JP3861083B2 - Capillary electrophoresis device - Google Patents

Description

  The present invention analyzes a DNA base sequence, a base length, a protein molecular weight, and the like by separating a biopolymer sample such as fluorescently labeled DNA, RNA, or protein by electrophoresis and detecting by laser excitation fluorescence. The present invention relates to a capillary electrophoresis apparatus.

  As a technique for separating and analyzing a biopolymer sample such as DNA, capillary electrophoresis using a quartz capillary having an inner diameter of about 50 to 100 μm is widely used. Since the capillary is excellent in heat dissipation, a higher voltage can be applied and the separation speed can be improved by 10 times or more compared to the slab gel. In addition, the capillary electrophoresis method has an advantage that the sample can be electrically injected from the reaction tube in which the sample is prepared without using a pipetting operation, and as a result, automation is easy. Currently, a fully automatic capillary electrophoresis apparatus using a “capillary array” in which 4 to 100 capillaries are arranged, a so-called capillary array electrophoresis apparatus is actively employed. For detection of sample molecules, laser-excited fluorescence detection is used exclusively because of its high sensitivity. In general, a capillary array electrophoresis apparatus is composed of an electrophoresis apparatus section that is a durable article incorporating a laser and a detector, and a capillary array that is a consumable that needs to be replaced in about one week.

  As a laser irradiation method for a plurality of capillaries in a capillary array electrophoresis apparatus, for example, as disclosed in Patent Document 1, a capillary arrayed on a plane is irradiated with a laser beam from the side surface of the array along the array axis, and the capillary lens The so-called multi-focus method, which irradiates all capillaries simultaneously by action, is the mainstream. The multi-focus method has the advantage that the excitation beam is used more efficiently than the beam scanning method that scans the laser beam and fluorescence detection can be performed with high sensitivity. However, the multi-focus method requires very high capillary array accuracy. In order to realize high alignment accuracy, in a conventional multi-focus system capillary array, as described in Patent Document 2, for example, capillaries are formed on an array member in which very precise V grooves are formed by photolithography. Arranged and bonded and fixed.

JP-A-9-152418

JP 2001-264293 A

  Although the method of Patent Document 2 can realize a high-precision capillary array, it is necessary to add expensive precision parts to the capillary array. In addition, even if the array member itself, which is a precision component, has high accuracy, it is necessary to precisely arrange the capillaries in the V-groove in the assembly / bonding process. This is not an easy task and requires a long time by skilled workers. As a result, it was difficult to reduce the manufacturing cost of the capillary array, and it was not easy to maintain a high yield. Capillary arrays are consumables that need to be replaced in weeks to months, unlike the electrophoresis device itself, which is a durable product. The high price of this part reduces the running cost of capillary array electrophoresis devices. Hinder. Furthermore, in the adhesive fixing method, the capillary arrangement accuracy can be influenced by the properties of the adhesive. In addition, the capillaries themselves have very small volumes and heat capacities, and if they are brought into contact with a heat bath at a predetermined temperature, the capillaries quickly reach the same temperature. There is a problem that becomes slow.

  The present invention realizes simple and highly accurate capillary arrangement, prevents deterioration of alignment accuracy due to adhesive swelling, which is a concern with the adhesive fixing method, and allows all of the plurality of capillaries to be immediately brought to a uniform temperature. A capillary array electrophoresis apparatus and a capillary array electrophoresis method are provided. As a configuration, between the laser beam irradiation position and each of the two constrained regions that are located in a range in which the capillaries in the capillary array are arranged in parallel and separated by the position where the laser beam is irradiated, The capillary is pressed against the electrophoresis apparatus with a movable pressing member.

  More specifically, the capillary electrophoresis apparatus of the present invention includes a plurality of capillaries for electrophoresis of a sample, voltage applying means for applying a voltage to both ends of the capillary, and laser beam irradiation for irradiating the capillary with a laser beam. And an electrophoretic device part having a detector for detecting light generated by the irradiation of the laser beam, and a pressing member for fixing a capillary to the electrophoretic device part, wherein the plurality of capillaries are the capillaries And having at least two constraining regions located between the irradiation positions of the laser beams along the axial direction of the plurality of capillaries, wherein the pressing member is located between the irradiation positions of the plurality of capillaries and the first constraining regions. The plurality of capillaries are respectively connected between the irradiation positions of the plurality of capillaries and the second constrained region. It is characterized by being pressed against the mounting portion.

  In the multi-focus method, which is a laser irradiation method for a plurality of capillaries, a deviation of several tens of μm in the center axis of the capillary from the reference plane where the center axis of the capillaries should be located causes significant variations in fluorescence detection sensitivity between the capillaries. In order to suppress the coefficient of variation of detection sensitivity between capillaries to 20% or less, it is desirable that the capillary central axis be within 10 μm with respect to the reference plane. By adopting the configuration described in the present invention, it is possible to greatly reduce the deviation of the distance between the central axis of the capillary from the reference plane to within 10 μm and to reduce the unevenness of detection sensitivity. By adopting the above configuration, in the capillary array electrophoresis apparatus, it is possible to use a low-cost capillary array in which the arrangement member and the assembly process thereof are omitted, and it is possible to provide a low running cost capillary array electrophoresis apparatus. . In addition, it is possible to obtain a high capillary array accuracy capable of multifocus irradiation. In addition, it is possible to quickly uniformize the temperature distribution of the capillary array, which is difficult when bonding the array members. As a result, the problem of non-uniform temperature distribution in the capillary array can be avoided even in measurements in which data is acquired while changing the temperature. Further, when a plurality of capillaries are fixed to each other with an adhesive or the like, the adhesive may be deformed with time, resulting in deterioration of arrangement accuracy over time. According to the configuration of the present invention, It is also possible to eliminate the deterioration of accuracy. Furthermore, when the capillary near the laser irradiation position is fixed with an adhesive, there is a risk that scattered light or fluorescence due to the adhesive may be generated, resulting in detection noise. Since the position control of the capillary in the very vicinity of the position is not performed by the adhesive but by the fixing tool for pressing, the above-described fear can be avoided.

  As described above, according to the present invention, a low-cost capillary array that omits the array member and its assembly process is used, and the temperature distribution of the capillary array becomes uniform quickly without degradation of the array accuracy over time. A capillary array electrophoresis apparatus with a low running cost can be realized.

  Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings.

  FIG. 1 is an explanatory diagram of a capillary array, an array member, and a pressing member in the first embodiment of the present invention. The capillary array 1 of this embodiment is composed of a sheet-like constraining member 3 and 16 capillaries 2-1 to 2-16 which are wired and bonded and fixed thereon according to a predetermined design pattern. Here, the binding member is a member for holding a plurality of capillaries. Instead of fixing the capillary on the binding member, it is of course possible to make a hole in the binding member and pass the capillary through the hole. Alternatively, the capillary may be embedded in the constraining member as a material that solidifies from a liquid state such as a plus chip in which the constraining member material is melted or a plastic dissolved in a volatile solvent. In FIG. 1, only the arrangement member 5 related to the installation of the capillary array is illustrated for the electrophoretic device section for operations such as electrophoresis, and other portions are omitted for the sake of simplicity. The capillary used in this example has an outer diameter of 250 ± 6 μm and an inner diameter of 50 ± 3 μm. The error of the capillary center axis position with respect to the design value in the design pattern is about ± 25 μm in any direction of xyz. The material of the constraining member 3 in this embodiment is polyimide, but any material having a high insulating property can be suitably used. The constraining member 3 has a rectangular opening 4 including an excitation beam irradiation position 11 therein, and capillaries located in the opening 4 are supported by upper and lower constraining regions 7a and 7b of the laser beam irradiation position 11, Meanwhile, they are exposed to the atmosphere without being bound. The capillaries located between the binding regions 7a and 7b are substantially parallel to each other. Since the upper end and the lower end of the capillary protrude from the binding member 3 as shown in the figure, they can be used for filling the separation medium and injecting the sample, respectively. The pitch at the lower end of the capillary is 4.5 mm, which is the same as the well pitch of a 384-well microtiter plate, facilitating direct sample injection from the microtiter plate. For all capillaries, the length (effective length) from the lower end to the beam irradiation position is 11 ± 0.05 cm for all capillaries 2-1 to 2-16. Of course, the effective length can be set to any length such as 21 cm, 36 cm, and 50 cm according to the required electrophoretic separation ability. The capillary in this embodiment is a quartz capillary coated with polyimide, and has a strength that does not easily break even in a portion that is not bonded to the binding member. However, at the irradiation position 11, the polyimide coating is removed by combustion over a width of 2 mm, and the quartz portion is exposed. However, since the upper and lower sides of the brittle quartz portion are supported by the constraining regions 7a and 7b located in the constraining member 3 connected to one sheet, they are not easily broken. The binding member 3 is provided with positioning holes 12a and 12b.

  When the capillary array 1 is attached to the main body of the electrophoresis apparatus, the capillaries 2-1 to 2-16 are arranged between the beam irradiation position 11 and the constraining regions 7a and 7b in the arrangement of the array members 5 fixed to the main body of the electrophoresis apparatus. The pressing surfaces 46a and 46b of the pressing member 8 are pressed against the surfaces 44a and 44b. Laser beams 6a and 6b are irradiated from two directions substantially perpendicular to the capillaries on the capillary array plane. The pressing member 8 includes a cover 9 and cushions 10 a and 10 b bonded to the cover 9. In FIG. 1, the arrangement member 5 and the pressing member 8 are shown only in the vicinity of the opening 4, and other portions are omitted. Details of the arrangement member and the periphery of the pressing member will be described below.

  FIG. 2A is a cross-sectional view around the beam irradiation position 11 taken along a plane that passes through the capillary 2-1 and is perpendicular to the capillary array plane. The array member 5 has the cross section shown in FIG. 2A and is screwed to the temperature control plate 13. As the material for the temperature control plate 13, a material having high insulation and thermal conductivity can be suitably used. In this example, Shapel M software (Sumikin Ceramics and Quartz) was used. The material of the array member 5 is preferably a substance having high rigidity and dimensional stability. For example, all metals can be used. By using such a material, the stability of the capillary array can be increased. The array member 5 in the present embodiment is an aluminum block whose surface is black anodized. Further, if the material of the array member 5 is a substance having high thermal conductivity, the temperature control of the capillaries via the array member becomes easy. Since aluminum used in this embodiment has a very high thermal conductivity, when the temperature control plate 13 is controlled to a predetermined temperature, the array member 5 also has a substantially predetermined temperature. Needless to say, the same effect can be obtained with copper, brass or the like other than aluminum.

  The capillary array 1 is first installed so that the binding member 3 contacts the temperature control plate 13. The array planes 44a and 44b are substantially on one reference plane, and the optical axes of the laser beams 6a and 6b have a distance from the reference plane that is half of the nominal value of the capillary outer diameter ± 3 μm (that is, in this embodiment). 125 ± 3 μm). This is approximately equal to the distance between the reference plane and the central axis of the capillary. However, in the state in which the binding member 3 is merely brought into contact with the temperature control plate 13, the center of the capillaries 2-1 to 2-16 is caused by the relative position error of the capillaries 2-1 to 2-16 and the deflection of the capillaries. A distance of several tens of μm can occur between the axis and the optical axis of the laser beams 6a and 6b. As shown by the arrows in FIG. 2 (a), the cover 9 of the pressing member 8 is provided with a recess 9 'for installing the cushions 10a and 10b, and at least 2 that are in contact with the binding regions 7a and 7b of the pressing member 8, respectively. The surfaces on the two covers 9 are positioned substantially on one plane. Further, when this flat surface is pressed against the binding member 3 of the capillary array 1 or at least the binding regions 7a and 7b, the cushions 10a and 10b are contracted by pressing the capillaries on the arrangement surfaces 44a and 44b. Specifically, it is in a contracted state by about 0.5 mm. When pressed, at least two surfaces on the cover 9 and the pressing surfaces 46a and 46b of the cushions 10a and 10b, which are in contact with the binding regions 7a and 7b of the binding member 8, respectively, are substantially located on one plane. It becomes the composition like this. As a result, the capillaries 2-1 to 2-16 are pressed against the arrangement surfaces 44a and 44b of the arrangement member 5 by the stress of the cushions 10a and 10b, and the outer periphery of the capillaries almost completely contacts the arrangement surface, thereby improving the arrangement accuracy. .

  On the other hand, the length of the section in a state where the capillaries between the array surfaces 44a and 44b are not in contact with the array surface is about 3 mm, and the capillary in this section can be regarded as a substantially straight line. In addition, since the diameter of the laser beam irradiated to the irradiation position is 0.2 mm, the beam is sufficiently smaller than the length of the section that is not in contact with the array surface, and the beam is not scattered by the array surface. If a capillary with an outer diameter of 0.25 mm is used as in this embodiment, the same result can be obtained even if the length of this non-contact section is about 5 mm. If the length of the section where the capillaries are not in contact with the array surface is in the range of 0.3 mm or more and 10 mm or less, beam irradiation can be performed appropriately. More generally, the same result can be obtained if the length of the section in which the capillary is not in contact with the array surface is set to a value larger than 1.5 times the diameter of the laser beam. Here, the diameter of the laser beam is defined by the diameter of the circumference where the power density of the beam cross section is 1 / e ^ 2 of the value on the optical axis. On the circumference where the power density is 1 / e ^ 2 to 1/8, the power is not sufficiently attenuated, and if there is an object at this position, the laser beam is scattered. Outside the circumference having a diameter 1.5 times the beam diameter defined above, the power density of the laser beam is attenuated to 1/100 or less. Therefore, outside this circumference, even if there is an object, the scattering of the beam can be ignored in practice. That is, if the length of the section in which the capillaries are not in contact with the array surface is set to 1.5 times or more of the beam diameter, the scattered light of the beam generated by the array surface can be ignored. Further, the same result can be obtained even when the length of the section in which the capillaries are not in contact with the array surface is 40 times or less the outer diameter of the capillaries. By providing this upper limit, it is possible to maintain the performance of the section such that the capillaries in the section where the capillaries are not in contact with the arrangement surface can be maintained substantially linear. In order to efficiently irradiate all the capillaries with a laser beam, it is ideal that the capillary central axis and the laser beam optical axis intersect. Although it is impossible to always completely intersect due to errors in the outer diameter of the capillary and errors in manufacturing the apparatus, it is desirable that the distance between the capillary central axis and the laser beam optical axis be kept at 10 μm or less. In this embodiment, the distance from the outer circumference of the capillary to the central axis of the capillary and the distance between the reference plane and the laser beam optical axis are both 125 ± 3 μm. Therefore, even when ± 3 μm for both errors is added, the maximum value of the error is 6 μm, and the distance between the capillary central axis and the optical axis of the laser beam is suppressed to 6 μm or less at the worst. The material of the cushions 10a and 10b is preferably an elastic body that reversibly expands and contracts, and for example, fluorocarbon rubber in general can be used. In this example, a black fluororubber Viton (registered trademark) was used. In this embodiment, polycarbonate is used as the material for the cover 9, but any solid material can be used.

  Further, since the thickness of the constraining member is as thin as 50 μm and the heat capacity is very small, the temperature of the capillary on the constraining member 3 quickly becomes substantially equal to the temperature of the temperature control plate by contacting the temperature control plate 13. The temperature of the capillaries pressed against the array member 5 quickly becomes equal to the temperature of the array member 5, but as described above, it is approximately equal to the temperature of the temperature control plate 13 in advance. As a result, uniform capillary temperature can be realized quickly throughout the entire capillary array.

  The array member 5 has an opening 14 in the vicinity of the optical axis of the laser beam, and the fluorescence 15 emitted from the sample in the capillary is observed through the opening 14 as shown in the figure. A substantially transparent window 45 is provided between the beam irradiation position 11 and the opening 14 for detecting fluorescence. Although any transparent material can be used as the material of the window, BK7 is adopted in this embodiment. By providing such a transparent window near the capillary, it is possible to reduce the risk that dust in the apparatus will adhere to the beam irradiation position during measurement. In this embodiment, BK7 that is substantially colorless and transparent is used as the window material. However, if colored glass that blocks light having a short wavelength is used, the scattered light of the irradiation beam can also be blocked.

  FIG. 2B is a cross-sectional view of the vicinity of the beam irradiation position 11 by a plane that passes through the capillary 2-1 and is perpendicular to the capillary array plane in another example relating to the first embodiment. Although the basic structure is the same as that of the first embodiment, in this embodiment, the cushions 10a and 10b are springs, plate members 49a and 49b are attached to the tips of the springs, and the surfaces of the plate members are pressing surfaces 46a and 46b. . By doing in this way, the possibility of plastic deformation generated when the cushions 10a and 10b are made of rubber can be eliminated. In FIG. 2B, the plate material having the pressing surface is separated into two parts 49a and 49b. However, as shown in FIG. 2C, a single plate material 49 having a U-shaped cross section may of course be used. . In FIG. 2 (c), the cushion 10 to which the plate member 49 is attached is one spring, but a plurality of springs may be used as shown in FIG. 2 (b).

  FIG. 3A is a front view of the array member 5. In the arrangement member shown in FIG. 3 (a), the arrangement surfaces 44a and 44b are simple planes. However, as a result of accurately arranging the central axes of the capillaries on one plane parallel to the plane, the gaps between the fluorescence detection capillaries Sensitivity unevenness can be suppressed to 20% or less. FIG. 3B is an enlarged view of the array member 5 with the array surface changed as seen from the front. This array member is made of silicon, and 16 V-grooves are precisely formed at a pitch of 250 μm on the capillary array surfaces 44a and 44b by etching. At the stage where the capillary array is simply placed on the temperature control plate, the capillary central axis has an error of about ± 25 μm with respect to the valley of the V groove. When the capillaries are pressed against the array member 5 by the pressing member 8, the capillary central axis is forcibly made to coincide with the center of the V-groove, and the capillaries are arrayed on the same plane within the range of the error of the capillary outer diameter. The effect inherent to the method of FIG. 3B is that a gap of about 25 μm that may occur in the capillary array plane disappears, and an extremely uniform fluorescence detection sensitivity with a coefficient of variation between capillaries of 10% or less becomes possible. is there. Details of a specific method for attaching the capillary array 1 to the temperature control plate 13 and the fluorescence detection method will be described below.

  FIG. 4A is a cross-sectional view of the capillary array electrophoresis apparatus by a plane orthogonal to the surface formed by the capillaries 2-1 to 2-16 at the beam irradiation position 11. FIG. The capillary array 1 is installed so that the positioning pins 16 a and 16 b on the temperature control plate 13 pass through the holes 12 a and 12 b of the binding member 3. Thereafter, the pressing member 8 is attached. In the cross section of FIG. 4A, the pressing member 8 is only the cover 9, and the cover 9 has holes for passing the positioning pins 16a and 16b. The capillary array 1 and the pressing member 8 are fastened to the temperature control plate 13 by screwing the screw holes provided in the positioning pins 16a and 16b and the bolts 17a and 17b. As a result, one side of the binding member of the capillary array 1 is pressed against the temperature control plate 13. At the same time, the capillaries are pressed against the array member 5 between the beam irradiation position 11 and the constraining region 7a and between the beam irradiation position 11 and the constraining region 7b. In this embodiment, the pressing member 8 is fixed to be detachable from the temperature adjusting plate 13, but it goes without saying that the pressing member may be connected to the temperature adjusting plate with a hinge as shown in FIG. 4 (b). good.

  Next, details of fluorescence excitation / detection will be described with reference to FIG. The laser 19 is a continuous wave helium neon laser with a wavelength of 594 nm and an output of 8 mW. The beam emitted from the laser is divided into two by the beam splitter 20 and irradiated to the capillary 2-n from both sides by the mirrors 21a, 21b, 21c and the prisms 18-1 to 18-4. As described in Patent Document 1, the light irradiated to the capillaries at both ends is irradiated to the adjacent capillaries one after another without diverging by the lens action of the capillary itself. By irradiating the beam into two parts and irradiating from both sides, the loss due to reflection is offset, and the variation in the amount of light transmitted through all 16 capillaries is within ± 20%. The light emitted from the capillary 2-n is converted into a parallel light beam by the camera lens 22-1, and after the background light other than the fluorescence from the sample is removed by the bandpass filter 23, the detector 25 is detected by the second camera lens 22-2. An image is formed on the photocathode 24 so that the ratio between the capillary body and the image size is 1: 1. Since the detector 25 is a CCD having a pixel pitch of 25 μm, fluorescence from each capillary can be separated and detected.

  FIG. 5 is an explanatory diagram of the electrophoresis system of this example. The temperature control plate 13 is maintained at a predetermined value by the Peltier element 30 attached to the back side thereof. The heat or cold generated in the Peltier element 30 is released to the side by the heat pipe 39 and discarded into the atmosphere by the fan 40. The upper ends of the capillaries of the capillary array 1 are inserted into a branch block 26 having a T-shaped channel and sealed with a rubber gasket 43. One of the flow paths of the branch block 26 is connected to the syringe 27 filled with the separation medium by the tube 29-2, and the other flow path is connected to the buffer block 28 through the tube 29-1. The tube is a Teflon (registered trademark) tube having an inner diameter of 1 mm, the branch block and the buffer block are made of substantially transparent acrylic, and the flow path has an inner diameter of 1 mm. The flow path in the buffer block 28 is bent in the block and directed vertically upward, and then connected to a buffer tank having a diameter of 30 mm and spreading in a bowl shape. The connecting portion between the flow path in the buffer block and the buffer tank is opened and closed by the upper and lower sides of the piston 31. The syringe 27 is pushed in a state in which the connecting portion is open, and the separation medium is filled in the tubes 29-1 and 29-2, the flow path of the branch block 26 connecting them, and the flow path in the buffer block 28. The The buffer tank is open at the top, and a predetermined amount of buffer is injected from there. Thereafter, with the piston 31 lowered and the flow path of the buffer block closed, the syringe 27 is pushed again, and the separation medium is now filled into the capillary of the electrophoresis member. The syringe is pushed by the electric stage 34, and the piston is moved up and down by the electric solenoid 32. As the separation medium, an aqueous solution of acrylamide, hydroxyethyl cellulose, or the like can be suitably used.

  The lower end of the capillary is immersed in the buffer in the buffer tank together with the electrode 37 having 16 protrusions corresponding to each capillary. An electrode 33 is immersed in the buffer in the buffer block 28. A high voltage is applied between the upper and lower ends of the capillary by a high voltage power supply 38 connected to the electrodes 33 and 37.

  FIG. 6 is a side view of the main part of the electrophoresis system of FIG. A buffer tank 36 and a microtiter plate 41 are mounted on the electric xz stage 35. When the electric xz stage 35 moves, the lower end of the capillary is immersed in the sample well to be measured on the microtiter plate 41, and then the sample is injected by the high voltage power source 38, and then the lower end of the capillary returns to the buffer 36 again. In this state, a high pressure is applied between the upper and lower ends of the capillary, and the sample injected into the capillary is subjected to electrophoresis and separation. Fluorescence is excited when the sample is electrophoresed and arrives at the beam irradiation position. A series of operations such as filling of the separation medium, sample injection, and electrophoresis are performed automatically by computer control.

  FIG. 7 is a diagram (electrophoretic diagram) showing temporal changes in fluorescence intensity simultaneously obtained from the capillaries 2-1 to 2-16 according to this example. The sample is GeneScan Size Standard 500 Rox from Applied Biosystems, and contains DNA labeled with ROX at the end of up to 500 bp. This sample was diluted 1/20 with formamide, and DNA was made into a single strand state and injected into the capillary. The numerical value attached to the peak in FIG. 7 is the base length of DNA corresponding to the peak. As a separation medium, POP7 of the same company was used, the electrophoretic electric field was 319 V / cm, and the temperature of the temperature control plate was 50 ° C. Here, since the injected sample is the same in all capillaries, it is desirable that the height of the peak attributed to the same fragment does not vary from capillary to capillary. If this variation is large, not only can quantitative comparison between capillaries be difficult, but a sample that can be analyzed in one capillary may not be analyzed because the signal-to-noise ratio is too low in another capillary. In the state where the pressing member 8 is not present, the capillary center axis varies about ± 25 μm with respect to the beam optical axis, so that the peak height variation coefficient between capillaries is 20% or more. By pressing the capillaries against the array surface with the holding member 8, the distance between the capillary center axis and the beam optical axis is suppressed to 10 μm or less. As a result, the inter-capillary variation coefficient of the peak height corresponding to the same base length is 15% or less. Is suppressed. Further, the horizontal axis in the graph of FIG. 7 corresponds to the line of fluorescence intensity = 0 with respect to the bottom electropherogram. The difference (background) between this line and the baseline of the electropherogram is due to Raman scattered light from the separation medium, scattered light from an object in the vicinity of the beam irradiation position, and fluorescence. When there is an adhesive in the vicinity of the beam irradiation position, the background due to it is much larger than the background due to Raman scattering. In this method, there is no adhesive near the beam irradiation position, and there is no scattered light and fluorescence, so the background is suppressed to a small value of 1/10 or less of the peak height. As a result, an electropherogram with less background noise was obtained. In addition, 15 fragments from 50 bases to 500 bases were completely separated despite the very short effective length and separation in a short time, and baseline separation with 10 base differences was obtained. Therefore, it can be seen that very high temperature uniformity is obtained over the entire surface of the capillary array and high resolution is obtained. The result of FIG. 7 is a measurement result immediately after mounting the capillary array on the electrophoresis apparatus, and shows that the temperature uniformity was obtained instantaneously immediately after mounting, and such high resolution was achieved. . In this example, fragment length analysis of ROX-labeled DNA is performed, but it is of course possible to determine a base sequence based on multicolor detection by using a diffraction grating instead of a filter to separate fluorescence. In that case, an argon ion laser that oscillates at two wavelengths of 488 nm and 514.5 nm can be suitably used as the light source 19.

  FIG. 8 shows a capillary array according to the second embodiment of the present invention. The capillary wiring pattern is the same as that of the first embodiment, but in this embodiment, the constraining members are separated into three, 3a, 3b and 3c. The binding members 3a and 3b bind the capillaries 2-1 to 2-16 only by the binding regions 7a and 7b, respectively. The binding member 3c keeps the capillary interval at 4.5 ± 0.1 mm in the vicinity of the lower end of the capillary. The outer diameter of the capillary used in this example is the same as that of the first example, and the core material is also quartz, but the coating is not a polyimide but a substantially colorless and transparent resin. As a result, it can be used without removing the coating even at the beam irradiation position, and even if the binding members 3a and 3b sandwiching the beam irradiation position 11 are separated, a sufficient strength can be maintained with only the capillary. The colorless and transparent resin in this embodiment is a fluorine resin AF1600 manufactured by DuPont, but an acrylic resin can of course be applied. Since there is no binding member at the side of the beam irradiation position in the beam irradiation direction, there is no need to reflect the beam by a prism near the capillary as in the first embodiment, and an optical system for side beam irradiation Can be simplified.

  A capillary array in the third embodiment of the present invention is shown in FIG. The structure of this capillary array is similar to that of the first embodiment, but instead of providing an opening having an outer periphery of a closed curve in the constraining member 3 around the beam irradiation position as in the first embodiment, One side is removed, and an opening is provided in the binding member 3 around the beam irradiation position. As a result of the constraining member having such a shape, it is possible to irradiate the excitation beam from one side of the beam irradiation position of the capillary without being reflected by the prism as in the second embodiment. In this embodiment, since the number of capillaries is reduced to four, the variation coefficient of fluorescence detection sensitivity between capillaries can be suppressed to 20% or less by irradiation on only one side. The number of capillaries is not limited to four and can be increased to about ten. If the capillary is filled with a medium having an intermediate refractive index between quartz and air, it can be expanded to about 50. The excitation beam after passing through the capillary is absorbed by the beam stopper 47 so as not to be scattered by the binding member. The beam stopper in this embodiment is a conical member made of black polycarbonate. As the material of the beam stopper 47, any black resin such as black bakelite can be used, and metal may be used as long as the surface is black-treated. Further, in this embodiment, since the front and rear of the beam irradiation position 11 are supported by the constraining members connected to each other, it is possible to detect fluorescence in the low background by removing the resin coating on the capillary at the irradiation position 11. .

The figure of an example of the capillary array of 1st Example, an arrangement | sequence member, and a pressing member. Sectional drawing by the plane perpendicular | vertical to a capillary arrangement plane through the capillary 2-1 central axis in the beam irradiation position periphery of 1st Example. Sectional drawing by the plane perpendicular to the capillary arrangement | positioning plane which passes along the capillary 2-1 central axis in the other example of a 1st Example. Sectional drawing by the plane perpendicular to the capillary arrangement | positioning plane which passes along the capillary 2-1 central axis in the other example of a 1st Example. The front view of the arrangement | sequence member in a 1st Example. The enlarged view and sectional drawing of the front view of the arrangement | sequence member in a 1st Example. Sectional drawing by the plane orthogonal to a capillary in the beam irradiation position of 1st Example. Sectional drawing by the plane orthogonal to a capillary in the beam irradiation position in the other example of a 1st Example. The conceptual diagram of the electrophoresis system in a 1st Example. 1 is a cross-sectional view of an electrophoresis system in a first embodiment. The electropherogram obtained in the first example. The front view of the capillary array in a 2nd Example. The front view of the capillary array in a 3rd Example.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Capillary array 2-1 to 2-16 Capillary 3, 3a, 3b, 3c Binding member 4 Opening 5 Array member 6a, 6b Excitation beam 7a, 7b Binding area 8 Holding member 9 Cover 10a, 10b, 10 Cushion 11 Beam irradiation position 12a, 12b Hole 13 Temperature control plate 14 Opening 15 Fluorescence 16a, 16b Positioning pins 17a, 17b Bolts 18-1 to 18-4 Prism 19 Laser 20 Beam splitters 21a, 21b, 21c Mirrors 22-1, 22-2 Camera lens 23 Bandpass filter 24 Detector 25 Photocathode 26 Branch block 27 Syringe 28 Buffer block 29-1, 29-2 Teflon (registered trademark) tube 30 Peltier element 31 Piston 32 Solenoid 33 Electrode 34 Automatic X stage 35 Automatic XZ stage 36 Buffer tongue 37 electrodes 38 high voltage power supply 39 heat pipe 40 fan 41 microtiter plate 42 flange 43 O-ring 44a, 44b arranged surface 45 windows 46a, 46b pressing surface 47 the beam stopper 48 hinges 49, 49a, 49b plate.

Claims (10)

A plurality of capillaries for electrophoresis of the sample;
An electrophoretic device section comprising a voltage applying means for applying a voltage to both ends of the capillary, a laser beam irradiating means for irradiating the capillary with a laser beam, and a detector for detecting light generated by the irradiation of the laser beam;
And a pressing member for fixing the capillary into the electrophoresis apparatus unit,
The plurality of capillaries have at least two constrained regions located across the irradiation position of the laser beam along the axial direction of the capillaries,
The pressing member is between the irradiation position of the plurality of capillaries and the first constraining region,
A capillary electrophoresis apparatus, wherein the plurality of capillaries are pressed against the electrophoresis apparatus unit at each of the irradiation positions of the plurality of capillaries and the second constrained region. The capillary electrophoresis apparatus according to claim 1, wherein the plurality of capillaries are arranged substantially in parallel at least at a part between the two constrained regions. The capillary electrophoresis apparatus according to claim 1, further comprising a binding member that binds the plurality of capillaries, wherein the plurality of capillaries are bonded and fixed to the binding member in the binding region. The capillary has a region where the mutual position is not constrained between the two constrained regions,
The capillary electrophoresis apparatus according to claim 1, wherein the pressing member presses the capillary against the electrophoresis apparatus unit in the unbound region. The capillary electrophoresis apparatus according to claim 1, wherein the laser beam irradiation unit irradiates the laser beam from a direction substantially parallel to a direction in which the capillaries are arranged. The capillary electrophoresis apparatus according to claim 1, wherein the electrophoresis apparatus unit includes a window member in the vicinity of the irradiation position. The pressing member further has a fixing bolt for fixing the position of the binding member,
The electrophoresis apparatus unit further includes at least one positioning pin,
The binding member further has a positioning hole for penetrating the positioning pin;
2. The capillary electrophoresis apparatus according to claim 1, wherein the pressing member and the plurality of capillaries are fixed in position by passing the positioning pin through a positioning hole and fixing with a fixing bolt in the previous period. The capillary electrophoresis apparatus according to claim 7, wherein the pressing member is connected to the electrophoresis apparatus unit by a hinge. At least two binding the plurality of capillaries in each of the at least two binding regions;
The capillary electrophoresis apparatus according to claim 1, further comprising one binding member, wherein the at least two binding members are separated from each other. The capillary electrophoresis apparatus according to claim 1, further comprising a binding member that binds the plurality of capillaries, wherein the binding member has an opening in one of two sides of the plurality of capillaries. .

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