction and the first direction with respect to the holding substrate together with the pair of electrodes. By setting the direction, the separation channel is used in the reciprocating direction.

  The second aspect of the present invention includes (a) a holding substrate partially including an optical window penetrating from the upper surface to the lower surface, and (b) an electrophoresis mounted on the upper surface and aligned with the optical window on the surface. And a sample reservoir connected to both ends of the separation channel, a microchip made of a transparent material, and (c) a pair of electrodes for applying an electrophoresis voltage to the separation channel, (D) The gist of the present invention is a bidirectional electrophoresis apparatus having an optical measurement system for measuring electrophoresis in a separation channel through an optical window. The bidirectional electrophoresis apparatus according to the second aspect of the present invention sets the direction of the microchip to the first direction and the second direction that is 180 degrees different from the first direction with respect to the holding substrate, The separation channel is used in the reciprocating direction by reversing the polarity of the pair of electrodes.

  The third aspect of the present invention includes (a) a holding substrate partially including a pair of optical windows penetrating from the upper surface to the lower surface, and (b) mounted on the upper surface and aligned with the pair of optical windows on the surface. A pair of electrophoretic separation channels, a sample reservoir connected to each end of the separation channel, a microchip made of a transparent material, and (c) a voltage for electrophoresis to the separation channel. And (d) a bidirectional electrophoresis apparatus having an optical measurement system that measures electrophoresis in a separation channel through one of a pair of optical windows. Then, the bidirectional electrophoresis apparatus according to the third aspect of the present invention provides the direction of electrophoresis in the separation channel by selecting one of the pair of optical windows and replacing the corresponding pair of electrodes or selecting the polarity. It is possible to select.

  According to a fourth aspect of the present invention, there is provided a holding substrate partially including an optical window penetrating from the upper surface to the lower surface, and a separation flow path for electrophoresis mounted on the upper surface and aligned with the optical window on the surface. And a bidirectional electricity having a microchip made of a transparent material, a pair of electrodes for applying a voltage for electrophoresis to the separation channel, and an optical measurement system for measuring the electrophoresis in the separation channel through an optical window. The gist is that the microchip is used in an electrophoresis apparatus. In the microchip according to the fourth aspect of the present invention, the microchip can invert the direction of the microchip with respect to the holding substrate, and the separation channel is 180 degrees with respect to the first direction and the first direction. A sample reservoir is provided at each end of the separation channel so that electrophoresis can be performed in different second directions, and a point-symmetric plane pattern is formed with the midpoint of the separation channel as the center of symmetry. To do.

  According to a fifth aspect of the present invention, there is provided a holding substrate partially including an optical window penetrating from the upper surface to the lower surface, an electrophoresis separation channel mounted on the upper surface and aligned with the optical window on the surface, and A sample reservoir connected to each end of the separation channel, a microchip made of a transparent material, a pair of electrodes for applying a voltage for electrophoresis to the separation channel, and an optical window through the separation channel The gist of the present invention is a bidirectional electrophoresis method using a bidirectional electrophoresis apparatus having an optical measurement system for measuring electrophoresis. And the bidirectional | two-way electrophoresis method which concerns on the 5th aspect of this invention WHEREIN: When the separation characteristic by the electrophoresis along a 1st direction falls, a 2nd direction which differs a microchip 180 degree | times from a 1st direction And the separation channel is used in the reciprocating direction.

  According to a sixth aspect of the present invention, there is provided a holding substrate partially including a pair of optical windows penetrating from the upper surface to the lower surface, and separation for electrophoresis mounted on the upper surface and aligned with the pair of optical windows on the surface. A sample reservoir connected to each end of the flow path and the separation flow path, a microchip made of a transparent material, a pair of electrodes for applying a voltage for electrophoresis to the separation flow path, and a pair of optical windows The gist of the present invention is a bidirectional electrophoresis method using a bidirectional electrophoresis apparatus having an optical measurement system for measuring electrophoresis in a separation channel via any one of them. The bidirectional electrophoresis method according to the sixth aspect of the present invention switches the selection of one of the pair of optical windows from one to the other when the separation characteristics due to electrophoresis along the first direction deteriorate. Correspondingly, by switching the pair of electrodes or switching the polarity, the direction of electrophoresis is reversed to the second direction 180 degrees different from the first direction, and the separation channel is used in the reciprocating direction. And

  According to the present invention, electrophoresis can be performed from both ends of the separation channel in the first direction and in the second direction that is 180 degrees different from the first direction. If problems such as adsorption to the inner wall of the separation channel or troubles on the injection side such as physical damage occur, use from the second direction can improve the number of reuses and reduce analysis costs. In addition, even when trouble occurs only on one side of the separation channel during production, the bidirectional electrophoresis device can be used from the opposite side and has a high production yield, and is suitable for use in this bidirectional electrophoresis device. A chip and a bidirectional electrophoresis method using the bidirectional electrophoresis apparatus can be provided.

  Next, first to third embodiments of the present invention will be described with reference to the drawings. In the following description of the drawings, the same or similar parts are denoted by the same or similar reference numerals. However, it should be noted that the drawings are schematic, and the relationship between the thickness and the planar dimensions, the ratio of the thickness of each layer, and the like are different from the actual ones. Therefore, specific thicknesses and dimensions should be determined in consideration of the following description. Moreover, it is a matter of course that portions having different dimensional relationships and ratios are included between the drawings.

  The first to third embodiments shown below exemplify apparatuses and methods for embodying the technical idea of the present invention, and the technical idea of the present invention is The material, shape, structure, arrangement, etc. are not specified below. The technical idea of the present invention can be variously modified within the technical scope described in the claims.

(First embodiment)
As shown in FIG. 1, the bidirectional electrophoresis apparatus according to the first embodiment of the present invention is detachably mounted on a flat holding substrate 21 and the holding substrate 21, and the separation channel 16 is mounted on the surface. A microchip (15, 17) made of a transparent material such as soda-lime glass or quartz glass, a resin upper lid 14 for protecting the microchip (15, 17), and a separation channel 16 Electrodes 11a and 11b for applying a high voltage necessary for electrophoresis are provided. The microchip (15, 17) is a capillary having a base plate 17 made of a flat plate having a thickness of about 1.5 to 2 mm and a U-shaped or U-shaped groove at the bottom of the base plate 17. Plate 15. The U-shaped or U-shaped groove provided at the bottom of the base plate 17 may be formed by a micro electro mechanical system (MEMS) technology that is an advancement of semiconductor manufacturing technology. The capillary plate 15 and the base plate 17 are each made of a transparent material such as soda-lime glass or quartz glass, and are joined to each other to form a monolithic microchip (15, 17). As a result, for example, a minute groove having a width of about 50 to 150 μm and a depth of about 20 to 50 μm is embedded as a microchannel in the upper portion near the surface of the microchip (15, 17), and the separation channel 16 is formed. . A U-shaped or U-shaped groove is provided on the surface of the base plate 17, and a flat capillary plate 15 is joined as a cover plate on the base plate 17 to thereby form a microchip (15, 17). A separation channel 16 may be embedded in the upper part near the surface, and a U-shaped or U-shaped groove is provided on the surface of the base plate 17, and a capillary having a groove on the lower surface of the base plate 17. The plate 15 may be joined so that the grooves are aligned with each other, and the separation channel 16 may be embedded in the upper part near the surface of the microchip (15, 17).

  The holding substrate 21 irradiates the substance to be measured (chemical species) to be electrophoresed in the separation channel 16 from the bottom of the microchip (15, 17) made of a transparent material with light such as ultraviolet rays. In order to measure absorption or fluorescence due to (), an optical window 22 penetrating vertically from the upper surface to the lower surface of the holding substrate 21 is provided. A chip fastener 23 for positioning the microchip (15, 17) at a predetermined position on the holding substrate 21 is provided on the upper surface of the holding substrate 21. The microchip (15, 17) is installed on the upper portion of the holding substrate 21 by the chip fastener 23 so as to determine the relative position with the optical window 22. Sample reservoirs 13 a and 13 b that store a substance to be measured (chemical species) and a buffer solution 61 are provided at both ends of the separation channel 16. The two sample reservoirs 13a and 13b are arranged symmetrically with the midpoint of the separation channel 16 coincident with the center of symmetry of the microchip (15, 17), and have a point-symmetric topology. FIG. 1 illustrates a case where the optical window 22 is located below the separation channel 16 and on the anode side. The holding substrate 21 includes a heater (not shown), and the heater is connected to the temperature control unit 32 so as to control the temperature of the substance (chemical species) to be electrophoresed in the separation channel 16 and the buffer solution 61. The output of the heater is controlled by the temperature control unit 32.

  Buffer reservoirs 12a and 12b are provided above the positions corresponding to the sample reservoirs 13a and 13b of the upper lid. The buffer reservoirs 12a and 12b are erected vertically with respect to the glass plate 15, and are positioned so that the centers of the circular sample reservoirs 13a and 13b are arranged at the centers of the circular buffer reservoirs 12a and 12b. .

  The electrodes 11a and 11b are inserted on the buffer reservoirs 12a and 12b side. A voltage control unit 31 that applies a high voltage necessary for electrophoresis to the separation channel 16 is connected to the electrodes 11a and 11b.

The optical measurement system 5 includes a light source 51, band pass filters 52 and 54, a dichroic mirror 53, and a detector 55. The light source 51 is disposed below the optical window 22, emits light toward the bottom of the upper microchip (15, 17), passes through the bottom of the microchip (15, 17), and passes through the separation channel 16. Light is irradiated to the substance (chemical species) to be electrophoresed. The band pass filter 52 is disposed above the light source 51 and perpendicular to the light traveling direction. A dichroic mirror 53 is arranged above the bandpass filter 52 so that fluorescence from the substance to be measured (chemical species) enters the bandpass filter 54. The light that has passed through the band pass filter 54 enters a detector 55 such as a photomultiplier or a semiconductor detector. A CCD camera may be used as the detector 55. The light source 51 and the detector 55 are connected to the data processing unit 33.

  When a substance to be measured (chemical species) and a buffer solution 61 are injected into a microchannel composed of the separation channel 16 and the glass plate 15 and a negative voltage is applied to the electrode 11a and a positive voltage is applied to the electrode 11b by the voltage control unit 31, The substance to be measured (chemical species) in 61 undergoes electrophoresis, and the substance to be measured (chemical species) is separated depending on its molecular weight. The separated substance (chemical species) to be measured is detected from the optical window 22 using the optical measurement system 5.

  As shown in FIG. 2, the holding substrate 21 is a rectangular flat plate whose upper surface has a sufficient area for detachably mounting the microchip (15, 17), and the optical window 22 is accurately positioned in the separation channel 16. In the longitudinal direction of the separation channel 16, it is located on the anode side from the center, that is, in the short side direction of the holding substrate 21. As shown in FIG. 2, the planar shape of the optical window 22 is not limited to a rectangle, and may be a polygon such as a hexagon or an octagon, or a circle. As shown in FIG. 2, the tip fastener 23 is L-shaped (saddle-shaped) when viewed from the upper surface, and the microchip (15, 17) can be rotated 180 degrees horizontally with respect to the holding substrate 21. The position of one corner of the microchip (15, 17) can be determined so that the bidirectional electrophoresis apparatus can be used. However, FIG. 2 is an example, and one of the microchips (15, 17) can be used even if the microchip (15, 17) is rotated 180 degrees horizontally with respect to the holding substrate 21. Any shape can be used as long as the positions of the two corners can be determined.

  Therefore, as shown in FIG. 3, the microchips (15, 17) have a two-fold rotationally symmetric (point symmetry) rectangular flat plate shape with the midpoint of the separation channel 16 as the rotation axis (center of symmetry). ing. In other words, the separation channel 16 is arranged on the microchip (15, 17) so that the midpoint of the separation channel 16 divided in the length direction is located on the rotational axis of two-fold rotational symmetry of the microchip (15, 17). It is located on the center line along the longitudinal direction. FIG. 3 shows an example in which one separation channel 16 is provided on the surface of the microchip (15, 17), but this is for simplifying the explanation, and actually two or more separation channels 16 are provided. The plurality of separation channels 16 can be formed by the MEMS technology, and may be, for example, about 30 to 400. The sample reservoirs 13 a and 13 b located at both ends of the separation channel 16 also maintain symmetry with a topology that is two-fold rotationally symmetric with the midpoint of the separation channel 16 as a rotation axis. When a plurality of separation channels 16 are formed on the surface of the microchip (15, 17), sample reservoirs 13a and 13b are provided at both ends of each separation channel 16. The sample reservoirs 13a and 13b are circular with the same dimensions when viewed from above, and the distances from the nearest short side of the microchip (15, 17) are equal. However, the shapes of the sample reservoirs 13a and 13b are not necessarily the same size and shape as long as they function as sample reservoirs, and do not need to be rotationally symmetrical twice with respect to the middle point of the separation channel 16. The glass plate 15 is a rectangular flat plate, and the lengths of the long side and the short side are the same as the microchip (15, 17), and are twice rotationally symmetric. The upper lid 14 is a rectangular flat plate and is similarly rotationally symmetric twice. The inner diameters of the buffer reservoirs 12a and 12b are larger than the inner diameters of the sample reservoirs 13a and 13b.

  As a result of the microchip (15, 17) having a two-fold rotational topology, the microchip (15, 17) is arranged so that the positions of the two sample reservoirs 13a, 13b are reversed as shown in FIG. Can also be used. The upper lid 14 is arranged so that the positions of the two buffer reservoirs 12a and 12b are reversed. At the same time as the direction of the microchip (15, 17) is reversed, the pair of electrodes 11a and 11b are also reversed in position. good. That is, by setting the microchip (15, 17) in the forward direction (first direction) and the reverse direction (second direction) with respect to the holding substrate 21, the separation channel 16 can be electrophoresed in the reciprocating direction. Can be used. Alternatively, when the direction of the microchips (15, 17) is set in the reverse direction (second direction) with respect to the holding substrate 21, the spatial positions of the pair of electrodes 11a, 11b are fixed to the platform on the apparatus side. Thus, the separation channel 16 can be electrophoresed in the reverse direction (second direction) by replacing the sample reservoirs 13a and 13b relative to each other and inverting the polarities of the pair of electrodes 11a and 11b. .

  FIG. 5 is a diagram showing a history of measurement results of resolution in a DNA sequence using the bidirectional electrophoresis apparatus according to the first embodiment of the present invention. Open diamonds (◇) indicate the degree of separation when the separation in the first direction (positive direction) is repeated multiple times and the degree of separation becomes significant. As shown in FIG. 5, it can be seen that the resolution decreases at 75 to 150 base pairs (bases). In this case, thereafter, as indicated by the black circles (●), the microchips (15, 17) are reversed (second direction) with respect to the separation channel direction, and the migration direction with respect to the separation channel is changed. It can be seen that using the reverse direction (second direction) improves the resolution at 75-150 base pairs. As shown in FIG. 5, the degree of resolution once lowered is obtained by rotating the microchip (15, 17) 180 degrees and performing the electrophoresis in the reverse direction (second direction) with respect to the separation channel. Thus, it can be seen that the number of times the microchip (15, 17) is used can be improved, and as a result, the analysis cost can be reduced.

  As already described in the Background Art section, in capillary electrophoresis, the protective coating of the capillary is removed by the optical detection unit in order to form a detection window in the capillary, so a protective mechanism is provided in the optical detection unit. It is necessary and it is difficult to make a structure that can be easily used in two directions. In the bidirectional electrophoresis apparatus according to the first embodiment of the present invention, minute grooves (microchannels) on the order of microns are formed on the surface of the microchip (15, 17) by the MEMS technique, and the separation flow path is formed. Therefore, mechanical strength can be sufficiently increased, and coating on the outer wall of the capillary for obtaining strength is not required as in capillary electrophoresis.

  In the bidirectional electrophoresis apparatus according to the first embodiment of the present invention, the sample reservoirs 13a and 13b are arranged symmetrically so that the midpoint of the separation channel 16 coincides with the symmetrical center of the microchip (15, 17). Therefore, the topology is point-symmetric and the separation channel 16 can be used in both the forward and reverse directions. In the bidirectional electrophoresis apparatus according to the first embodiment, since the chip fastener 23 is provided on the upper surface of the holding substrate 21, when the microchip (15, 17) is set on the holding substrate 21. The optical measurement system 5 and the electrodes 11a and 11b can be aligned accurately, easily and in a short time to correspond to a dispensing mechanism such as the buffer reservoirs 12a and 12b.

  When the microchip (15, 17) having a structure capable of performing electrophoretic separation from both directions with respect to the separation channel is used in the bidirectional electrophoresis apparatus, the bidirectional electrophoresis is performed on the microchip (15, 17) once set. The detection system of the apparatus, the electrode mechanism, or the holding mechanism of the microchip (15, 17) is made movable, and a mechanism capable of performing migration from both directions without re-installing the microchip (15, 17) is provided.

  Therefore, according to the bidirectional electrophoresis apparatus according to the first embodiment of the present invention, deterioration of the inner wall coating of the separation channel 16 for separation, adsorption of proteins or the like to the inner wall of the separation channel 16, and Even if the performance deteriorates due to a trouble on the injection side such as physical breakage, the number of reuse is improved by reversing the direction and using the separation channel 16 from the reverse direction (second direction), and the analysis cost Can be reduced. According to the bidirectional electrophoresis apparatus according to the first embodiment of the present invention, if a trouble occurs only on one side of the separation channel 16 at the time of manufacture, it can be used from the opposite side of the separation channel 16, so that the production yield is improved. Can be made.

(Modification of the first embodiment)
The configuration shown in FIG. 1 is an example. For example, in the optical measurement system 5, a light source 51 is arranged below the optical window 22 as shown in FIG. The optical system is not limited to the optical system in which the fluorescence emitted toward is reflected by the dichroic mirror 53 disposed below the optical window 22 and detected by the detector 55. That is, as shown in FIG. 6, the irradiation optical system 5a for irradiating light to the separation channel 16 of the microchip (15, 17) above the microchip (15, 17) and the microchip (15, 17). A detection optical system 5b for measuring fluorescence or transmitted light from the separation channel 16 of the microchip (15, 17) is provided below, and the optical measurement system is sandwiched from both sides of the microchip (15, 17). You may divide into two.

  In the bidirectional electrophoresis apparatus according to the modification of the first embodiment of the present invention shown in FIG. 6, the irradiation optical system 5a is disposed above the microchip (15, 17), and the microchip (15, 17) a light source 51 that emits light downward toward the separation channel 16, and an optical path under which the light emitted from the light source 51 travels toward the separation channel 16 of the microchip (15, 17). A band-pass filter 52 is provided therein. In a location located above the separation channel 16 of the microchip (15, 17) of the upper lid 14 so that the light emitted from the light source 51 can be irradiated to the separation channel 16 of the microchip (15, 17), An incident optical window 28a is provided. The upper lid 14 is symmetrical so that the two incident optical windows 28a and 28b have a point-symmetric topology with the midpoint of the separation channel 16 coincident with the center of symmetry of the microchip (15, 17). Has been placed. The incident optical windows 28 a and 28 b may be simple openings provided in the upper lid 14. The light emitted from the light source 51 is transmitted through the incident optical window 28a, is transmitted through the capillary plate 15 of the microchip (15, 17) disposed below the incident optical window 28a, and is electrically connected to the separation channel 16. Light is irradiated to the substance (chemical species) to be migrated.

  The detection optical system 5 b is reflected by the dichroic mirror 53 disposed below the optical window 22 provided below the separation channel 16 of the microchip (15, 17) of the holding substrate 21 and the dichroic mirror 53. A band-pass filter 54 that transmits light and a detector 55 that detects fluorescence or transmitted light emitted from the separation channel 16 of the microchip (15, 17) via the band-pass filter 54 are provided. As described with reference to FIG. 1, the detector 55 can be a photomultiplier, a semiconductor detector, or the like. The dichroic mirror 53 of the detection optical system 5b is omitted, and the fluorescence or transmitted light emitted from the separation channel 16 of the microchip (15, 17) is directly incident on the detector 55 via the bandpass filter 54. Any optical arrangement may be used. When the detector 55 detects the transmitted light emitted from the separation channel 16 of the microchip (15, 17), an absorption measurement is performed. The light source 51 of the irradiation optical system 5a and the detector 55 of the detection optical system 5b are connected to the data processing unit 33, and the data processing unit 33 performs data processing necessary for fluorescence measurement or absorbance measurement.

  In contrast to FIG. 6, an irradiation optical system 5a for irradiating light to the separation channel 16 of the microchip (15, 17) is provided below the microchip (15, 17), and the microchip (15, 17). A detection optical system 5b for measuring fluorescence or transmitted light from the separation channel 16 of the microchip (15, 17) may be provided above the above.

  The microchip (15, 17) of the bidirectional electrophoresis apparatus according to the modification of the first embodiment has a rotation axis (a center of symmetry) with the midpoint of the separation channel 16 as shown in FIG. ) And a two-fold rotational symmetry (point symmetry) rectangular flat plate shape. In addition to the two incident optical windows 28a and 28b described above, the sample reservoirs 13a and 13b located at both ends of the separation channel 16 also have a two-fold rotational symmetry with the midpoint of the separation channel 16 as the rotation axis. Maintains symmetry. As a result of the microchip (15, 17) having a two-fold rotationally symmetric topology, the microchip (15, 17) is such that the positions of the two sample reservoirs 13a, 13b and the two incident optical windows 28a, 28b are reversed. It is possible to use it even if it is arranged. By setting the microchip (15, 17) in the forward direction (first direction) and the reverse direction (second direction) with respect to the holding substrate 21, the separation channel 16 is used for electrophoresis in the reciprocating direction. Can do. Alternatively, when the direction of the microchips (15, 17) is set in the reverse direction (second direction) with respect to the holding substrate 21, the spatial positions of the pair of electrodes 11a, 11b are fixed to the platform on the apparatus side. Thus, the separation channel 16 can be electrophoresed in the reverse direction (second direction) by replacing the sample reservoirs 13a and 13b relative to each other and inverting the polarities of the pair of electrodes 11a and 11b. .

(Second Embodiment)
The bidirectional electrophoresis apparatus according to the second embodiment of the present invention includes a holding substrate 21, a microchip (15, 17), an upper lid 14, as in the first embodiment shown in FIGS. Although the glass plate 15 and the electrodes 11a and 11b are provided, as shown in FIGS. 7 and 8, the center of symmetry of the separation channel 16 is shifted from the position of the center of symmetry of the microchip (15, 17). Yes. That is, the microchip (15, 17) does not have a two-fold rotational symmetry (point symmetry) topology with the midpoint of the separation channel 16 as the rotation axis.

  Therefore, when the holding substrate 21 according to the second embodiment is in the positive direction (first direction), the positioning end portions of the microchips (15, 17) are arranged in the first direction as shown in FIG. It is fitted to the tip fastener 23a, aligned so that the center line of the separation channel 16 of the microchip (15, 17) passes through the center of the optical window 22, and rotated in the reverse direction (second direction). 8), as shown in FIG. 8, the positioning end of the microchip (15, 17) is fitted to the second chip fastener 23b, and the separation flow path of the microchip (15, 17) is obtained. The 16 center lines are aligned so that they pass through the center of the optical window 22. 7 and 8 show an example in which one corner of a rectangle (rectangle) is used as the “positioning end of the microchip (15, 17)”, the “positioning end” is limited to the corner. It may be a circular or arc-shaped convex part.

  The first and second chip fasteners 23a and 23b are disposed at a pair of positions that are substantially diagonal to each other on the surface of the microchip (15, 17) that contacts the holding substrate 21. Since the description of the other part of the structure of the bidirectional electrophoresis apparatus according to the second embodiment is the same as the description of the structure of the first embodiment, a duplicate description is omitted. Further, the explanation of the operation of the electrophoretic separation and analysis is the same as the explanation of the operation of the bidirectional electrophoretic device according to the first embodiment, and thus the duplicate description is omitted.

  In the bidirectional electrophoresis apparatus according to the second embodiment of the present invention, a micron is formed on the surface of the microchip (15, 17) by the MEMS technology, similarly to the bidirectional electrophoresis apparatus according to the first embodiment. Since the separation channel 16 is formed by forming an order microchannel, the mechanical strength can be sufficiently increased, and there is no need to coat the outer wall of the capillary to obtain strength, as in capillary electrophoresis. is there.

  Unlike the bidirectional electrophoresis apparatus according to the first embodiment, the bidirectional electrophoresis apparatus according to the second embodiment of the present invention is not a point-symmetric topology of the microchips (15, 17). Since the two chip fasteners 23 a and 23 b are provided on the upper surface of the holding substrate 21, when setting the asymmetric microchip (15, 17) on the holding substrate 21, the optical measurement system 5 and the electrodes 11 a and 11 b In addition, it is possible to perform alignment accurately, easily, and in a short time in order to cope with a dispensing mechanism such as the buffer reservoirs 12a and 12b.

  Therefore, according to the bidirectional electrophoresis apparatus according to the second embodiment of the present invention, as in the bidirectional electrophoresis apparatus according to the first embodiment, the inner wall coating of the separation channel 16 for separation is performed. Even if the performance deteriorates due to problems on the injection side such as degradation of the protein, adsorption of proteins, etc. to the inner wall of the separation flow path 16 and physical damage, the flow is reversed from the reverse direction (second direction). By using the path 16, the number of reuses can be improved, and the analysis cost can be reduced. According to the bidirectional electrophoresis apparatus according to the second embodiment of the present invention, if a trouble occurs only on one side of the separation channel 16 at the time of manufacture, it can be used from the opposite side of the separation channel 16, so that the manufacturing yield is improved. Can be made.

(Modification of the second embodiment)
Although not shown, as shown in FIG. 6, an irradiation optical system for irradiating light to the separation channel of the microchip above the microchip, and a separation channel of the microchip below the microchip. The optical measurement system may be divided into two so as to be sandwiched from both sides of the microchip by providing a detection optical system that measures fluorescence or transmitted light from the microchip. Or, conversely, an irradiation optical system for irradiating light to the separation channel of the microchip is provided below the microchip, and a detection optical system for measuring fluorescence or transmitted light from the separation channel of the microchip is provided above the microchip. May be provided.

  In the bidirectional electrophoresis apparatus according to the modified example of the second embodiment of the present invention, the symmetrical center of the separation channel is the position of the symmetrical center of the microchip, as shown in FIGS. It is off. That is, the microchip does not have a two-fold rotational symmetry (point symmetry) topology with the midpoint of the separation channel as the rotation axis. However, the two optical windows for incidence are symmetrically arranged on the upper lid so as to have a point-symmetric topology with respect to the middle point of the separation channel.

  For this reason, when the holding substrate according to the second embodiment is in the positive direction (first direction), the positioning end of the microchip is used as the first chip fastener as shown in FIG. FIG. 8 shows the case of the reverse direction (second direction) rotated 180 degrees after fitting and positioning so that the center line of the separation channel of the microchip passes through the center of the optical window. In the same manner as above, the positioning end of the microchip is fitted to the second chip fastener, and the center line of the separation channel of the microchip is aligned so as to pass through the center of the optical window.

  In the bidirectional electrophoresis apparatus according to the modification of the second embodiment of the present invention, the microchip is not a point-symmetric topology, but two chip fasteners are provided on the upper surface of the holding substrate. When an asymmetric microchip is set on a holding substrate, alignment for dealing with a dispensing mechanism such as an optical measurement system, electrodes, and a buffer reservoir can be performed accurately, simply, and in a short time.

  Therefore, according to the bidirectional electrophoresis apparatus according to the modification of the second embodiment of the present invention, degradation of the inner wall coating of the separation channel for separation, adsorption of proteins and the like to the inner wall of the separation channel, Even if the performance deteriorates due to injection-side troubles such as physical damage, the number of reuses is improved by reversing the direction and using the separation channel from the reverse direction (second direction), and the analysis cost Can be reduced.

(Third embodiment)
The bidirectional electrophoresis apparatus according to the third embodiment of the present invention is the first in that it includes a holding substrate 21, a microchip (15, 17), an upper lid 14, a glass plate 15, and electrodes 11a, 11b. As in the bidirectional electrophoresis apparatus according to the second embodiment, as shown in FIG. 9, two holding optical windows 22a and 22b through which the holding substrate 21 passes vertically from the upper surface to the lower surface of the flat plate are provided. This is different from the bidirectional electrophoresis apparatus according to the first and second embodiments. The optical windows 22a and 22b are two-fold rotationally symmetric (point symmetric) with respect to the center of symmetry of the separation channel 16 provided in the microchip (15 and 17), and the optical windows 22a and 22b are respectively close to the holding substrate 21. The distance from the short side is equal, and is located in the center of the short side.

  In the bidirectional electrophoresis apparatus according to the third embodiment of the present invention, the direction of electrophoresis in the separation channel 16 is changed by 180 degrees by the selection of the optical windows 22a and 22b, so the microchip (15, 17). Need not be detachably mounted on the holding substrate 21, and once the position of the microchip (15, 17) with respect to the holding substrate 21 is set, the position may be fixed. Tip fasteners 23, 23a, and 23b as shown in FIGS. 1 to 4 and 7 are not particularly required on the upper surface, but they may be provided.

Selection of the optical windows 22a and 22b in the bidirectional electrophoresis apparatus according to the third embodiment of the present invention is performed with the microchip (15, 17) fixed to the holding substrate 21:
(A) A method of rotating the holding substrate 21 together with the microchip (15, 17) with respect to the optical measurement system 5;
(B) A method of fixing the holding substrate 21 and moving the optical measurement system 5;
Either of these can be adopted.

  Depending on the selection of the optical windows 22a and 22b, the polarities of the electrodes 11a and 11b also need to be reversed. However, the reversal of the polarities of the electrodes 11a and 11b may be electrically reversed. , 22b may be physically (spatially) replaced with 22b. Thus, in the bidirectional electrophoresis apparatus according to the third embodiment of the present invention, the direction of the separation channel 16 can be reversed 180 degrees with the microchip (15, 17) fixed to the holding substrate 21. The positioning mechanism of the microchip (15, 17) with respect to the holding substrate 21 is not necessary.

  The description of the other parts of the structure of the bidirectional electrophoresis device according to the third embodiment is the same as the description of the structure of the bidirectional electrophoresis device according to the first and second embodiments. Description to be omitted is omitted. Since the explanation of the operation of the electrophoretic separation and analysis is the same as the explanation of the operation of the bidirectional electrophoresis apparatus according to the first embodiment, a duplicate description is omitted.

  In the bidirectional electrophoresis apparatus according to the third embodiment of the present invention, as in the bidirectional electrophoresis apparatus according to the first and second embodiments, the microchip (15, 17) is formed by MEMS technology. A microchannel of micron order is formed on the surface and the separation flow path 16 is formed, so that the mechanical strength can be sufficiently increased, and the outer wall of the capillary is coated to obtain the strength as in capillary electrophoresis. Is unnecessary.

  In the bidirectional electrophoresis apparatus according to the third embodiment of the present invention, the two optical windows 22a and 22b are rotated twice with respect to the symmetrical center of the separation channel 16 provided in the microchip (15, 17). Since the holding substrate 21 is provided so as to be symmetric (point symmetric), the separation channel 16 can be used in both forward and reverse directions by switching the two optical windows 22a and 22b. That is, in the bidirectional electrophoresis apparatus according to the third embodiment, the optical measurement system 5 of the bidirectional electrophoresis apparatus, the electrode 11a, the microchip (15, 17) once set on the holding substrate 21. Electrophoresis from both directions of the separation channel 16 can be performed easily and in a short time without making the main body side mechanism such as 11b movable and re-installing the microchip (15, 17) on the holding substrate 21.

  Therefore, according to the bidirectional electrophoresis apparatus according to the third embodiment of the present invention, similarly to the bidirectional electrophoresis apparatus according to the first and second embodiments, the separation channel 16 for separation is used. Even if the performance deteriorates due to the deterioration of the inner wall coating, the adsorption of protein or the like to the inner wall of the separation channel 16 and the trouble on the injection side such as physical breakage, the two optical windows 22a and 22b are switched to reverse the performance. By using the separation channel 16 from the direction (second direction), the number of reuse can be improved, and the analysis cost can be reduced. According to the bidirectional electrophoresis apparatus according to the third embodiment of the present invention, when a trouble occurs only on one side of the separation channel 16 at the time of manufacturing, it can be used from the opposite side of the separation channel 16, so that the manufacturing yield is improved. It is possible to make the same as in the bidirectional electrophoresis apparatus according to the first and second embodiments.

(Modification of the third embodiment)
The configuration shown in FIG. 9 is an example. For example, as shown in FIG. 10, an irradiation optical system 5a that irradiates light onto the separation channel 16 of the microchip (15, 17) above the microchip (15, 17). In addition, a detection optical system 5b for measuring fluorescence or transmitted light from the separation channel 16 of the microchip (15, 17) may be provided below the microchip (15, 17). Alternatively, contrary to FIG. 10, an irradiation optical system 5a for irradiating light to the separation channel 16 of the microchip (15, 17) is provided below the microchip (15, 17), and the microchip (15, 17). A detection optical system 5b for measuring fluorescence or transmitted light from the separation channel 16 of the microchip (15, 17) may be provided above the above. As shown in FIG. 10, the upper lid 14 has two incident optical windows 28a and 28b with the midpoint of the separation channel 16 aligned with the center of symmetry of the microchip (15, 17). They are arranged symmetrically. Furthermore, the optical windows 22a and 22b provided on the holding substrate 21 are twice rotationally symmetric (point symmetric) with respect to the symmetry center of the separation channel 16 provided on the microchip (15 and 17).

  In the bidirectional electrophoresis apparatus according to the modification of the third embodiment of the present invention shown in FIG. 10, the irradiation optical system 5a includes a light source 51 disposed above the microchip (15, 17), a light source Below 51, it has the band pass filter 52 provided in the optical path which the light radiate | emitted from the light source 51 advances toward the separation flow path 16 of a microchip (15, 17). The detection optical system 5b includes a dichroic mirror 53 disposed below the optical window 22, a bandpass filter 54 through which light reflected by the dichroic mirror 53 passes, and a microchip (15, 17) via the bandpass filter 54. ) To detect fluorescence or transmitted light emitted from the separation channel 16. When the detector 55 detects the transmitted light emitted from the separation channel 16 of the microchip (15, 17), an absorption measurement is performed. The light source 51 of the irradiation optical system 5a and the detector 55 of the detection optical system 5b are connected to the data processing unit 33, and the data processing unit 33 performs data processing necessary for fluorescence measurement or absorbance measurement.

  In the bidirectional electrophoresis apparatus according to the modification of the third embodiment of the present invention, two optical windows 22a and 22b and two incident optical windows 28a and 28b are provided in the microchip (15 and 17), respectively. Since the holding substrate 21 is provided so as to be twice rotationally symmetric (point symmetric) with respect to the center of symmetry of the separation channel 16 formed, the two optical windows 22a and 22b and the corresponding two incident optical windows 28a are provided. , 28b, the separation channel 16 can be used in both the forward and reverse directions. That is, in the bidirectional electrophoresis apparatus according to the modification of the third embodiment, the optical measurement system 5 of the bidirectional electrophoresis apparatus is applied to the microchip (15, 17) once set on the holding substrate 21. Electrophoresis from both directions of the separation channel 16 can be carried out easily and in a short time without making the main body side mechanisms such as the electrodes 11a and 11b movable and re-installing the microchip (15, 17) on the holding substrate 21.

  Therefore, according to the bidirectional electrophoresis apparatus according to the modification of the third embodiment of the present invention, the inner wall coating of the separation channel 16 for separation is deteriorated, the protein or the like is applied to the inner wall of the separation channel 16. Even if the performance deteriorates due to a trouble on the injection side such as adsorption and physical damage, the two optical windows 22a and 22b and the corresponding two incident optical windows 28a and 28b are switched in the reverse direction (second By using the separation channel 16 from the (direction), the number of reuses can be improved and the analysis cost can be reduced.

(Other embodiments)
As described above, the present invention has been described according to the first to third embodiments. However, it should not be understood that the description and drawings constituting a part of this disclosure limit the present invention. From this disclosure, various alternative embodiments, examples and operational techniques will be apparent to those skilled in the art.

  In the description of the first and second embodiments already described, the positioning mechanism using the bowl-shaped (L-shaped) chip fasteners 23, 23 a, and 23 b has been described. , 23a and 23b, and are provided at predetermined locations on the microchip (15, 17) by etching or the like so as to match the markers provided at corresponding locations on the holding substrate 21 side. Thus, the separation channel 16 and the optical window may be aligned.

  In the description of the first to third embodiments, the electrodes 11a and 11b that apply a high voltage necessary for electrophoresis to the separation channel 16 reach the bottoms of the sample reservoirs 13a and 13b from the buffer reservoirs 12a and 12b side. However, as shown in FIG. 11, the electrodes 11 a and 11 b may be embedded in the microchips (15 and 17) below the separation channel 16. In this case, as shown in FIG. 11, a high voltage necessary for electrophoresis is supplied from the voltage control unit 31 to the electrodes 11a and 11b through vias (through holes) provided inside the holding substrate 21. The

  As shown in FIG. 11, when the electrodes 11a and 11b are embedded in the microchip (15, 17), the selection of the optical windows 22a and 22b in the bidirectional electrophoresis apparatus according to the third embodiment is as follows. When the microchip (15, 17) is rotated in the holding substrate 21 and the holding substrate 21 is rotated with the microchip (15, 17) with respect to the optical measurement system 5, or the holding substrate 21 is fixed and the optical measurement system 5 is fixed. Is moved, the polarity of the electrodes 11a and 11b may be reversed.

  As described above, the present invention naturally includes various embodiments not described herein. Therefore, the technical scope of the present invention is defined only by the invention specifying matters according to the scope of claims reasonable from the above description.

1 is a block diagram showing a basic configuration of a bidirectional electrophoresis apparatus according to a first embodiment of the present invention, centering on a cross-sectional view of a microchip. It is a perspective view which shows the holding substrate used for the bidirectional | two-way electrophoresis apparatus which concerns on the 1st Embodiment of this invention. It is a perspective view explaining the assembly state of the microchip used for the bidirectional | two-way electrophoresis apparatus which concerns on the 1st Embodiment of this invention, a holding substrate, a glass plate, and an upper cover. It is a perspective view explaining the state which arrange | positions a microchip with respect to a holding substrate in the direction opposite to the direction shown in FIG. 3 by 180 degrees. The relationship between the resolution and the number of base pairs obtained using the bidirectional electrophoresis apparatus according to the first embodiment of the present invention is the forward direction (first direction) and the reverse direction (second direction). It is a figure compared with. It is a block diagram which shows the basic composition of the bidirectional | two-way electrophoresis apparatus which concerns on the modification of the 1st Embodiment of this invention centering on sectional drawing of a microchip. FIG. 7A is a perspective view showing a holding substrate used in the bidirectional electrophoresis apparatus according to the second embodiment of the present invention, and FIG. 7B is a microchip on the holding substrate shown in FIG. It is a perspective view explaining the state which installed. FIG. 8A shows the same holding substrate as FIG. 7A, and FIG. 8B shows a state where the microchip is inverted 180 degrees and installed on the holding substrate shown in FIG. 8A. It is a perspective view explaining. It is a block diagram which shows the basic composition of the bidirectional | two-way electrophoresis apparatus which concerns on the 3rd Embodiment of this invention centering on sectional drawing of a microchip. It is a block diagram which shows the basic composition of the bidirectional | two-way electrophoresis apparatus which concerns on the modification of the 3rd Embodiment of this invention centering on sectional drawing of a microchip. It is a block diagram which shows the fundamental structure of the bidirectional | two-way electrophoresis apparatus which concerns on other embodiment of this invention centering on sectional drawing of a microchip.

Explanation of symbols

11a, 11b ... Electrodes 12a, 12b ... Buffer reservoirs 13a, 13b ... Sample reservoir 14 ... Upper lid 15 ... Glass plate 16 ... Separation channel 17 ... Microchip 21 ... Holding substrate 22, 22a, 22b ... Optical windows 23a, 23b ... Chip fasteners 28a, 28b ... Optical window for incidence 31 ... Voltage control unit 32 ... Temperature control unit 33 ... Data processing unit 5 ... Optical measurement system 5a ... Irradiation optical system 5b ... Detection optical system 51 ... Light source 52 ... Band pass filter 53 ... Dichroic mirror 54 ... Bandpass filter 55 ... Detector 61 ... Buffer

Claims (9)

A holding substrate having in part an optical window penetrating from the upper surface to the lower surface;
A microchip made of a transparent material, mounted on the upper surface, having a separation reservoir for electrophoresis on the surface and a sample reservoir connected to both ends of the separation passage;
A pair of electrodes for applying an electrophoresis voltage to the separation channel;
A bi-directional electrophoresis apparatus comprising: an optical measurement system that measures the electrophoresis in the separation channel through the optical window;
By setting the direction of the microchip together with the pair of electrodes to the holding substrate in a first direction and a second direction that is 180 degrees different from the first direction, the separation channel is reciprocated. A bidirectional electrophoresis apparatus characterized by being used in a direction. A holding substrate having in part an optical window penetrating from the upper surface to the lower surface;
A microchip made of a transparent material, mounted on the upper surface, having a separation reservoir for electrophoresis on the surface and a sample reservoir connected to both ends of the separation passage;
A pair of electrodes for applying an electrophoresis voltage to the separation channel;
A bi-directional electrophoresis apparatus comprising: an optical measurement system that measures the electrophoresis in the separation channel through the optical window;
By setting the direction of the microchip to a first direction and a second direction that is 180 degrees different from the first direction with respect to the holding substrate, the polarity of the pair of electrodes is reversed, and thus the separation is performed. A bidirectional electrophoresis apparatus characterized by using a flow path in a reciprocating direction.   The bidirectional electrophoresis apparatus according to claim 1 or 2, wherein the microchip forms a point-symmetric plane pattern with a center point of the separation channel as a center of symmetry.   The bidirectional electrophoresis apparatus according to claim 3, wherein the microchip has a planar pattern having corners.   The said holding | maintenance board | substrate is provided with the chip | tip fastener which fits the said corner | angular part to the said upper surface, The said optical window and the said separation flow path are aligned by this chip | tip fastener. The bidirectional electrophoresis apparatus as described. The holding substrate includes first and second chip fasteners on the upper surface, and the microchip defines a positioning end on the periphery of the microchip on a planar pattern,
By fitting the positioning end portion to the first chip fastener, the separation flow path for electrophoresis in the first direction and the optical window are aligned, and the positioning end portion is moved to the second tip. 3. The separation channel and the optical window that are electrophoresed in a second direction 180 degrees different from the first direction by positioning with a tip fastener are aligned with each other. The bidirectional electrophoresis apparatus according to 1. A holding substrate having in part a pair of optical windows penetrating from the upper surface to the lower surface;
A microchip made of a transparent material, which is mounted on the upper surface and has a separation channel for electrophoresis which is aligned with the pair of optical windows on the surface, and a sample reservoir connected to both ends of the separation channel. ,
A pair of electrodes for applying an electrophoresis voltage to the separation channel;
A bi-directional electrophoresis apparatus having an optical measurement system for measuring the electrophoresis in the separation channel through one of the pair of optical windows,
A bidirectional electrophoresis apparatus characterized in that the direction of electrophoresis of the separation channel can be selected by selecting one of the pair of optical windows and replacing a corresponding pair of electrodes or selecting a polarity. A holding substrate having in part an optical window penetrating from the upper surface to the lower surface, a separation reservoir for electrophoresis mounted on the upper surface, and a sample reservoir connected to both ends of the separation channel on the surface; Both having a microchip made of a transparent material, a pair of electrodes for applying a voltage for electrophoresis to the separation channel, and an optical measurement system for measuring the electrophoresis in the separation channel through the optical window A bidirectional electrophoresis method using a electrophoretic apparatus,
When the separation characteristics by electrophoresis along the first direction are deteriorated, the microchip is inverted with respect to the holding substrate in a second direction that is 180 degrees different from the first direction, and the separation flow path is reciprocated. A bidirectional electrophoresis method, characterized by being used in the above. A holding substrate having a pair of optical windows penetrating from the upper surface to the lower surface, a separation channel for electrophoresis mounted on the upper surface and aligned with the pair of optical windows on the surface, and the separation channel Sample reservoirs connected to both ends, a microchip made of a transparent material, a pair of electrodes for applying a voltage for electrophoresis to the separation flow path, and the pair of optical windows A bidirectional electrophoresis method using a bidirectional electrophoresis apparatus having an optical measurement system for measuring the electrophoresis in a separation channel,
When the separation characteristics by electrophoresis along the first direction are deteriorated, the selection of one of the pair of optical windows is switched from one to the other, and the pair of electrodes is switched or the polarity is switched accordingly. Thus, the bidirectional electrophoresis method is characterized in that the direction of electrophoresis is reversed in a second direction 180 degrees different from the first direction, and the separation channel is used in a reciprocating direction.

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