JP3967817B2 - Capillary cassette preparation method and apparatus - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
In the present invention, a plurality of capillary columns are arranged, a sample is injected into each capillary column, a multi-capillary array electrophoretic part that is electrophoresed simultaneously in all capillary columns, and a capillary light is irradiated to the capillary in the multi-capillary array electrophoretic part The present invention relates to a multicapillary electrophoresis apparatus having an optical measurement unit for measuring the absorbance of the irradiated portion and the fluorescence from the sample, and a method for producing a capillary cassette constituting the multicapillary array migration unit and the apparatus Is.
[0002]
[Prior art]
The electrophoresis apparatus is used for separating and analyzing nucleic acids, proteins, peptides, sugars, and the like, and particularly plays an important role in analysis of DNA base sequences.
In order to determine the base sequence of a DNA having a long base sequence such as the human genome, a DNA sequencer having high sensitivity, high speed, and large throughput is required. As one of the methods, a multicapillary DNA sequencer in which a plurality of capillary columns packed with gels are arranged instead of using a flat slab gel has been proposed. Capillary columns are not only easier to handle and inject samples than slab gels, but also can be migrated at high speed and detected with high sensitivity. In other words, if a high voltage is applied with a slab gel, problems such as band expansion or temperature gradients occur due to the effect of Joule heat, but such problems are few with capillary columns. Even if it is made, it is possible to perform highly sensitive detection with little band spread.
[0003]
When attaching multiple capillary columns to the electrophoresis device, it is desirable that the capillary column be easily attached to and detached from the device, so it is fixed to the cassette holder (capillary column fixing means) at two locations on the injection side and the detection side. It is desirable that
It is conceivable that the plurality of capillary columns are two-dimensionally arranged on the injection side and arranged in a line on a plane on the detection side, and are fixed to cassette holders having different shapes.
[0004]
[Problems to be solved by the invention]
Capillary columns are thin, so they are difficult to break manually and are easy to break. When fixing a capillary column to a cassette holder, bundling a plurality of capillary columns is a difficult operation requiring skill and takes time.
An object of the present invention is to simplify and quickly produce a capillary cassette.
[0005]
[Means for Solving the Problems]
In the capillary cassette manufacturing method of the present invention, a support at a predetermined fixing position of a capillary column fixing means having a support made of a rubber plate is penetrated by a needle having an inner diameter larger than that of the capillary column from the vertical direction, and the capillary column is inserted into the needle. After guiding and penetrating the support, the capillary column is clamped by the support by pulling out only the needle, and this operation is repeated while changing the fixing position on the support. It is a method of dimensionally arranging and fixing.
The capillary column guided through the needle and penetrating the support of the rubber plate of the injection side cassette (capillary column fixing means) is clamped and fixed by the elastic force of the rubber after only the needle is pulled out of the support.
[0006]
The capillary cassette making apparatus of the present invention is provided with a capillary column fixing means for holding a capillary column fixing means having a support made of a rubber plate and moving and fixing the support in the in-plane direction of the support, and the capillary column fixing means. A needle that penetrates the support body from the vertical direction of the surface and guides the capillary column to a fixed position, a capillary column holder that holds the capillary column and inserts into the needle, and a guide that guides the tip of the capillary column held by the capillary column holder into the needle Slide guide means for moving the needle, guide and capillary column holder in a linear direction perpendicular to the surface of the support, cutting means for cutting the capillary column at a predetermined length, and a roller unit for sequentially feeding the capillary column in the insertion direction, With There.
[0007]
The capillary column fixing means is attached to the arrangement position determining means, and the arrangement position determining means is moved up, down, left and right to determine the fixed position of the capillary column. Along the slide guide means, the needle is linearly moved in the direction of the capillary column fixing means, and the support at the fixing position is penetrated. The tip of the capillary column wound around the capillary drum is guided into the needle by the guide that follows the needle along the slide guide means and the first capillary column holder, and protrudes from the tip of the needle. The tip of the capillary column is held by a second capillary column holder provided on the needle protruding side of the arrangement position determining means, and only the needle is pulled out from the support of the capillary column fixing means. The tip of the capillary column remains in the support, and is fixed to the capillary column fixing means by the elastic force of rubber as a support that works in the direction of closing the hole opened by the needle. The capillary column is cut to a predetermined length by a cutting means. The arrangement position determining means moves to bring the next fixed position on the movement line of the needle, guide and capillary column holder. The capillary column wound around the drum is sent timely by a roller unit having a motor as power.
[0008]
DETAILED DESCRIPTION OF THE INVENTION
In order to form a detection window at the same time as the capillary cassette is created, the capillary cassette creation device includes a detection window creation means for creating a detection window by removing the capillary column film at a predetermined position from the tip of the capillary column, and a capillary column that has been cut. And arranging means for arranging the end sides in a line in a predetermined order.
After the capillary column is guided through the needle and penetrates the capillary column fixing means, a voltage is applied to the detection window creating means to create a detection window at a predetermined position from the tip of the capillary column.
The end of the capillary column cut into a predetermined length is sequentially dropped between the planes and arranged in a line by the arranging means composed of two planes having parallel gaps slightly wider than the outer diameter of the capillary column.
[0009]
【Example】
FIG. 1 shows a schematic perspective view of an example of a multi-capillary electrophoresis apparatus to which a multi-capillary cassette prepared by the present invention and in which a plurality of capillary columns are arranged is applied.
The capillary cassette 2 includes a plurality of capillary columns 102 arranged and fixed by cassettes 4 and 6 to form a capillary array, and one end 2a of the capillary array serves as an injection (sample injection) side and is an injection side cassette holder. 4 is arranged and fixed in a two-dimensional manner and comes into contact with the buffer solution of the electrophoresis reservoir 62 after sample injection. A capillary column 102 is arranged in a row at the end 2b of the capillary array, and comes into contact with the buffer solution in the reservoir 56 at the tip. On the end side of the capillary array, a detected portion 2c in which capillary columns 102 are arranged in a line and supported by a detection-side cassette holder 6 is provided. The capillary column 102 is covered with a film to protect it from breakage. When the fluorescence detection method is used to detect the sample that has migrated, when the coating emits fluorescence, the film to be detected is removed in the detected portion 2c. If a capillary column 102 that is coated with a non-fluorescent material coating is used, the capillary column 102 coating need not be removed even in the detected portion 2c. Different samples are injected into each capillary column 102, and electrophoresis is performed simultaneously.
[0010]
For example, an argon gas laser device 8 is provided as an excitation light source for exciting the sample or the fluorescent substance labeled with the sample. Reference numeral 10 denotes an excitation / light-receiving optical system, which irradiates the capillary column 102 of the detection target portion 2c with an argon laser beam of excitation light to detect fluorescence from the sample. The detection target is detected by a scanning mechanism (not shown). Scanning is performed in a scanning direction that is parallel to the array plane of the capillary cassette in 2c and orthogonal to the migration direction. In order to prevent the excitation light beam from the laser device 8 from being shifted by the scanning of the excitation / light receiving optical system 10, here, as an example, the excitation light beam is excited through an optical fiber 9 coupled by a coupler. The light is guided to the light receiving optical system 10.
[0011]
An electrophoresis buffer solution is placed in the lower reservoir 62, the lower end 2a of the capillary array is immersed in the buffer solution, and an electrophoresis voltage is applied to the capillary end of the lower end 2a of the capillary cassette 2 through the buffer solution. The upper electrode 54 is immersed in contact with the buffer solution in the upper reservoir 56, and the lower electrode 58 is immersed in contact with the buffer solution in the lower reservoir 62. The electrophoresis voltage is applied. The power supply voltage is, for example, 30 kV, and the current capacity is 10 to 30 mA.
The sample to be migrated is a protein sample, a DNA fragment sample labeled with a fluorescent substance, or the like.
[0012]
FIG. 2 is a perspective view schematically showing the configuration of an embodiment of a capillary cassette production apparatus, and an example of a method for producing a capillary cassette according to the present invention will be described with reference to this.
In the embodiment, a quartz glass capillary column having an outer diameter of 300 μm and an inner diameter of 100 μm is used. As the injection side cassette holder, the injection side cassette holder 1 that holds 16 × 24 two-dimensional capillaries with a pitch of 4.5 mm is used in accordance with the 384-hole microplate used at the time of sample introduction.
[0013]
The injection-side cassette holder 1 has a structure in which a support made of a silicone rubber plate is sandwiched between two metal plates or resin plates provided with holes at positions corresponding to the 384-hole microplate. A needle 3 is provided that moves linearly in a direction perpendicular to the surface of the support and penetrates the support. The needle 3 has an inner diameter (for example, gauge 22) larger than the outer diameter of the capillary column. A chuck 7 is provided for grasping a predetermined position from the tip of the capillary column and inserting the tip into the needle 3. The tip of the capillary column gripped by the chuck 7 is guided into the needle 3 and is larger than the outer diameter of the capillary column. A guide pipe 5 having an inner diameter is provided. A nichrome wire coil 13 is provided to create a detection window at a predetermined position from the tip of the capillary column. The guide pipe 5, chuck 7 and nichrome wire coil 13 move on the same straight line as the needle 3. Further, a cutter 15 that cuts a capillary column, one end of which is fixed to the injection-side cassette holder 1, with a predetermined length, and a capillary feed 17 that draws the capillary column wound around the capillary drum 19 to a certain length are also provided.
The needle 3, the guide pipe 5, the chuck 7, and the nichrome wire coil 13 are reciprocated between the position on the left side of the solid line and the position on the broken line through which the needle 3 penetrates the support of the cassette holder 1 on the injection side. Can be moved.
[0014]
The method of the present invention comprises the following procedures.
(1) Hold the injection-side cassette holder 1 and slide it up, down, left and right in the figure. Place the injection-side cassette holder 1 on the XY stage (not shown) with the support surface of the injection-side cassette holder 1 perpendicular to the needle 3. 1 is attached. The hole provided in the injection side cassette holder 1 is aligned with the moving line of the needle 3. The hole becomes the fixed position of the capillary column.
(2) With the chuck at the position of the broken line, the capillary column is pulled out from the capillary drum 19 by the capillary feed 17 by the same length as the capillary column provided in the capillary cassette. The drawn capillary column is stored in a slack state between the chuck 7 and the capillary feed 17.
[0015]
(3) The needle 3 passes through the support in the injection side cassette holder 1.
(4) The capillary column gripped by the chuck 7 at a predetermined position from the tip is pushed leftward, and the tip passes through the guide pipe 5 and the needle 3 to penetrate the support. When the support 3 is clogged with the needle 3, it is pushed out by a capillary column. At this time, since the capillary column between the needle 3 and the chuck 7 is protected by the guide pipe 5, it is possible to prevent the capillary column from being bent and bent by a force when pushing the debris of the support in the needle 3. .
[0016]
(5) The tip of the capillary column that protrudes through the needle 3 to the opposite side of the injection-side cassette holder 1 is gripped by the chuck 11.
(6) The capillary column coating at a predetermined position from the tip of the capillary column is removed by the heat of the nichrome wire coil 13 to create a detection window.
(7) Pull out only the needle 3 from the support of the injection-side cassette holder 1. The capillary column passing through the support is fixed to the injection-side cassette holder 1 by the elastic force of the support that works in the direction of closing the hole.
[0017]
(8) With the tip of the capillary column held by the chuck 11, the needle 3, guide pipe 5, chuck 7 and nichrome wire coil 13 are moved in the direction of the capillary feed 17 to the position of the broken line while sliding along the capillary column. Thereafter, the capillary column is cut into a predetermined length from the tip by the cutter 15.
(9) The cut side (detection window side) of the capillary column, one end of which is fixed to the injection-side cassette holder 1, consists of two flat surfaces having a parallel gap slightly wider than the outer diameter (300 μm) of the capillary column. Are arranged in the order of being cut into the slit holder 21a and cut (fixed to the injection-side cassette holder 1).
[0018]
(10) The XY stage is moved so that the next fixed position of the capillary or the set 1 is located on the moving track of the needle 3.
(11) Repeat steps (2) to (10) 384 times to fix the capillary column to all the fixed positions of the capillary or the set 1, and then align the end face on the detection window side of the capillary column aligned with the slit holder 21a. Install and fix the cassette holder.
By carrying out the method for producing a capillary cassette according to the present invention, a capillary cassette can be produced simply and quickly.
[0019]
FIG. 3 and FIG. 4 show a configuration diagram of an embodiment of a capillary cassette making apparatus for performing the above method more reliably. 3 is a plan view, and FIG. 4 is a front view. Parts having the same role as in FIG.
Three columns 25a, 25b and 25c are provided on the substrate 23 in a direction perpendicular to the plane of the substrate 23. A slide plate 27 provided on a side surface with a rail 27a for sliding a needle unit 3a, a guide unit 5a, a holder unit 7a, and a detection window creation unit 13a, which will be described later, in a direction parallel to the substrate 23, includes three columns 25a, 25b. And 25c. An XY stage 29 is provided on a substrate 23 on the extension in the longitudinal direction of the slide plate 27 with its moving direction (XY plane) being a vertical direction. An injection-side cassette holder 1 having a silicone rubber plate as a support is attached to the XY stage 29 with the support surface vertical, and can be moved up, down, left and right. A chuck 11 that holds the tip of the capillary column is provided on the opposite side of the XY stage 29 (on the side opposite to the slide plate 27) on the extension of the slide plate 27 in the longitudinal direction.
[0020]
The needle unit 3 a that slides on the rail 27 a provided on the slide plate 27 has a needle 3 that has an inner diameter larger than the outer diameter of the capillary column and penetrates the silicone rubber plate that is a support for the injection-side cassette holder 1. It has been. The guide unit 5a is provided with a guide pipe 5 having an inner diameter larger than the outer diameter of the capillary column so that the capillary column between the needle unit 3a and a holder unit 7a described later is not bent. The holder unit 7a is provided with a chuck 7 that holds a predetermined position from the tip of the capillary column. The detection window creation unit 13a is provided with a nichrome wire coil 13 for burning and removing the film of the capillary column. The capillary column passes through the center of the nichrome wire coil 13, and a ceramic pipe that blocks heat transfer from the nichrome wire coil 13 to the capillary column is provided between the capillary column and the nichrome wire coil 13 so as to be movable along the capillary column. A pipe unit 31 that guides the capillary column to the holder unit 7a is fixed in the vicinity of the column 25c on the side surface of the slide plate 27 so that the capillary column sent from the capillary drum 19 described later is linear along the slide plate 27. .
[0021]
The chuck 11, the needle 3, the guide pipe 5, the chuck 7, and the nichrome wire coil 13 are provided on a straight line.
A capillary drum 19 for winding and storing one long capillary column is provided on the substrate 23 opposite to the XY stage.
The support column 25b is provided with a cutting unit 15a for cutting the capillary column held by the injection-side cassette holder 1 to a predetermined length by moving the cutter up and down.
[0022]
A detection-side cassette creation unit 21 is provided on the substrate 23 between the columns 25a and 25b. On the rotating plate 21b provided in parallel with the substrate 23, there are provided two slit holders 21a composed of two planes having parallel gaps slightly wider than the outer diameter of the capillary column. The rotating plate 21b is in contact with the substrate 23 via a fulcrum plate 21c and a roller 21d, and during the operation of fixing the capillary column to the injection side cassette 1, the longitudinal direction of the rotating plate 21b is parallel to the slide plate 27. At that time, the gap between the slit holders 21 a is also directed in the direction parallel to the slide plate 27, that is, in the direction of the XY stage 29. A structure in which the detection side cassette creating unit 21 can rotate 90 ° in the direction parallel to the substrate 23 in the opposite direction to the slide plate 27 around a rotation shaft (not shown) provided inside the fulcrum plate 21c when the detection side cassette is created. It has become. By rotating the detection-side cassette creation unit 21 by 90 °, the capillary column is bent by 90 ° as shown in FIG. 1, and then fixed to the detection-side cassette holder.
[0023]
FIG. 5 shows a flowchart of the operation procedure of the above embodiment, and the operation will be described with reference to FIGS.
The XY stage is moved so that the position at which the capillary column is fixed at the beginning of the injection side cassette holder 1 is aligned with the straight line of the chuck 11, the needle 3, the guide pipe 5, the chuck 7 and the nichrome wire coil 13. The positions of the needle unit 3a, the guide pipe 5a, the holder unit 7a, and the detection window creation unit 13a shown in FIGS. 3 and 4 are in the initial state. In this state, the tip of the capillary column wound around the capillary drum 19 is sent to the pipe unit 31 via a capillary feed (not shown) having a motor as power. Further, the tip is sent into the guide pipe 5 via the nichrome wire coil 13 and the chuck 7, and when the tip comes out from the guide pipe 5, the capillary column is held by the chuck 7. Although current is passed through the nichrome wire coil 13 and heated, since a ceramic pipe is interposed between the nichrome wire coil 13 and the capillary column, the heat of the nichrome wire coil 13 is not transmitted to the capillary column.
[0024]
(STEP 1) The needle unit 3a and the guide unit 5a are moved leftward with the knob provided on the guide unit 5a. The needle unit 3a is moved to the left along the rail 27a until the needle 3 penetrates and penetrates the silicone rubber that is the support for the injection-side cassette holder 1 installed on the XY stage 29. At this time, the holder unit 7a and the detection window creating unit 13a move along the rail 27a following the guide unit 5a while maintaining a predetermined interval. Further, since the capillary column is held by the chuck 7 of the holder unit 7a, it is simultaneously pulled leftward.
[0025]
(STEP2) The guide unit 5a is further moved leftward to contact the needle unit 3a, the tip of the capillary column coming out from the guide pipe 5 is guided into the needle 3, and the tip of the capillary column protrudes from the tip of the needle 3. The silicon rubber debris in the needle 3 is pushed out.
(STEP 3) The holder unit 7a is further moved to the left to further protrude the tip of the capillary column from the tip of the needle 3. At this time, the ceramic pipe between the nichrome wire coil 13 and the capillary column of the detection window creating unit 13a is moved for a certain time from the position of the nichrome wire coil 13, and the nichrome wire coil 13 covers the coating film at a predetermined position from the tip of the capillary column. Heat and start creating detection window. Since it takes time to create the detection window, STEP 4 and STEP 5 are performed in parallel.
[0026]
(STEP 4) The tip of the capillary column protruding from the tip of the needle 3 is held by the chuck 11.
(STEP 5) The needle unit 3a and the guide unit 5a are moved rightward until the needle 3 comes out of the silicone rubber, and the distance from the holder unit 7a is shortened. At this time, the capillary column is held by the chucks 7 and 11 and remains in a state of penetrating the silicone rubber, and is fixed to the injection side cassette holder 1 by the elastic force of the silicone rubber.
[0027]
(STEP 6) The chuck 7 is opened, the needle unit 3a, the guide unit 5a, the holder unit 7a, and the detection window creation unit 13a are moved to the right, and the holder unit 7a and the detection window creation unit 13a are returned to their initial positions. At this time, the distance between the guide unit 5a and the holder unit 7a is shorter than the initial state.
(Step 7) The holder unit 7a closes the chuck 7 at the initial position, and holds the capillary column.
[0028]
(STEP 8) The cutting unit 15 is moved upward, and the capillary column is cut by the provided cutter. The cut portion becomes the tip of the next capillary column. One end of the capillary column cut to a predetermined length is fixed to the injection-side cassette holder 1, and the other end is dropped into the gap of the slit holder 21a. The capillary columns fixed to the injection-side cassette holder 1 are sequentially dropped into the gap between the slit holders 21a and are arranged in a line in a plane.
[0029]
(STEP 9) The needle unit 3a and the guide unit 5a are moved to the left to return the distance between the guide unit 5a and the holder unit 7a to the initial position. Thereby, the needle unit 3a, the guide unit 5a, the holder unit 7a, and the detection window creation unit 13a are returned to the initial positions.
(STEP 10) The chuck 11 is opened, and the tip of the grasping capillary column is opened. Thereafter, the XY stage is moved, and the position where the capillary column of the injection side cassette holder 1 is fixed next is aligned with the straight line of the chuck 11, the needle 3, the guide pipe 5, the chuck 7 and the nichrome wire coil 13. When all the capillary columns have been fixed, the process proceeds to STEP11, and otherwise returns to STEP1.
[0030]
(STEP 11) To create a capillary cassette, the XY stage 29 is moved to the lowest position, and the injection side cassette holder 1 is moved to a predetermined position.
(STEP 12) A rotating shaft (not shown) provided inside the fulcrum plate 21c with the rotating plate 21b of the detection side cassette creating unit 21 in a state where 384 capillary columns are arranged in a line on the plane in the two slit holders 21a. Is rotated 90 ° using the roller 4d in the opposite direction to the slide plate 27. At this time, each capillary column is bent 90 °.
[0031]
(STEP 13) The end faces of 384 capillary columns coming out of the slit holder 21a are aligned with a predetermined jig (angle). At this time, the detection window provided in each capillary column is located between the two slit holders 21a.
(STEP 14) The 384 capillary columns are squeezed and fixed by a clamp provided in the slit holder 21a that holds the capillary column located in the gap between the slit holders 21a.
(STEP 15) The detection-side cassette is aligned between the two slit holders 21a and screwed.
[0032]
By operating the lever in accordance with the above procedure, capillary columns having a predetermined length are aligned in a predetermined order, and a capillary cassette can be created.
Although the operation in the embodiment is an example of manual operation, it is preferable to automate using a drive source such as a motor or an air cylinder. As a result, the capillary cassette can be easily created without failure.
In the embodiment, the capillary columns are loaded one by one, but it is preferable to move a plurality of, for example, 16 needles and capillary columns side by side. As a result, the capillary cassette can be created in a shorter time.
[0033]
【The invention's effect】
According to the present invention, by operating a lever according to a predetermined procedure, capillary columns having a predetermined length can be arranged in a predetermined order and a capillary cassette can be created. Will go up significantly.
Capillary cassettes required for capillary electrophoresis devices that are multiplexed for the purpose of significantly reducing measurement time can be easily created, and by attaching a drive source, it is possible to easily shift to automation. Capillary cassettes can be created easily and with few failures.
As described above, the present invention can easily and quickly produce a capillary cassette.
[Brief description of the drawings]
FIG. 1 is a schematic perspective view of an example of a multi-capillary electrophoresis apparatus to which a capillary cassette prepared according to the present invention is applied.
FIG. 2 is a schematic perspective view of an embodiment of a capillary cassette making apparatus.
FIG. 3 is a plan view of the same embodiment.
FIG. 4 is a front view of the same embodiment.
FIG. 5 is a flowchart of the operation procedure of the embodiment.
[Explanation of symbols]
1 Injection side cassette holder 3 Needle 5 Guide pipe 7, 11 Chuck 13 Nichrome wire coil

Claims (3)

The support at a predetermined fixing position of the capillary column fixing means having a support made of a rubber plate is penetrated by a needle having an inner diameter larger than that of the capillary column from the vertical direction, and the capillary column is guided into the needle so that the support is After penetrating, the capillary column is clamped and fixed by the support by pulling out only the needle, and this operation is repeated while changing the fixing position on the support, thereby sequentially arranging a plurality of capillary columns two-dimensionally. Capillary cassette making method, characterized by An array position determining means for holding a capillary column fixing means having a support made of a rubber plate and moving and fixing in the in-plane direction of the support;
A needle for penetrating the support provided in the capillary column fixing means from a direction perpendicular to the surface thereof to guide the capillary column to a fixed position;
A capillary column holder that grabs the capillary column and inserts it into the needle;
A guide for guiding the tip of the capillary column held by the capillary column holder into the needle;
Slide guide means for moving the needle, the guide, and the capillary column holder in a linear direction perpendicular to the surface of the support;
Cutting means for cutting the capillary column at a predetermined length;
And a roller unit that sequentially feeds the capillary column in the insertion direction. A detection window creating means for creating a detection window by removing the coating of the capillary column at a predetermined position from the tip of the capillary column;
The capillary cassette manufacturing apparatus according to claim 2, further comprising: an arrangement unit that arranges the end sides of the cut capillary columns in a line in a predetermined order.

Images