JP4431421B2 - Capillary electrophoresis device - Google Patents

Description

  The present invention belongs to a technical field related to an apparatus for separating and analyzing biological substances such as DNA, RNA, or protein, and in particular, a capillary array electrophoresis using a plurality of capillaries in a sample separation column and using a fluorescence detection method as a sample measurement method It belongs to the technical field related to equipment.

  Electrophoresis is a basic means for separating and analyzing DNA and RNA. Conventionally, a slab gel electrophoresis apparatus using a slab gel in which a gel is sandwiched between two glass plates has been used as an electrophoresis separation column. However, as the importance of large-scale separation analysis represented by the Human Genome Project has increased, high-speed, high-throughput performance has been demanded of the electrophoresis apparatus. An apparatus developed to realize this is a capillary electrophoresis apparatus.

  With regard to capillary electrophoresis, for example, Patent Document 1 describes a capillary array electrophoresis apparatus that performs on-column fluorescence measurement in which a plurality of capillaries are simultaneously irradiated with laser light to detect fluorescence from a sample component. As shown in FIG. 1, this apparatus includes a capillary 1, a laser beam 2, a glass flat plate 3, a first lens 4, an image dividing prism and a combination filter 5, a second lens 6, a two-dimensional CCD camera 7, and the like. Yes. Non-Patent Document 1 describes that multiplex PCR (polymerase chain reaction) -mini-sequencing is performed by capillary gel electrophoresis and liquid core waveguide fluorescence detection. Patent Document 2 describes a capillary electrophoresis apparatus that includes a capillary pre-filled with a gel and a buffer tank provided at the lower end of the capillary, and performs fluorescence detection from the lower end surface of the capillary. Patent Document 3 discloses an electrophoresis apparatus in which lower portions of a plurality of gel plates arranged side by side are positioned in a transparent lower buffer tank, and a photo detector of a fluorescence detector is installed facing the bottom surface in the buffer tank. Are listed. Patent Document 4 describes the use of a capillary container as a waveguide cell. Patent Document 5 describes the use of a gel injection mechanism that injects a gel into a capillary. As shown in FIG. 2, the gel injection mechanism includes a block 17, an injection syringe 14 for injecting the gel 10 into the capillary 111, a refilling syringe 15 for refilling the injection syringe 14 with the gel 10, and a check valve. 16

JP-A-9-96623 JP-A-10-19846 JP-A-8-261988 US Pat. No. 5,570,447 Japanese Patent No. 3389547 Johan Roeraade et al, Electrophoresis 2002 23 1467-1472 Anazawa, T., Takahashi, S., Kambara, H., Anal. Chem., 1996, 68, 2699-2704

  Capillary array electrophoresis apparatuses are required to perform simultaneous fluorescence measurement on a large number of capillaries with high sensitivity. In addition, it is required to continuously analyze a large number of samples. However, the conventional capillary array electrophoresis apparatus cannot meet such a request.

  An object of the present invention is to provide a capillary array electrophoresis apparatus that meets the above-mentioned demand and can perform simultaneous fluorescence measurement on a large number of capillaries with high sensitivity and can automatically analyze a large number of samples continuously. And

  In order to realize continuous automatic analysis using a capillary array electrophoresis apparatus, it is necessary to automatically fill a capillary with a polymer solution. Patent Document 5 describes a gel injection mechanism for injecting a gel into a capillary as described above. On the other hand, when examining a method for performing simultaneous fluorescence measurement with high sensitivity using a large number of capillaries at once, the method of performing fluorescence detection described in Patent Document 2 from the lower end surface of the capillary (hereinafter referred to as end detection) is This is advantageous because the capillary ends can be arranged two-dimensionally.

  Therefore, consider applying end detection to the apparatus of Patent Document 5. FIG. 3 is a cross-sectional view of the polymer filled block 400. The polymer is filled into a capillary 101 in which a large number of polymers are bundled through a flow path 403 by a syringe 401. When end detection is performed using the polymer filling block 400, fluorescence detection must be performed from the long axis direction of the terminal end portion 103 of the capillary 101, that is, through the left side surface 400 a of the polymer filling block 400. The fluorescence detection optical system includes a first camera lens 301, an optical filter 303, a second camera lens 302, and a two-dimensional CCD camera 300. However, since the material of the polymer-filled block 400 is an acrylic resin, the fluorescence or scattered light generated from the acrylic resin becomes background light, which causes a problem that the fluorescence detection sensitivity is lowered. In addition, an opaque flow path 403 that is bent between the capillary detection end face 105 and the side face 400a exists inside the polymer filling block 400, so that the fluorescence emitted from the capillary detection end face 105 is refracted until it reaches the fluorescence detection optical system. , Reflected and scattered, making fluorescence detection difficult.

In general, in order to perform highly sensitive fluorescence detection, the brightness of the first camera lens 301 is preferably F ≦ 1.2. However, the focal length of a commercially available camera lens that satisfies this condition is f <50 mm. is there. In order to make the fluorescence emitted from the capillary detection end face 105 by the first camera lens 301 into a parallel light beam, the distance L between the first camera lens 301 and the capillary detection end face 105 is set to the focal length f of the first camera lens 301. It needs to be equal. That is, the relationship of the following formula (1) must be satisfied.
L = f <50mm (1)

On the other hand, since the distance between the capillary detection end face 105 and the side face 400a of the polymer filling block 400 is 70 mm or more, the relationship of the following formula (2) must be satisfied in terms of structure.
L> 70mm (2)

  Since the above equations (1) and (2) are clearly incompatible, it is impossible to perform highly sensitive capillary end detection using the polymer packed block 400.

  As described above, the continuous automatic analysis of Patent Document 5 and the capillary end detection of Patent Document 2 cannot be compatible.

  As described above, it has been reported that a buffer tank is provided at the end of the capillary or at the bottom of the gel plate, and fluorescence detection is performed from the bottom. However, in that case, the capillary or the gel plate is pre-filled with a gel, and no means for injecting the gel, that is, the polymer has been required. In addition, in the case of continuous automatic analysis having a polymer injection means, the end of the capillary cannot be detected because of the configuration shown in FIG. The present invention provides a novel means for achieving both continuous automatic analysis and capillary end detection when a configuration having a polymer injection means is used. Accordingly, it is possible to provide a high-throughput capillary array electrophoresis apparatus capable of simultaneously measuring a large number of capillaries with high sensitivity and automatically analyzing a large number of samples.

The capillary array electrophoresis apparatus of the present invention irradiates a capillary with laser light, and detects emitted fluorescence by a fluorescence detection optical system through a polymer container (polymer-filled block) provided near the end of the capillary. It is characterized by doing. The polymer filled block desirably has the following characteristics.
(1) The material of the polymer-filled block in the region between the end face of the end of the capillary and the fluorescence detection optical system is made of a non-fluorescent substance. In particular, the material is preferably made of quartz.
(2) The ends of the capillaries are arranged so that the end faces of all the capillaries are substantially aligned on a plane, and the plane and all planes in which the refractive index within the polymer-filled block in the region changes are substantially equal to each other. The parallelism is within the accuracy error.
(3) The distance between the end face of the end of the capillary and the outer face of the polymer-filled block in the region is equal to or shorter than the focal length of the lens closest to the polymer-filled block in the fluorescence detection optical system. In particular, the distance is desirably 50 mm or less.
(4) A polymer or a buffer solution is contained, and fluorescence detection is performed through the polymer filling block.

  The fluorescence detection optical system includes an objective lens that is provided outside the detection window and detects fluorescence, and the distance between the end face of the capillary end and the outer surface of the detection window is equal to or less than the working distance of the objective lens. Also good.

  The capillary may be detachable from the polymer container. The end of the capillary may have positioning means for determining the position of the end face of the capillary end with respect to the outer surface of the detection window, and it may be a spacer provided in the detection window. Alternatively, one end of the capillary may come into contact with a solution having the same refractive index as that of the polymer filled in the capillary. The plurality of capillaries form a plurality of sheets at least partially arranged on a plane at an irradiation position where light is irradiated, and the plurality of sheets are substantially parallel to the plane. It may be arranged in. Further, a mechanism for removing Joule heat generated in the capillary when a voltage is applied to the capillary may be provided in the vicinity of one end of the capillary. Further, at least a part of the irradiation position irradiated with light is arranged on a substantially plane, and the light irradiates at least a part of the plurality of capillaries from a direction substantially parallel to the plane. It may be a thing.

  In another capillary array electrophoresis apparatus according to the present invention, the polymer accommodating portion introduces and discharges a polymer or a buffer in a direction substantially perpendicular to the direction in which the polymer moves inside the capillary.

  With the above apparatus configuration, fluorescence emitted from the end face of the terminal end of the capillary can be detected with high sensitivity via the polymer filling block. As a result, not only simultaneous fluorescence measurement of a large number of capillaries can be performed with high sensitivity, but also continuous automatic analysis of a large number of samples becomes possible.

  In addition, what was made into the polymer accommodating part or the polymer filling block here is named in this way for convenience, and the function is not restricted to accommodating a polymer or filling a polymer. A sheet formed by a plurality of capillaries indicates a state in which a plurality of capillaries are arranged.

  According to the present invention, high-sensitivity simultaneous fluorescence measurement of a large number of capillaries is possible, and a continuous automatic analysis of a large number of samples is possible.

  Embodiments of the present invention will be described below with reference to the drawings.

Example 1
FIG. 4 shows an overview of an example of a fully automatic capillary array electrophoresis apparatus using the present invention. In this example, a method for performing electrophoretic analysis of 20 capillaries simultaneously will be described. Twenty capillaries are bundled to form a capillary array 101a. Each capillary 101 is made of quartz and has a polyimide coating on the outer surface, a total length of 40 cm, an outer diameter of 360 μm, and an inner diameter of 50 μm. The side of each capillary 101 into which the sample is introduced is referred to as a start end portion 102, and the side where the sample migrates inside the capillary by electrophoresis and is eluted is referred to as a termination portion 103. A position 30 cm from the end face of the start end 102 of each capillary 101 (10 cm from the end face of the terminal end 103) is set as a laser irradiation position, and the polyimide coating on the capillary in that portion is removed. The laser irradiation positions of five capillaries 101 are arranged, and four sets of capillary arrays are formed. At the laser irradiation position, the capillaries 101 are arranged substantially parallel to each other, and the laser irradiation positions of the capillaries are arranged substantially in a straight line. Here, the fact that the capillaries are substantially parallel to each other means that the capillaries have a degree of parallelism within a precision error. Further, the capillaries are arranged so that at least a part thereof is arranged substantially on a plane at the irradiation position. This flat surface has a flatness that is within an accuracy error. Furthermore, it is arranged in a plurality of sheets so as to form a plurality of sheets. Each sheet is disposed substantially parallel to a plane formed by arranging the capillaries. This parallel refers to a degree of parallelism that falls within an accuracy error. Laser light 202 (wavelengths 488 nm and 515 nm, output 100 mW) oscillated from an argon ion laser light source 200 is divided into four by a beam splitter 206 and a mirror 207, and irradiates four sets of capillary arrays. Each laser beam 202 is adjusted so as to be substantially parallel to each capillary 101 and is applied to the capillary array. Here, the laser beam is applied to each capillary substantially in parallel means that the direction in which the laser beam is applied is substantially parallel to a substantial plane formed by arranging the capillaries. Say. In order to suppress a decrease in the separation performance of electrophoresis, it is desirable that the laser width irradiated to the capillary array is not more than the inner diameter (50 μm) of the capillary 101. When the above laser irradiation is performed while the inside of each capillary 101 is filled with a polymer solution (Applied Biosystems POP-4) as a separation medium, the laser beam propagates through the capillary array. At the same time, it can be irradiated efficiently.

  FIG. 5 is a perspective view of the vicinity of the end portion 103 of the capillary array 101a. At the terminal end portion 103 of the capillary array 101a, 20 capillaries 101 are bundled to form a capillary array head 107. The capillary array head 107 is composed of a terminal portion 103 of 20 capillaries 101, a cylindrical fixing jig 106, and an adhesive 108. Here, the end face (capillary detection end face 105) of the end portion 103 of each capillary 101, the edge of the fixing jig 106, and the surface of the adhesive 108 are arranged substantially in a plane to form a detection plane 109. The capillary detection end faces 105 are arranged in a 5 × 4 lattice pattern on the detection plane 109. Here, the position at the start end 102 of each capillary 101 and the position at the capillary detection end face 105 are made to correspond to each other. The material of the fixing jig 106 is preferably a hard material having low electrical conductivity. In addition, since the material of the adhesive 108 is in contact with the polymer solution, it is preferable that the adhesive 108 is not easily decomposed into water so as not to affect the separation of electrophoresis and has high chemical stability. In this example, polyether ether ketone was used as the material for the fixing jig 106, and a silicone-based adhesive (cemedine super X black, cemedine) was used as the adhesive 108.

  As shown in FIG. 4, the capillary array head 107 is connected to the polymer filling block 404. A cross-sectional view of the polymer filling block 404 with the capillary array head 107 connected thereto is shown in FIG. By tightening the screw 110, the ferrule 111 is pressed against the inner wall surface of the polymer filling block 404. At the same time, the ferrule 111 tightens the capillary array head 107, and the gap between the polymer filling block 404 and the capillary array head 107 is sealed by the ferrule 111. The This structure has pressure resistance, and this sealing is maintained even when a high pressure is applied inside the polymer filling block 404. As the material of the ferrule 111 and the screw 110, polyether ether ketone which is hard and has low electric conductivity was used. The polymer filling block 404 is made of acrylic resin, and a flow path 405 is formed inside. The flow path 405 is connected to a pressure-resistant syringe 401 and a pressure-resistant tube 501, and these are filled with a polymer solution. The tube 501 is connected to a buffer tank 502 containing a buffer (3700 buffer, Applied Biosystems) through the valve 402 shown in FIG.

  A part of the flow path 405 is in contact with the detection plane 109, and the surface of the detection plane 109 is filled with the polymer solution. Thus, by performing fluorescence detection in a state where the flow path 405 is filled with the polymer solution, there is no refractive index change boundary surface between the capillary 101 and the flow path 405 at the capillary detection end face 105, and the capillary detection end face 105 There is no reflection while the emitted fluorescence enters the channel 405. For this reason, an effect of suppressing a decrease in sensitivity when the fluorescence emitted from the capillary detection end face 105 is detected by the two-dimensional CCD camera 300 can be obtained. The same effect can be obtained by filling the channel 405 with a solution other than the polymer solution having the same refractive index as that of the polymer solution. Of course, fluorescence detection may be performed by filling the channel 405 with a buffer solution. Further, at a portion of the polymer filling block 404 adjacent to the detection plane 109 of the capillary array, the polymer or the buffer solution is introduced and discharged in a direction substantially perpendicular to the direction in which the polymer moves inside the capillary. Here, the term “substantially vertical” specifically means that an angle in the range of about 85 degrees to about 95 degrees is appropriate. By adopting such an arrangement, it is possible to efficiently introduce and discharge the polymer or buffer solution, and it is possible to reduce the size of the apparatus due to the ease of pipe arrangement.

  The fluorescence emitted from the capillary detection end face 105 passes through the flow path 405 filled with the polymer solution and the detection window 306, and the first camera lens 301 (F = 1.2, f = 50) from below the polymer filling block 404. ), The optical filter 303, the diffraction grating 304, the second camera lens 302 (F = 1.2, f = 50), and the detection unit 307 configured by the two-dimensional CCD camera 300 of 512 × 512 pixels.

  In order to reduce fluorescence and scattered light from materials constituting the polymer-filled block 404 other than the fluorescence from the sample, the detection window 306 is made of non-fluorescent quartz glass. As the detection window 306, an optical filter that can exclude back fluorescence and excitation light may be used. Alternatively, the entire polymer filling block 404 may be made of a non-fluorescent and transparent material, and the polymer filling block 404 and the detection window 306 may be integrated. In this embodiment, the distance from the detection plane 109 of the capillary array head 107 to the outer surface of the detection window 306 is 20 mm, which is smaller than the focal length of 50 mm of the first camera lens. The detection window 306 is located between the detection plane 109 of the capillary array head 107, that is, a region including each end face of the capillary, and the opening of the first camera lens 301. The shape of the detection window 306 in a plane substantially parallel to the included region may be a shape substantially similar to the included shape. Further, the area of the included area, the detection window 306, and the opening of the first camera lens 301 may increase in this order. In this case, light can be efficiently detected by the first camera lens 301.

  The capillary array head 107 can be positioned using a spacer. Specifically, with the detection window 306 as shown in FIG. 7, the detection plane 109 is pressed against the four positioning pins 310 on the inside so that the detection plane 109 is substantially parallel to the detection window 306. To be able to position. As a result, even when the capillary array is replaced, the detection plane 109 and the detection window 306 can be connected substantially in parallel and at the above distance with good reproducibility. As another method for positioning the detection plane 109, a step is formed on the outer shape of the fixed jig 106 in the vicinity of the detection plane 109, and a step is formed on the inner wall of the portion where the capillary array head 107 of the polymer filling block 404 is inserted, You may match | combine by pressing the level | step difference of the fixing jig 106 on the level | step difference of the polymer filling block 404. FIG. Thereby, the position of the detection plane 109 of the capillary array head 107 with respect to the outer surface of the detection window can be determined.

  In this embodiment, the thickness of the detection window 306 is 19 mm, and the distance L between the detection plane 109 and the inner surface of the detection window 306 is 1 mm. When the distance L is smaller than the inner diameter of the capillary 101, a large electric resistance is generated in the flow path 405 between the detection plane 109 and the detection window 306, and bubbles are generated from the polymer solution in the flow path 405 by Joule heat when a voltage is applied. It tends to occur and electrophoresis becomes impossible. Therefore, the distance L needs to be equal to or greater than the inner diameter of the capillary 101. When the capillary array 101a is connected to the polymer filling block 404, if bubbles remain in the flow path 405, it is necessary to open the valve 402, pressurize the syringe 401, and push the bubbles into the buffer solution tank 502. . If the distance L is too large, the polymer solution cannot be swept away at high speed in the flow path 405 when the syringe 401 is pressurized, it becomes difficult to discharge the bubbles, and the amount of the polymer solution consumed increases. Therefore, the distance L needs to be within 2 mm. As described above, the distance L is set so that the inner diameter of the capillary 101 <L <2 mm, thereby suppressing the generation of bubbles from the polymer solution in the upper air flow path 405 when a voltage is applied to the capillary 101. When the array 101 a is connected to the polymer filling block 404, it is possible to easily discharge bubbles remaining in the flow path 405 to the buffer solution tank 502.

  The process of analyzing the sample is as follows. First, the valve 402 is closed, and the polymer solution inside the polymer filling block 404 is pressurized by applying a load to the piston of the syringe 401, and the capillary 101 is filled with the polymer solution from the end portion 103 toward the start end portion 102. After filling a certain amount of polymer solution into the capillary 101, the valve 402 is opened, and the starting end 102 of each capillary 101 is immersed in the sample solution in each well. In this state, a constant voltage is applied between the buffer solution tank 502 and the sample solution by a high voltage power source 506 for a certain period of time, and the sample labeled with the phosphor in each well is electrically injected into each capillary 101. . After the sample injection, the buffer solution tank 505 containing the buffer solution is moved to the position of the start end portion 102 of the capillary 101, the start end portion 102 is immersed in the buffer solution, and the high voltage power source 506 is interposed between the buffer solution tank 502 and the buffer solution tank 505. A voltage is applied by, and the sample injected into each capillary 101 is electrophoresed toward the terminal portion 103. At this time, there was no difference in level between the buffer solution in the buffer solution tank 502 and the buffer solution in the buffer solution tank 505 so that the polymer solution in the capillary 101 would not move due to the pressure difference.

  A sample to be electrophoresed in each capillary 101 is irradiated with a laser at a laser irradiation position of the capillary 101. The fluorescent substance labeled on the sample is excited by laser irradiation, and a part of the fluorescence is totally reflected on the inner surface of the capillary 101 and propagates inside the capillary 101 and is emitted from the detection end face 105 of each capillary 101. The emitted fluorescence is converted into a parallel light flux by the first camera lens 301 through the detection window 306 of the polymer filling block 404, the back fluorescence and excitation light are excluded by the optical filter 303, and wavelength-dispersed by the diffraction grating 304. The image is formed on the two-dimensional CCD camera 300 by the camera lens 302. Here, an optical prism may be used in place of the diffraction grating 304 as the fluorescence wavelength dispersion means. An objective lens may be used instead of the first camera lens 301. When an objective lens is used, the distance from the detection plane 109 of the capillary array head 107 to the outer surface of the detection window 306 is set to be equal to or smaller than the working distance of the objective lens, and the distance between the objective lens and the detection plane 109 is set to the objective plane. By setting the working distance of the lens, a similar high-sensitivity fluorescence detection system can be constructed. The distance between the first camera lens and the capillary detection end face 105 was 50 mm. Images in which the fluorescence from each capillary 101 is wavelength-dispersed are formed at different positions on the two-dimensional CCD camera 300. Thereby, the fluorescence from each capillary 101 can be detected independently and collectively. Moreover, the time change of the fluorescence from each capillary 101 is measured by repeating this detection continuously. A plurality of types of samples can be analyzed by recording and analyzing the obtained measurement results in a computer.

  Since all of the above steps can be automatically repeated, unattended continuous analysis of a large number of samples becomes possible.

  If stray light from the outside enters the detection unit 307 at the time of fluorescence detection, the sensitivity of fluorescence detection emitted from the capillary detection end face 105 is reduced. Therefore, it is desirable to shield the areas of the capillary array head 107, the polymer filling block 404, and the detection unit 307 from the outside from the laser irradiation position of the capillary 101. In this example, the entire area was covered with a dark box. It may be divided into the above three areas and covered with a dark box. Alternatively, the polymer filling block 404 may be made of black acrylic resin or black plastic to block stray light from the outside.

  In this embodiment, the number of capillaries to be measured simultaneously is 20. However, with the apparatus using the present invention, a larger number of capillaries can be detected simultaneously and with high sensitivity, and continuous unattended operation of the sample becomes possible. . In order to measure fluorescence from a plurality of capillaries 101 independently and with high sensitivity, the imaging distance on the two-dimensional CCD camera 300 of the adjacent capillaries 101 needs to be at least 5 pixels or more away. 102 capillaries can be arranged in the first dimension on the CCD camera 300. If the number of pixels required in the wavelength dispersion direction per capillary 101 is 100, it is possible to arrange five or more capillaries 101 in the second dimension on the two-dimensional CCD camera 300. Therefore, for example, in the present embodiment, if a capillary array in which five capillary arrays are arranged into 96 capillary arrays and the capillary arrays are arranged in four rows is used, simultaneous measurement of 384 capillaries becomes possible.

(Example 2)
In this embodiment, the polymer filling block 404 in FIG. 4 is divided into two parts, a part to which the syringe 401 is attached and a part to which the capillary array head 107 is attached, thereby facilitating operations such as washing and replacement of the detection window. Aimed to get.

  The configuration of the apparatus is different from that of the first embodiment only in the configuration of the polymer filling block 404, and is otherwise the same. FIG. 8 shows a configuration of a polymer filling block 406 and a detection block 407 as an attachment portion of the capillary array head 107 according to this embodiment. The capillary array head 107 is connected to the detection block 407 using screws 110 and a ferrule 111. The method for connecting the capillary array head 107 to the detection block 407 and the connection position alignment of the capillary array head 107 are the same as in the first embodiment. The detection block 407 is made of acrylic resin, and as shown in FIG. 8, a detection window 306 is attached to a portion of the capillary array head 107 that is located directly below the detection plane 109. The detection window 306 is non-fluorescent quartz glass. A flow path 408 formed in the detection block 407 is filled with a polymer solution. The surface of the detection plane 109 of the capillary array head 107 is in contact with the polymer solution in the flow path 408. The distance between the detection plane 109 of the capillary array head 107 and the outer surface of the detection window 306 is 20 mm, and the detection plane 109 and the inner and outer surfaces of the detection window 306 are substantially parallel to each other.

  One of the flow paths 408 in the detection block 407 is connected to a flow path 410 formed in the polymer filling block 406 via a tube 409 filled with the polymer solution, and the flow path is filled with the polymer solution. It is connected to the syringe 401. The other side of the channel 408 is connected to a tube 501 filled with the polymer solution, and is connected to the buffer solution tank 502 shown in FIG. The tube 409 and the tube 501 are connected to the detection block 407 with a pressure-resistant connector, and the attachment and detachment of the tubes 409 and 501 and the detection block 407 are simplified.

  With the above configuration, even if either the polymer filling block 406 or the detection block 407 is contaminated, the contamination can be removed by washing only the contaminated block. Further, in order to prevent external stray light from entering the detection unit 307, the method of covering the entire polymer-filled block 404 with a dark box is employed in the first embodiment, but in this embodiment, the light shielding region can be reduced in size. Furthermore, in Example 1, the polymer filling block 404 needs to be enlarged so that there is no steric hindrance between the capillary array 101a and the syringe 401. However, in this example, the steric hindrance can be avoided, so The filling block can be downsized. Also in Example 2, the polymer or buffer solution is introduced in a direction substantially perpendicular to the direction in which the polymer moves inside the capillary at a portion adjacent to the detection plane 109 of the capillary array of the detection block 407, and Discharged. Here, the term “substantially vertical” specifically means that an angle in the range of about 85 degrees to about 95 degrees is appropriate. By adopting such an arrangement, it is possible to efficiently introduce and discharge the polymer or buffer as in the first embodiment, and it is possible to reduce the size of the apparatus due to the ease of arrangement of the tubes.

  9, there is a method of integrating the flow path 408 and the detection window 306 sandwiched between the detection window 306 and the detection plane 109 in FIG. 8 and the capillary array head 107 as shown in FIG. 9. As in FIG. 8, the tube 409 and the tube 501 are connected to the capillary array head 107 by a pressure-resistant connector, and the attachment and detachment of the tubes 409 and 501 and the capillary array head 107 are simplified. Even in this method, the same effect as the method shown in FIG. 8 can be obtained.

(Example 3)
In Example 1 or Example 2, when a voltage is applied to each capillary 101 for electrophoresis, Joule heat is generated in the capillary 101. In the capillary array head 107 shown in FIG. 5, since the capillary 101 is surrounded by the adhesive 108, the fixing jig 106, the polymer filling block 404 or the detection block 410, the effect of radiating Joule heat generated in the capillary is low. Therefore, the vicinity of the capillary array head 107 becomes high temperature. As a result, the polymer solution in the capillary 101 near the capillary array head 107 and the polymer solution in the flow path formed between the detection plane 109 and the detection window 306 may boil and bubbles may be generated. When falling into such a state, not only the performance of electrophoresis is remarkably lowered, but also fluorescence detection cannot be performed. In this embodiment, an object is to effectively remove Joule heat generated in the capillary array head 107 during electrophoresis.

  In this embodiment, the configuration other than the capillary array head 107 is the same as that of the first or second embodiment. A perspective view of the vicinity of the capillary array head 107 is shown in FIG. The capillary array head 107 is formed of four sets of capillary arrays 112 formed by arranging five capillaries 101 in a substantially planar shape, a fixing jig 106, an adhesive 108, and five metal plates 113. Yes. The four sets of capillary arrays 112 and metal plates 113 are arranged alternately and substantially parallel to each other. The materials of the fixing jig 106 and the adhesive 108 are the same as those in the first embodiment. A gap other than the metal plate 113 and the capillary 101 in the fixing jig is filled with the adhesive 108. In FIG. 10, the metal plate 113 is disposed so as not to protrude from the detection plane 109 to the lower side of the fixing jig 106 and to protrude to the upper side of the fixing jig 106. In this embodiment, an aluminum material is used for the metal plate 113, but in order to prevent the flow of electricity from the polymer solution in contact with the detection plane 109 to the metal plate 113, the periphery of the aluminum material was coated with polyimide. As a material of the metal plate 113, ceramics having low electrical conductivity and high thermal conductivity may be used. In the upper part of the fixing jig 106, all the five metal plates 113 are connected so as to be sandwiched between the two heat sinks 114. During electrophoresis, Joule heat generated from the capillary 101 is dissipated into the air from the heat sink 114 via the metal plate 113.

  According to this embodiment, Joule heat generated from the capillary 101 during electrophoresis can be effectively removed, generation of bubbles from the polymer solution in the flow channel in contact with the detection plane 109 can be suppressed, and stable electrophoresis and fluorescence detection can be achieved. It can be carried out. When the number of capillaries to be analyzed simultaneously is increased and the voltage applied to the capillaries 101 is increased, the amount of Joule heat generated in the capillary array head 107 is increased. Therefore, this embodiment is very effective in suppressing the generation of the bubbles, particularly under the above conditions.

  In the present embodiment, the heat sink 114 is used as a method for removing Joule heat generated from the capillary 101 during electrophoresis. However, instead of the heat sink 114, a Peltier is attached to the metal plate 113 to control the temperature in the vicinity of the capillary array head 107. You may do it. Thereby, in addition to the effective removal of the Joule heat, the temperature of the polymer solution in each capillary 101 in the vicinity of the capillary array head 107 is stabilized, so that a stable electrophoresis result is always obtained without being affected by the outside air temperature. can get.

Example 4
In the laser irradiation method to the capillary 101 in the first embodiment, the variation in the distance from the laser optical axis of the capillary 101 shown in FIG. 4 can greatly affect the laser irradiation efficiency to each capillary 101.

  The purpose of this embodiment is to irradiate all capillaries with the same laser intensity regardless of the capillary variation.

  This embodiment is the same as the first embodiment or the second embodiment except for the irradiation position and irradiation method of the capillary 101 shown in FIG. In the vicinity of the laser irradiation position where the laser of the capillary is irradiated, the capillaries are arranged substantially in parallel and on a plane. Here, the fact that the capillaries are substantially parallel to each other means that the capillaries have a degree of parallelism within a precision error. In addition, the plane here refers to a plane having a degree of flatness that is within an accuracy error. A laser oscillated from a laser light source is spread by a beam expander, converged in a line shape by a cylindrical lens, and all the capillaries are irradiated simultaneously from a direction perpendicular to the arrangement surface of the capillaries. This makes it possible to irradiate all capillaries with substantially the same laser intensity regardless of the variation in the capillaries.

  Another method of irradiating the capillary with the laser is described below. The laser light emitted from the laser light source is reflected by a mirror, condensed by an objective lens, and irradiated to the laser irradiation position of the capillary. Since the mirror and the objective lens constitute a drive unit and can be reciprocated at high speed in the same direction as the direction in which the capillaries are arranged, it is possible to scan each capillary one after another with a laser. This laser irradiation method can achieve the same effect as described above.

(Example 5)
As a method of simultaneously irradiating a large number of capillaries in a capillary array electrophoresis apparatus with laser, there is a method (expand method) in which laser light emitted from a laser light source is spread by a beam expander or the like and irradiated onto the capillaries. In this method, for example, when the outer diameter of the capillary is 150 μm, the inner diameter is 50 μm, and the number of capillaries to be irradiated is 20, the irradiation efficiency per capillary is about 1.7%. On the other hand, there is a method in which the capillaries are arranged in parallel and on a plane, and a laser beam is irradiated from the side surface direction of the plane in which the capillaries are arranged (multi-focus method) (Non-patent Document 2). In this case, under the same conditions as in the above example, the irradiation efficiency per capillary is about 50%. The width of the capillary array is 3 mm or more, and the visual field range for condensing at a time becomes wide. Therefore, the fluorescence of the sample is condensed using a camera lens having an F value of about 1.4. The condensing efficiency of the lens is obtained by 1 / (16 × F ² ), and is about 3.2% when the F value is 1.4. The efficiency when the fluorescence emitted from the sample is received by a detector such as a CCD camera is determined by the irradiation efficiency × the condensing efficiency of the lens. The efficiency per capillary is about 0.054% for the expand method and about 1.6% for the multi-focus method.

  The present embodiment aims to increase the light receiving efficiency with a multi-focus method with high irradiation efficiency.

FIG. 11 shows the laser irradiation and detection method. In this embodiment, the capillary used, except the capillary end edge vicinity and the detection unit is equivalent to the first embodiment. The capillary 101 is made of quartz, and the outer surface is coated with a low refractive index fluororesin and has a total length of 40 cm, an outer diameter of 150 μm, and an inner diameter of 50 μm. The capillary is filled with a cross-linked gel (polyacrylamide gel) which is an electrophoretic separation medium. A fluid polymer solution may be used as the separation medium. The position 10 cm from the end face of the terminal portion 103 of each capillary 101 is set as the laser irradiation position, and the fluororesin coating of the capillary in that portion is removed. The laser irradiation positions of the 20 capillaries 101 are arranged to form a capillary array. The capillaries 101 are arranged in parallel to each other, and the laser irradiation positions are arranged perpendicular to the capillaries 101 and in a straight line.

  The laser beam 202 oscillated from the argon ion laser light source 200 is adjusted so as to be perpendicular to the capillaries 101 and is applied to the capillary array. As described above, in this irradiation method, the irradiation efficiency per capillary is about 50%.

  The end portions 103 are arranged in a lattice shape having a size of 0.75 mm × 0.6 mm and 5 × 4, and the fluorescence detection area becomes very small. A small value can be used. The end portion 103 is immersed in a buffer tank 502 filled with a buffer (3700 buffer, Applied Biosystems). The buffer bath may be filled with the same cross-linked gel as the separation medium. Moreover, you may fill with a fluid polymer. Fluorescence emitted from the end face of the capillary terminal portion 103 passes through the buffer solution from below in the buffer solution tank 502, the first objective lens 308 (magnification 20 times, numerical aperture 0.75, working distance 1 mm), optical filter 303, a second objective lens 309 (magnification 20 times, numerical aperture 0.75, working distance 1 mm), and detected by a detection unit 307 constituted by a two-dimensional CCD camera 300 of 256 × 256 pixels.

  In order to reduce fluorescence and scattered light other than the fluorescence from the sample, the material of the bottom surface of the buffer solution tank 502 is made of non-fluorescent quartz glass. As a material for the bottom surface of the buffer solution tank 502, an optical filter that can exclude background light and excitation light may be used. In this embodiment, the distance from the end face of the capillary terminal portion 103 to the bottom surface of the buffer solution tank 502 is adjusted to 0.1 mm, and the thickness of the bottom surface of the buffer solution tank is set to 0.5 mm. The working distance was smaller than 1 mm.

  A sample to be electrophoresed in each capillary 101 is irradiated with a laser at a laser irradiation position of the capillary 101. The fluorescent substance labeled on the sample is excited by the laser irradiation, and a part of the fluorescence is totally reflected on the inner surface of the capillary 101 and propagates inside the capillary 101 and is emitted from the end face of the capillary terminal portion 103. The emitted fluorescence is converted into a parallel light beam by the first objective lens 308 from below the buffer solution tank 502 through the buffer solution, the background light and the excitation light are excluded by the optical filter 303, and two-dimensionally by the second objective lens 309. The image is formed on the CCD camera 300. A diffraction grating or a prism may be inserted between the optical filter 303 and the second objective lens 309, and the fluorescence emitted from the capillary may be spectrally detected. The distance between the first objective lens 308 and the end face of the capillary terminal portion 103 was 1 mm. Since the light collection efficiency of the objective lens is 1 / {16 × (1 / (2 × numerical aperture))}, the light collection efficiency of the lens in this embodiment is about 14%. Therefore, the light receiving efficiency in the detector of fluorescence emitted from the sample per capillary is 7%, and this embodiment makes it possible to make the electrophoresis apparatus very sensitive.

(Example 6)
The laser irradiation method to the capillary in Patent Document 2 that measures fluorescence from the end of the capillary is the expanding method described in Example 5. When 100 capillaries having an outer diameter of 150 μm and an inner diameter of 50 μm are simultaneously measured, the irradiation efficiency per capillary is about 0.33%.

  The purpose of this embodiment is to increase the irradiation efficiency of a large number of capillaries such as 100, and simultaneously measure with high sensitivity.

In this embodiment, the capillary used, except the structure of the vicinity of the capillary end edge is equivalent to Example 1. FIG. 12 shows the laser irradiation and detection method. In this example, a method for performing electrophoretic analysis of 100 capillaries simultaneously will be described. 100 capillaries are bundled to form a capillary array 101a. Each capillary 101 is made of quartz and has an outer surface coated with a low refractive index fluororesin, and has a total length of 40 cm, an outer diameter of 150 μm, and an inner diameter of 50 μm. The capillary is filled with a cross-linked gel (polyacrylamide gel) which is an electrophoretic separation medium. A fluid polymer solution may be used as the separation medium. The position 10 cm from the end face of the terminal portion 103 of each capillary 101 is set as the laser irradiation position, and the fluororesin coating of the capillary in that portion is removed. The laser irradiation positions of 20 capillaries 101 are arranged to form 5 sets of capillary arrays. At the laser irradiation position, the capillaries 101 are arranged in parallel to each other, and the laser irradiation positions are arranged perpendicular to the capillaries 101 and in a straight line. The laser beam 202 oscillated from the argon ion laser light source 200 is divided into five by a beam splitter 206 and a mirror 207, and irradiates five sets of capillary arrays from the side. Each laser beam 202 is adjusted so as to be perpendicular to each capillary 101 and applied to the capillary array. The end portions 103 are arranged in a lattice shape having a size of 6 mm × 6 mm and 20 × 5. The end portion 103 is immersed in a buffer tank 502 filled with a buffer (3700 buffer, Applied Biosystems). The buffer bath may be filled with the same cross-linked gel as the separation medium. Moreover, you may fill with a fluid polymer. The fluorescence emitted from the end face of the capillary end portion 103 is detected by the detection unit 307 from below the buffer solution tank 502 through the buffer solution.

  According to this embodiment, the irradiation efficiency per capillary is about 10%, and it is possible to obtain an irradiation efficiency about 30 times that of the expanded system, and a large number of capillaries of 100 can be detected by one two-dimensional detector. Therefore, the device configuration becomes very easy.

The figure which shows the external appearance of the conventional capillary electrophoresis apparatus. The figure which shows the general view of the capillary electrophoresis apparatus which has a conventional gel injection | pouring mechanism. The figure which shows the cross section of the conventional polymer filling block. The figure which shows the general view of an example of the fully automatic capillary electrophoresis apparatus by this invention. FIG. 3 is a perspective view of the vicinity of a capillary array end portion in Embodiment 1 of the present invention. The figure which shows the cross section of the polymer filling block in Example 1 of this invention. The perspective view of the detection window in the polymer filling block in Example 1 of this invention. The figure which shows the cross section of the polymer filling block and detection block in Example 2 of this invention. The figure which shows the cross section of the capillary array head in Example 2 of this invention. The perspective view of the capillary array terminal part vicinity in Example 3 of this invention. The perspective view of the laser irradiation area | region and detection area vicinity in Example 5 of this invention. The perspective view of the laser irradiation area | region and the capillary terminal part vicinity in Example 6 of this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Capillary 2 ... Laser beam 3 ... Glass plane plate 4 ... 1st lens 5 ... Image division prism and combination filter 6 ... 2nd lens 7 ... Two-dimensional CCD camera 101 ... Capillary 101a ... Capillary array 102 ... Start end 103 ... End Part 104 ... Laser irradiation part 105 ... Capillary detection end face 106 ... Fixing jig 107 ... Capillary array head 108 ... Adhesive 109 ... Detection plane 110 ... Screw 111 ... Ferrule 112 ... Capillary array 113 ... Metal plate 114 ... Heat sink 200 ... Laser light source 202 ... Laser beam
206 ... Beam splitter 207 ... Mirror 300 ... Two-dimensional CCD camera 301 ... First camera lens 302 ... Second camera lens 303 ... Optical filter 304 ... Diffraction grating 305 ... Prism 306 ... Detection window 307 ... Detection unit 308 ... First objective lens 309 ... second objective lens 400 ... polymer filling block 400a ... polymer filling block side surface 401 ... syringe 402 ... valve 403 ... channel 404 ... polymer filling block 405 ... channel 406 ... polymer filling block 407 ... detection block 408 ... channel 409 ... Tube 410 ... Flow path 501 ... Tube 502 ... Buffer solution tank 505 ... Buffer solution tank 506 ... High voltage power supply

Claims (10)

A capillary filled with a polymer;
A polymer container provided in the vicinity of one end of the capillary;
A polymer injection part for injecting the polymer into the polymer storage part and into the capillary via the polymer storage part;
A sample container provided in the vicinity of the other end of the capillary;
Voltage applying means for applying a voltage between the one end of the capillary and the other end;
An irradiator for irradiating the capillary with light;
The fluorescence generated from the sample, the capillary electrophoresis apparatus according to claim Rukoto which have a a fluorescence detection means for detecting through the polymer housing part. Capillary chromatography electrophoresis apparatus according to claim 1, characterized in that the capillary is provided a plurality. The one end of a plurality of the capillaries is arranged substantially on a plane, and the polymer container has a detection window located between the plane and the fluorescence detection means. capillary chromatography electrophoresis apparatus according to 2. An optical lens that is provided in the vicinity of the polymer container and detects the fluorescence; and the detection window includes a region including an end face of the one end of each of the plurality of capillaries; and the optical located between the aperture of the lens, capillary chromatography electrophoresis apparatus according to claim 3, characterized in that in the region the entailment and has a substantially similar shape.   The capillary electrophoresis apparatus according to claim 3, wherein the capillary is detachable from the polymer container. The irradiation positions where the light from the plurality of capillaries is irradiated are at least partially arranged on a plane, and the light is at least part of the plurality of capillaries substantially parallel to the plane. capillary chromatography electrophoresis apparatus according to claim 2, characterized in that it is irradiated on. The capillary electrophoresis apparatus according to claim 1, wherein the voltage applying unit applies the voltage when the polymer filled in the capillary does not move. A first buffer tank that includes a connecting portion that connects to the polymer container and holds a first buffer solution;
A second buffer solution tank provided near the other end of the capillary and holding a second buffer solution;
The polymer container includes a flow path, the polymer container, the capillary, and the connecting part are connected via the flow path, and the voltage applying unit includes a liquid surface of the first buffer solution, the first buffer solution, and the first buffer solution. when no difference in height between the liquid surface of the second buffer solution, capillary electrophoresis device according to claim 1, wherein applying the voltage. From one end of the capillary to be connected to the port Rimmer accommodating portion into the interior of the capillary, and filling a polymer,
Electrophoresing a sample from the other end of the capillary to the one end of the capillary;
Capillary chromatography electrophoresis method characterized by have a fluorescence generated by irradiating a laser to the sample, and detecting through the polymer housing part. The capillary electrophoresis method according to claim 9, wherein in the step of electrophoresis, the polymer solution in the capillary is installed so as not to move.

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