JP2005338100A - Capillary array electrophoresis apparatus and electrophoresis method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To solve the problem wherein reflected signals have entered a laser source to destabilize laser oscillation, when a laser beam is irradiated to a multicapillary array, in a direction in which the optical axis of the laser beam intersects with a side surface of the array at right angles and that feedback light has entered the laser source to destabilize the laser source, when laser is irradiated from both side surfaces of the array. <P>SOLUTION: When laser is irradiated from a side surface of the capillary array, by inclining the optical axis of the laser beam 43, in such a direction as to intersect a plane of the capillary array a right angles, it is possible to prevent reflected waves from entering the laser source and feedback light from entering a light receiving element and the laser source. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to an electrophoresis apparatus that separates and analyzes samples such as DNA and proteins by electrophoresis using a capillary array in which a plurality of capillaries are configured collectively.

For the purpose of determining the base sequence and base length of DNA, an electrophoresis method using a capillary is used. A sample containing DNA to be measured is injected into a gel such as polyacrylamide in a glass capillary, and a voltage is applied to both ends of the capillary. The DNA synthesis product in the sample moves in the capillary, and is separated according to the molecular weight or the like to generate a DNA band in the capillary. A fluorescent dye is added to each DNA band, which develops color when irradiated with laser light, which is read by a fluorescence measuring means to determine the DNA sequence. Protein separation and analysis can be performed in the same manner to examine the structure of the protein.

  One of the laser beam irradiation methods is as follows. In a capillary array composed of a plurality of capillaries, the surface of the capillaries such as polyimide is removed to form a detection unit. There is a system in which the capillaries on both sides or one end of the detection unit are respectively irradiated with laser light, and each of the laser beams irradiated on one or both sides crosses a plurality of capillaries.

  In the conventional laser beam irradiation method, when the laser beam is irradiated from one end of the capillary array, the reflected light from the capillary array surface returns to the laser oscillator, which makes the laser oscillation unstable. is there. In addition, when laser light is irradiated from both ends of the capillary array, not only reflected light from the capillary array surface but also light that has passed through the capillary array returns to the laser oscillator, which makes laser oscillation unstable. There's a problem.

In the present invention, there are at least the following three methods for solving the problem of return light and reflected light from the capillary array. In this specification, the capillary array does not have a plane, but a plurality of capillaries are aligned in parallel, and the central axes of the capillaries are arranged substantially on the plane, so that “the plane of the capillary array (array surface)” Expressed.
(1) The irradiation optical axis incident in a direction parallel to the plane of the capillary array is inclined in a direction perpendicular to the longitudinal direction of the capillary. As a result, the laser light axis and the reflected light from the capillary do not overlap, so noise is not picked up.
(2) The irradiation optical axis incident in the direction parallel to the plane of the capillary array is inclined with respect to the plane of the array. In this case, since irradiation light is irradiated from both ends of the array, when one irradiation light spot is seen, the light passing through the array and the incident light are adjacent to each other.
(3) The incident angles of the two irradiation optical axes that intersect in parallel to the plane from both ends of the capillary array plane are different from each other in the longitudinal direction of the capillary.

  The present invention employs at least one of the above configurations (1) to (3). In particular, by combining the above two or three configurations, the obstacle caused by the return light of the irradiation light can be solved very well.

  In view of the above, in one embodiment of the present invention, in order to solve the above-described problem, a laser beam is applied to one or both ends of a capillary array in a capillary array electrophoresis apparatus, and the laser beam includes a plurality of laser beams. A capillary array electrophoresis apparatus configured to traverse the capillaries, in which laser light is incident between the laser condensing means located on the optical axis between the capillaries and the laser and farthest from the capillaries and the laser There is provided a capillary array electrophoresis apparatus characterized in that there is no overlap between laser reflected light and incident laser light by a surface.

  This condition is satisfied when the capillary and the optical axis of the incident laser beam are not perpendicular. In some electrophoresis apparatuses having only one capillary, the optical axis of incident light and the capillary are not substantially perpendicular. However, the purpose of this configuration is a single capillary electrophoresis apparatus, and there are cases where direct reflected light is prevented from entering the detection system by tilting the optical axis. The purpose of tilting the laser optical axis in the present invention is fundamentally different from this prior art. In this embodiment, it is necessary to incline so much that the directly reflected light deviates from the fluorescent condenser lens. For example, when the F value of the detection system is 1.4, it is necessary to have an incident angle of about 20 ° or more. However, in the case of the present invention, when the focal length of the condenser lens is 50 mm, an inclination of about 1 ° to 2 ° is sufficient.

  Further, the present invention provides a capillary array in which the capillaries on both ends of the capillary array in the capillary array electrophoresis apparatus are each irradiated with laser light, and each of the two laser lights traverses a plurality of capillaries. In the electrophoresis apparatus, there is provided a capillary array electrophoresis apparatus characterized in that a plane formed by the capillary array and the incident laser light are not parallel. When the laser beams are basically branched and arranged so as to face the same axis, the laser return light becomes a problem. However, in the capillary array, the light passing through the capillaries is the intersection of two planes: the plane formed by the capillary array and the plane formed by the optical axis traveling through the capillaries and the incident light inclined as described above. It has an optical axis centered on a straight line. Therefore, in this respect, in the capillary array according to the present invention, it is possible to create a situation in which the opposing laser light is not coaxial in the space outside the capillary, although it is coaxial in the capillary.

  Further, the present invention provides a capillary array in which the capillaries on both ends of the capillary array in the capillary array electrophoresis apparatus are each irradiated with laser light, and each of the two laser lights traverses a plurality of capillaries. In the electrophoresis apparatus, there is provided a capillary array electrophoresis apparatus characterized in that orthogonal projections of the two incident laser beams with respect to a plane formed by the capillary array are not substantially parallel.

  Here, as described above, when the orthogonal projections of the two incident laser beams with respect to the plane formed by the capillary array are not substantially parallel, the following may be problematic. Compared to the case where the two laser beams are coaxial, the laser beam diameter when the two laser beams are added is increased, and thus the spatial resolution in fluorescence detection may be reduced. That is, in electrophoresis, a DNA synthesis product in a sample moves in the capillary and is spatially separated in the capillary depending on the size of the molecular weight, and the DNA band is generated in the capillary. There is a possibility that the ability to detect and decompose the degradation of In order to avoid such a possibility, it is desirable that the centers of the two laser beams overlap in the vicinity of the center of the capillary array. By doing so, the expansion of the laser beam diameter can be minimized.

  The method for realizing the laser optical axis described above is as follows. First, the respective condensing lenses for the two opposed laser beams for condensing the laser on the capillary are removed. Then, the two laser beams facing each other are adjusted so as to be substantially parallel and substantially perpendicular to the capillary axis. Thereafter, a condensing lens for condensing the laser on the capillary is inserted into each of the two laser beams. The position of the condenser lens is adjusted so that the laser light is incident on the fluorescence detection part of the capillary.

  In the above laser optical axis realization method, the position of the lens is adjusted in order to introduce the laser light into an appropriate location of the capillary. Therefore, it is desirable that the condenser lens has a position fine adjustment function. Regarding the laser optical axis direction, for example, when the focal length of the condenser lens is 50 mm, the positional accuracy of the lens required for this direction is about 1 mm. Not necessary. However, for the two axes orthogonal to the laser optical axis, a position adjustment function of about 10 μm is required when the inner diameter / outer diameter of the capillary = 5 μm / 300 μm. In this case, this requirement can be satisfied if a screw with a pitch of about 0.5 mm is used as a screw for adjusting the position of the condenser lens.

  Further, in the laser optical axis realization method described above, in order to make the two opposed laser beams substantially parallel to each other at an appropriate position, the diameter of the laser beam is set at each position where the two substantially parallel laser beams pass. This adjustment can be easily performed by using a plate-like group in which holes of the same size as those are used as a laser optical axis adjustment jig.

  In the present invention, since the laser optical axis and the capillary axis are not orthogonal, the laser optical axis is not in the vertical direction when the capillary is installed horizontally. As a multi-capillary detection means, a two-dimensional CCD camera is often used. One of the two dimensions is lined up in the capillary array direction, and an axis for detecting a signal from each capillary is set, and the other axis is set in the wavelength dispersion direction of the fluorescence emitted from each capillary. That is, using a diffraction grating, a prism or the like, the light emission from the single capillary is dispersed in the latter direction. In the present invention, the capillary and the laser optical axis are not substantially perpendicular, but in an electrophoretic apparatus using a CCD (Charge Coupled Device) camera in the fluorescence detection means, the pixel grid of the CCD is substantially parallel to the capillary. Instead, it is more convenient for taking data from the CCD to be substantially parallel to the laser optical axis passing through the capillary.

  Further, in an electrophoresis apparatus having a wavelength dispersion means such as a diffraction grating or a prism in the fluorescence detection means, the direction from the CCD is that the wavelength dispersion direction by the wavelength dispersion means and the laser optical axis passing through the capillary are substantially perpendicular. Convenient for data acquisition.

  According to the present invention, it is possible to prevent laser oscillation from becoming unstable due to reflection or return light when a multi-capillary array is irradiated with laser.

[Example 1]
An overview of the electrophoresis apparatus of the present invention is shown in FIG. An electrode (sample introduction end) 2 is formed at one end of the capillary array 1 so that a negative voltage can be applied. When injecting DNA, the negative electrode 2 is immersed in a solution containing a DNA sample, and when electrophoresis of the injected sample is performed, the negative electrode is immersed in the buffer solution 3 and a voltage is applied. At the other end of the capillary array 1, there is formed a connection portion 5 to a gel block 4 which is a means for injecting a gel as an electrophoresis medium into the capillary. When filling the gel, which is a migration medium inside the capillary, into the capillary, the valve 6 is closed and the syringe 10 is pushed in, thereby injecting the gel in the syringe 10 into the capillary 1. When performing electrophoresis, the valve 6 is opened, and a voltage is applied between the negative electrode 2 immersed in the buffer 3 and the ground electrode 7 immersed in the buffer 12. The capillary 1 is kept at a constant temperature by the gas circulation type thermostatic chamber 11.

FIG. 3 shows a schematic diagram of the vicinity of the detection part of the capillary array and the laser light introduction path. Laser shutters, filters, and the like are well-known matters in this field, and are not a direct object of the present invention. FIG. 3A is a schematic front view of the main part of the electrophoresis apparatus of the present invention, FIG. 3B is a top view of the detection part of the capillary array, and FIG. 3C is a pinhole attached to the laser body emission port. It is a board. Sixteen capillaries 21 are fixed on the array table 20 to form a capillary array. A plane formed by the central axes of the 16 capillaries on the array table 20 and a virtual plane obtained by extending the plane to the entire space are hereinafter referred to as an array surface 22. Further, an imaginary straight line that is in the array plane and is perpendicular to the 16 capillary axes and penetrates the center of the detection unit is hereinafter referred to as an optical axis basic axis 28 (FIG. 3A). In the capillary, a quartz glass tube is covered with a polymer thin film. However, in the detection unit 23, the polymer film is removed and the quartz is exposed. The inner diameter / outer diameter of the quartz tube is 50/320 μm, and the outer diameter of the capillary including the polymer thin film is 363 μm. The pitch of the capillaries is equal to the outer diameter of the capillaries is 363 μm, and the width of the array is 363 μm × 16 = 5.8 mm.

  DNA is detected by irradiating the fluorescence detector 23 in the capillary array with laser light 24 from one side surface of the array and observing the fluorescence emitted from the detector 23. The laser beam 24 is condensed by a laser condenser lens 25 (f = 50 mm). The capillary located at the end of the array and into which the laser is introduced is hereinafter referred to as a first capillary 26. The distance between the laser condenser lens 25 and the first end capillary 26 is 50 mm, and the laser light introduced into the first capillary 26 propagates one after another to adjacent capillaries and crosses 16 capillaries.

  Incident laser reflection occurs at the air / capillary outer wall, capillary inner wall / gel interface. In particular, at the former interface, the reflected light intensity increases due to the large refractive index. Since there are two air / outer wall interfaces per capillary, there are 32 air / outer wall interface reflections from 16 capillaries.

In the present invention, as shown in FIG. 3A, the light enters the first capillary 26 at an angle 30 of about 2 ° with respect to the normal of the 16 capillary axes. The laser beam is on the array surface 22 and is incident on the first capillary at an angle on the array surface 22. As a result of this oblique incidence, the reflected light 29 from the capillary array is shifted from the incident laser light 24. The reflected light 29 becomes substantially parallel to the incident laser light 24 when passing through the condenser lens 25. The distance between the incident laser light 24 and the reflected light 29 was about 4 mm. A pinhole plate 32 having a pinhole 34 having a diameter of 1.4 mm was attached to the laser beam emission port of the laser body 31, and the center of the pinhole and the center of the laser beam were aligned. The reflected light spot 35 on the pinhole plate 32 is as shown in FIG. 3C, and it can be seen that the reflected light 29 is blocked by the pinhole plate 32 and does not return to the laser body 31. By the above method, stable laser oscillation could be obtained.
[Example 2]
The signal intensity distribution from 16 capillaries in Example 1 is shown in FIG. As can be seen from FIG. 18, when laser light is introduced only from one side surface of the capillary array, the signal intensity ratio from 16 capillaries increases. In the present embodiment, laser light is introduced from both side surfaces of the capillary array to reduce variation in signal intensity from the 16 capillaries.

  A schematic diagram of Example 2 of the present invention is shown in FIG. Only the vicinity of the detection part of the capillary array and the laser beam introduction path are displayed, and the laser shutter, filter and the like are not displayed. The configuration of the capillary array is the same as that of the first embodiment. In addition, definitions relating to part names and terms not particularly defined in the present embodiment are the same as those in the first embodiment. The laser beam 40 was divided into two equal parts by a half mirror 41, and these two laser beams were irradiated to the capillary array 42 from both side surfaces. The light reflected by the half mirror 41 is laser light 43 and the transmitted light is laser light 44. The condenser lens for the laser beam 43 is a condenser lens 45, and the condenser lens for the laser beam 44 is a condenser lens 46.

  The capillary located at the end of the array and into which the laser beam 43 is introduced is hereinafter referred to as a first capillary 65, and the capillary into which the laser beam 44 is introduced is hereinafter referred to as a sixteenth capillary 66. The optical axis configuration of the laser beam 43 is the same as that in the first embodiment. The optical axis configuration of the laser beam 44 is symmetrical to the laser beam 43 with respect to the capillary array. The laser beams 43 and 44 are coaxial, and the optical axis is adjusted so that the light that has passed through the capillary out of one laser beam passes coaxially with the other incident laser beam and returns to the laser body. The reflected lights 47 and 48 from the capillaries of the laser beams 43 and 44 are not coaxial with either of the two laser beams as shown in FIG. As in Example 1, a pinhole plate 51 having a pinhole with a diameter of 1.4 mm was attached to the laser beam exit of the laser body 49 to prevent the reflected light from returning to the laser oscillator. Although the light transmitted through the capillary returns to the exit of the laser body, the reflected light is prevented from returning to the laser body, so that relatively stable laser oscillation can be obtained.

  A schematic diagram of the fluorescence detection system in the present invention is shown in FIG. The light emission 53 in the capillary array 42 is made into substantially parallel light by the light-emission condensing lens 54 of f = 1.4, and this is introduced into the transmission diffraction grating 55. Lights 56 and 57 separated by the diffraction grating 55 are focused on the two-dimensional CCD 59 by the imaging lens 58. The wavelength dispersion direction by the diffraction grating 55 is substantially perpendicular to the laser optical axis. As a result, of the two orthogonal axes on the two-dimensional CCD 59, one axis represents the spatial coordinates in the arrangement direction of the 16 capillaries, and the other axis represents the emission spectrum from each capillary.

  4B schematically illustrates the rotation angles of the capillary array 42, the laser optical axis 61, the diffraction grating 55, and the CCD 59 when viewed in the direction from the capillary array 42 toward the diffraction grating 55. Show. The diffraction grating and the CCD were installed so that the grating of the diffraction grating and the grating of the CCD pixel were not substantially perpendicular to the capillary axis but substantially parallel to the laser optical axis. Thereby, signals of the same wavelength from the respective capillaries are arranged on the same column 62 of CCD pixels, and emission spectra from the single capillaries are arranged on the same row 63 of CCD pixels. Such a relationship between an image and a pixel is advantageous for binning processing of a CCD pixel or image analysis.

  The optical axis of the present invention can be realized by the set of pinhole plates shown in FIG. The two laser condensing lenses 45 and 46 are removed from the electrophoresis apparatus, and pinhole plates 67, which are formed with pinholes as shown in FIG. 68 is installed. Hereinafter, the intersection of the pinhole plate and the array surface 22 is defined as the array reference line 64, a straight line that is substantially perpendicular to the pinhole plate and passes through the center position of the detection portion of the first capillary, and the intersection of the pinhole plate and the pinhole reference point. Let's call it 69.

The position of the pinhole in the pinhole plates 67 and 68 is determined as follows according to the incident angle to the capillary to be set. The pinhole plate has two pinholes (the pinhole plate 67 has pinholes 70 and 71, and the pinhole plate 68 has pinholes 72 and 73). The center of these pinholes is placed on the array reference line 64. The midpoint of the two pinholes is set to the pinhole reference point 69. The distance 74 from the pinhole reference point 69 to the center of the pinhole is X, the incident angle 78 to the capillary is T1, and the [distance between the condenser lens 45 and the first capillary 65] + [width of the capillary array] / 2. In case of L1,
X = L1 × tanT1
The relationship is established. That is, in this embodiment, L1 = 52.9 mm (the distance between the condenser lens 45 and the first capillary 65 is 50 mm, the width of the capillary array is 5.8 mm), T1 = 2.1 °, and X is 1. 0.9 mm.

The capillary array is removed from the capillary array mounting location 75, and in the pinhole plates 67 and 68, the laser beam 43 passes through the pinholes 70 and 72, the laser beam 44 passes through the pinholes 71 and 73, and the two laser beams are substantially parallel. As shown in FIG. 6A, the laser optical axis is adjusted. The pinhole plate 68 is removed, and the condensing lens 46 and the capillary array 42 into which the electrophoresis medium has been injected are installed at each mounting location (FIG. 6B). At this time, the position of the lens 46 was determined as follows.

As shown in FIG. 6B, the X, Y, and Z axes are taken. The Z-axis direction is determined so that the distance between the condenser lens 46 and the sixteenth capillary 66 is 50 mm. With respect to the Y-axis direction, the position of the condenser lens 46 is adjusted so that the intensity of transmitted light through which the laser light 44 has passed through the capillary array is maximized. By moving the position of the condenser lens 46 in the Y-axis direction, the irradiation position of the laser light 44 on the sixteenth capillary 66 moves in the Y-axis direction.

In this embodiment, a lens position adjusting function of about 10 μm is required. With respect to the X-axis direction, the center of the transmitted light 77 through which the laser beam 44 has passed through the capillary array is set to the center of the pinhole 70 in the pinhole plate 67. The pinhole plate 67 is removed, and the condenser lens 45 is set (FIG. 6C). The Y and Z axes are set in the same manner as the condenser lens 46. Regarding the X-axis direction, the position is determined on the eighth eighth capillary counted from the first capillary so that the two irradiation laser beams overlap. At this time, Raman scattering having a narrow spectral width in the eighth capillary is observed with a CCD, and the Raman band by the laser beam 43 and the Raman band by the laser beam 44 may be adjusted so as to overlap. The above optical axis can be realized by the above procedure.

In the present invention, the reflected laser light is reflected once by the capillary surface and is incident on the lens of the fluorescence detection system. In the present invention, this directly reflected light is removed by an optical filter.
[Example 3]
A schematic diagram of Example 3 of the present invention is shown in FIG. FIG. 9A is a front view, and FIG. 9B is a side view. Only the vicinity of the detection part of the capillary array and the laser beam introduction path are displayed, and the laser shutter, filter and the like are not displayed. There is no incident laser light on the array surface 22, and the laser is incident at an angle 99 of about 2 ° with respect to this plane. The configuration of the capillary array is the same as that of the second embodiment. Also, the definitions relating to part names and terms not particularly defined in the present embodiment are the same as those in the first or second embodiment.

  In order to realize such an optical axis, the optical axis was adjusted as follows using the pinhole shown in FIG. The two laser condensing lenses 45 and 46 are removed from the electrophoresis apparatus, and pinhole plates 90 and 91 each having a pinhole shown in FIG. Install.

  The position of the pinhole in the pinhole plate is determined as follows according to the incident angle to the capillary to be set. The pinhole plate has two pinholes (the pinhole plate 90 has pinholes 92 and 93, and the pinhole plate 91 has pinholes 94 and 95). A straight line that is substantially parallel to the array reference line that is separated from the array reference line 64 by a distance Y97 (the side opposite to the diffraction grating of the detection system with respect to the array reference line 64) is hereinafter referred to as an elevation angle line 96. The intersection of the perpendicular of the array reference line 64 passing through the pinhole reference point 69 and the elevation angle line 96 is hereinafter referred to as an elevation angle reference point 98. The center of the pinhole 92 is set to the pinhole reference point 69, and the center of the pinhole 93 is set to the elevation angle reference point 98.

When the angle between the plane formed by the center of the pinhole 92 and the central axis of the first capillary 65 and the array surface is T2, and the distance between the condenser lens and the first capillary array is L2,
Y = L2 × tanT2
The relationship is established. That is, in this embodiment, L2 = 50 mm, T2 =
The angle was 2.2 °, and Y was 1.9 mm. The pinholes 94 and 95 on the pinhole plate 91 are similarly set.

  In the pinhole plate set in two places, both the laser beams 43 and 44 pass through the pinholes 93 and 95, and the laser beams are adjusted as shown in FIG. 8 so that the two laser beams are coaxial. FIG. 8A is a front view, and FIG. 8B is a side view. The pinhole plate 91 is removed and the condenser lens 46 is set. At this time, the position of the lens was determined as follows. As shown in FIG. 7, the X, Y, and Z axes are taken. The Y axis and Z axis are optimized in the same manner as in the second embodiment. With respect to the X-axis direction, the center of the transmitted light 100 through which the laser light 44 has passed through the capillary array is made to be the center of the pinhole 92 in the pinhole plate 90 (FIG. 7B). The pinhole plate 90 is removed and the condenser lens 45 is set. For the X, Y, and Z axes, the lens position is optimized in the same manner as in the second embodiment. With the above procedure, the incident angle to the capillary can be arbitrarily set by appropriately setting X and Y by using a pinhole.

Further, as in Example 1, a pinhole plate 32 having a pinhole 34 having a diameter of 1.4 mm was attached to the laser emission port of the laser body. A spot 101 of light that passed through the capillary and returned to the laser body was observed on the pinhole plate as shown in FIG. As can be seen from the light intensity distribution on the broken line 102 in FIG. 7 (c) shown in FIG. 7 (d), according to the present invention, the region where the intensity of transmitted light through the capillary is the strongest is prevented from returning to the laser body. be able to. In this way, the return light intensity can be reduced and the laser can be prevented from becoming unstable.

  In the present invention, a combination of Example 2 and Example 3 is provided. Two laser condensing lenses are removed from the electrophoresis apparatus, and a pinhole plate is installed in each of the places where the laser condensing lenses are installed.

The position of the pinhole in the pinhole plate is determined as follows according to the incident angle to the capillary to be set. There are four pinholes in the pinhole plate. Place the pinhole center on a straight line that is approximately parallel to the array reference line at a distance from the array reference line (opposite to the diffraction grating in the detection system with respect to the array reference line, this straight line is hereinafter referred to as the elevation angle line). The midpoint of the two pinholes is made to be the intersection of the perpendicular of the array reference line passing through the pinhole reference position and the elevation angle line (hereinafter, this point will be referred to as the elevation angle reference point). Determine the intersection of the perpendicular to the elevation line passing through each pinhole and the array reference line. X is the distance from the elevation reference point to the center of the pinhole, T1 is the angle formed by the orthogonal projection of the incident laser beam onto the array surface and the capillary, and the plane and array formed from the elevation reference point and the central axis of the first capillary When the angle between the surfaces is T2, and the distance between the condenser lens and the first capillary array is L2,
X = L1 × tanT1
Y = L2 × tanT2
The relationship is established. That is, in this embodiment, L1 = 53 mm, L2 =
50 mm, T1 = 2.1 °, T2 = 2.2 °, X was 1.9 mm, and Y was 1.9 mm.
[Example 4]
FIG. 10 shows a schematic diagram of the fourth embodiment of the present invention (only the vicinity of the detection section of the capillary array and the laser beam introduction path are displayed, and the laser shutter, filter, etc. are not displayed). FIG. 13A shows a method of adjusting the laser optical axis by two pinhole plates, FIG. 13B shows a case where one pinhole plate is replaced with a condenser lens, and FIG. FIG. 13D is a front view when two condenser lenses are inserted, and FIG. Similar to the second embodiment, the two laser beams 43 and 44 are both in the plane of the array surface 22. However, unlike the second embodiment, these two laser beams are not coaxial and are shifted by 0.86 °. The central axes of the transmitted light 124 in which the incident laser light 43 has passed through the capillary, the transmitted light 122 in which the laser light 44 has passed through the capillary, the reflected light 125 of the laser light 43 by the capillary, and the reflected light 126 of the laser light 44 by the capillary are all. The incident laser beams 43 and 44 are not coaxial. Outside the condenser lenses 45 and 46 with respect to the capillary array 42, these incident laser light, transmitted light and reflected light are substantially parallel light. The configuration of the capillary array is the same as that of the second embodiment. Further, the definitions relating to the part names and terms not particularly defined in the present embodiment are the same as those in the first, second, and third embodiments.

  In order to realize such an optical axis, the optical axis was adjusted as follows using the pinhole shown in FIG. The two laser condensing lenses 45 and 46 are removed from the electrophoresis apparatus, and the pinhole plate 130 in which pinholes as shown in FIG. 131 is installed.

The position of the pinhole in the pinhole plate is determined as follows according to the deviation angle between the two lasers to be set. The pinhole plate has two pinholes (the pinhole plate 130 has pinholes 132 and 133, and the pinhole plate 131 has pinholes 134 and 135). The midpoint of two pinholes is set to the pinhole reference point 69. The distance 136 from the pinhole reference point 69 to the center of the pinhole is dX, the deviation angle 137 between the two lasers is 2 × dT1, [the distance between the condenser lens 45 and the first capillary 65] + [width of the capillary array] When / 2 is L1,
dX = L1 × tan (dT1)
The relationship is established. That is, in this embodiment, L1 = 52.9 mm (the distance between the condenser lens 45 and the first capillary 65 is 50 mm, the width of the capillary array is 5.8 mm), dT1 = 0.86 °, and dX is 0. It was set to 4 mm. The diameter of each pinhole is 0.5
mm.

  The capillary array is removed from the capillary array mounting location 75, and in the pinhole plate set at two locations, both laser beams 43 and 44 pass through the pinholes 133 and 135 so that the two laser beams are coaxial. The laser beam is adjusted as shown in FIG. The pinhole plate 131 is removed, and the condensing lens 46 and the capillary array 42 into which the electrophoresis medium has been injected are installed at each mounting location (FIG. 13B). At this time, the position of the condenser lens 46 was determined as follows. As shown in FIG. 13, the X, Y, and Z axes are taken. The Y and Z axes are the same as in the second embodiment.

  With respect to the X-axis direction, the center of the transmitted light 122 through which the laser beam 44 has passed through the capillary array is set to be the center of the pinhole 132 in the pinhole plate 130 (FIG. 14B). The pinhole plate 130 is removed and the condenser lens 45 is set. For the X, Y, and Z axes, the lens position is optimized in the same manner as in the second embodiment. With the above procedure, the deviation angle between the two lasers can be arbitrarily set by using a pinhole in which dX is appropriately set.

  As shown in FIG. 13 (c), slits 138 and 139 having a width of 3 mm were attached at two locations opposite to the capillary with respect to the lens. On the slit 138, spots of transmitted light 122 and reflected light 125 as shown in FIG. 14C are observed. The slit is installed so as to transmit the incident laser beam 43 and to block the transmitted light 122 and the reflected light 125. The slit 139 is installed so as to transmit the incident laser light 44 and to block the transmitted light 124 and the reflected light 126. In this way, the return light intensity can be reduced and the laser can be prevented from becoming unstable.

  Further, since the incident laser light, transmitted light, and reflected light are substantially parallel to the capillary array outside the laser condensing lens, the slit can be inserted anywhere outside the laser condensing lens. In this embodiment, the slit position was easier to adjust near the laser condenser lens than the laser exit port. Therefore, there is an advantage that the return light intensity can be easily reduced.

  In the present invention, since the deviation angle between the two laser beams is not 0, the following effects are exhibited. Due to the characteristics of the diffraction grating, the image 141 on the CCD of the monochromatic light source 140 parallel to the grooves of the diffraction grating is the center of the image (the center of the fluorescent condenser lens 54 in the second embodiment) as shown in FIG. As the distance from the axis increases, the long wavelength side is distorted. FIG. 12B schematically shows the light intensity distribution 143 of the incident light laser on the capillary array in this embodiment.

  In other words, when an 8 M urea aqueous solution having a refractive index of 1.41 is injected into all capillaries and attention is paid to a specific Raman band using the incident laser light 43 as an excitation light source, the light is split by a diffraction grating. In the image, the imaging position of the Raman band from the first capillary is on the shorter wavelength side than the imaging position of the Raman band from the sixteenth capillary, and the incident laser light 44 is used as the excitation light source. When focusing on a specific Raman band, the image formation position of the Raman band from the 16th capillary is compared with the image formation position of the Raman band from the first capillary in the image dispersed by the diffraction grating. It can be said that it is on the short wavelength side.

This is because the deviation angle between the two laser beams 43 and 44 is not zero, and the intensity of each laser beam decreases as it propagates through the 16 capillaries. An image 144 on the CCD of such a light source cancels with the distortion of the diffraction grating and becomes as shown in FIG. (Note that the image is inverted.) By eliminating the distortion of the image, it was possible to obtain the merit that analysis of data taken out from the CCD becomes easy.
[Example 5]
FIGS. 13 (c) and 13 (d) show schematic diagrams of the sixth embodiment of the present invention (only the vicinity of the detection portion of the capillary array and the laser beam introduction path are displayed, and the laser shutter, filter, etc. are not displayed). (FIG. 13A is a front view and FIG. 13B is a side view). The present embodiment can be said to be a combination of the second embodiment and the fourth embodiment. Similar to the fourth embodiment, the two laser beams 43 and 44 are both in the plane of the array surface 22, and the two laser beams are not coaxial and are shifted by 0.86 °. However, unlike the fourth embodiment, the optical axis basic axis 28 is not a bisector of two laser beams, and the rotation angles of the diffraction grating and the CCD in the fluorescence detection system are the same as in the second embodiment. is there.

  The central axes of the transmitted light 124 in which the incident laser light 43 has passed through the capillary, the transmitted light 122 in which the laser light 44 has passed through the capillary, the reflected light 125 of the laser light 43 by the capillary, and the reflected light 126 of the laser light 44 by the capillary are all. The incident laser beams 43 and 44 are not coaxial. Outside the condenser lenses 45 and 46 with respect to the capillary array 42, these incident laser light, transmitted light, and reflected light are substantially parallel light. The configuration of the capillary array and the definitions relating to the part names and terms not specifically defined in the present embodiment are the same as those in the first to fourth embodiments.

  In order to realize such an optical axis, the optical axis was adjusted as follows using the pinhole shown in FIG. The two laser condensing lenses 45 and 46 are removed from the electrophoresis apparatus, and pinhole plates 160 and 160 having pinholes as shown in FIG. 161 is installed.

The position of the pinhole in the pinhole plate is determined as follows according to the incident angle to the capillary to be set. The pinhole plate has four pinholes (pinhole plate 160 has pinholes 162, 163, 164, 165, pinhole plate 161 has pinholes 166, 167, 168, 169) and two marks (pinhole plate 160 includes a mark 170,
171 and the pinhole plate 161 have marks 172 and 173). These four pinholes and two marks are all on the array reference line 64. Let the midpoint of the two marks be the pinhole reference point 69, and let X be the distance 174 from the pinhole reference point to each mark.

The midpoint of the pinholes 162 and 164 is the mark 170, and the midpoint of the pinholes 163 and 165 is the mark 171. The distance between the pinholes 162 and 164 and the mark 170, and the distance between the pinholes 163 and 165 and the mark 171 Let 175 be dX. The pinholes 162, 166, 163, and 167 have a diameter of 0.5 mm, and the pinholes 164, 168, 165, and 169 have a diameter of 0.2 mm. An angle 176 formed by the laser beam 43 and the optical axis basic axis 28 is (T1-
dT1), and the angle formed by the laser beam 44 and the optical axis basic axis 28 is (T1 + dT1) 177. The deviation angle between the two lasers is 2dT1. When [Distance between condenser lens 45 and first capillary 65] + [Width of capillary array] / 2 is L1,
X = L1 × tan (T1)
dX = L1 × tan (dT1)
The relationship is established. That is, in this embodiment, L1 = 52.9 mm (the distance between the condenser lens 45 and the first capillary 65 is 50 mm, the width of the capillary array is 5.8 mm), T1 = 2.1 °, dT1 = 0. 43 °, X was 1.9 mm, and dX was 0.4 mm. The pinholes 166, 167, 168, and 169 on the pinhole plate are similarly set.

The capillary array is removed from the capillary array mounting location 75, and in the pinhole plate set at two locations, the laser beam 43 passes through the pinholes 162 and 166, the laser beam 44 passes through the pinholes 163 and 167, and the two lasers The laser beam is adjusted as shown in FIG. 13A so that the beams are parallel. The pinhole plate 161 is removed and the condenser lens 46 is set. At this time, the position of the lens was determined as follows. As shown in FIG. 13, the X, Y, and Z axes are taken. The Y axis and Z axis are optimized in the same manner as in the fourth embodiment.

  With respect to the X-axis direction, the center of the transmitted light 122 through which the laser beam 44 has passed through the capillary array is set to be the center of the pinhole 164 in the pinhole plate 160 (FIG. 14B). The pinhole plate 160 is removed and the condenser lens 45 is set. For the X, Y, and Z axes, the lens position is optimized in the same manner as in the third embodiment. With the above procedure, the incident angle to the capillary can be arbitrarily set by using a pinhole in which X and dX are appropriately set.

  Further, as in Example 5, slits 138 and 139 were attached. On the slit 138, spots of transmitted light 122 and reflected light 125 as shown in FIG. 14C are observed. The slit is installed so as to transmit the incident laser beam 43 and to block the transmitted light 122 and the reflected light 125. The slit 139 is installed so as to transmit the incident laser light 44 and to block the transmitted light 124 and the reflected light 126. In this way, the return light intensity can be reduced and the laser can be prevented from becoming unstable.

In addition, according to this invention, the structure which combined Example 3 and Example 4 can be proposed, and this is also within the scope of the present invention.
[Example 6]
The optical axis of Example 6 of the present invention is shown in FIG. The present embodiment is a combination of the second, third, and fourth embodiments. The central axes of the transmitted light 124 in which the incident laser light 43 has passed through the capillary, the transmitted light 122 in which the laser light 44 has passed through the capillary, the reflected light 125 of the laser light 43 by the capillary, and the reflected light 126 of the laser light 44 by the capillary are all. The incident laser beams 43 and 44 are not coaxial. The configuration of the capillary array is the same as that of the second embodiment. In addition, definitions relating to part names and terms that are not particularly defined in the present embodiment are the same as those in the first to fifth embodiments.

  In order to realize such an optical axis, the optical axis was adjusted as follows using the pinhole shown in FIG. The two laser condensing lenses 45 and 46 are removed from the electrophoresis apparatus, and the pinhole plate 210 and the pinhole plate 210 having pinholes as shown in FIG. 211 is installed. The position of the pinhole in the pinhole plate is determined as follows according to the incident angle to the capillary to be set. The pinhole plate has four pinholes (one pinhole plate has pinholes 212, 213, 214, 215, the other pinhole plate has pinholes 216, 217, 218, 219) and four marks (pin The hole plate 210 has marks 220, 221, 222, and 223, and the pinhole plate 211 has marks 224, 225, 226, and 227). The centers of the pinholes 212 and 213 and the marks 220 and 221 are on an elevation line 96 that is a distance Y away from the array reference line 64. The centers of the pinholes 214 and 215 and the marks 222 and 223 are on the array reference line 64. The midpoints of the marks 220 and 221 are set to the elevation angle reference point 98, and the midpoints of the marks 222 and 223 are set to the pinhole reference point 69. The distance 228 from the elevation reference point 98 to the marks 220 and 221 is equal to the distance from the pinhole reference point 69 to the marks 222 and 223, and this is X. The distance 229 between the pinhole 212 and the mark 220, the pinhole 213 and the mark 221, the pinhole 214 and the mark 222, and the pinhole 215 and the mark 223 is equal to dX.

The angles formed by the orthogonal projection of the incident laser light onto the array surface 22 and the optical axis reference line 28 are (T1-dT1) for the laser light 43 and (T1 + dT1) for the laser light 44. An angle 230 formed by the plane formed by the elevation reference point and the central axis of the first capillary and the array surface is T2, and [Distance between the condenser lens 45 and the first capillary 65] + [Width of the capillary array] / 2 is L1. When the distance between the condenser lens 45 and the first capillary 65 is L2,
X = L1 × tan (T1)
dX = L1 × tan (dT1)
Y = L2 × tan (T2)
The relationship is established. That is, in this embodiment, L1 = 52.9 mm, T1 = 2.1 °, dT1 = 0.43 °, X is 1.9 mm, dX is 0.4 mm, and Y is 1.9 mm. The diameters of the pinholes 212 and 213 are 0.5 mm, and the diameters of the pinholes 214 and 215 are 0.2 mm. The same applies to the pinholes 216, 217, 218, and 219 on the pinhole plate.

In the pinhole plate set in two places, the laser beam 43 passes through the pinholes 212 and 216, the laser beam 44 passes through the pinholes 213 and 217, and the two laser beams are parallel to each other in FIG. The laser beam is adjusted as shown in FIG. The pinhole plate 211 is removed, and the condenser lens 46 is set. At this time, the position of the lens was determined as follows. As shown in FIG. 17, the X, Y, and Z axes are taken. The Y axis and Z axis are optimized in the same manner as in the fifth embodiment. With respect to the X-axis direction, the center of the transmitted light 122 through which the laser light 44 has passed through the capillary array is set to be the center of the pinhole 214 in the pinhole plate 210 (FIG. 16B).

  The pinhole plate 210 is removed and the condenser lens 45 is set. For the X, Y, and Z axes, the lens position is optimized in the same manner as in the fifth embodiment. With the above procedure, the incident angle to the capillary can be arbitrarily set by using a pinhole in which X, dX, and Y are appropriately set.

  Further, as in Example 5, slits 138 and 139 were attached. On the slit 138, spots of transmitted light 122 and reflected light 125 as shown in FIG. 16C are observed. The slit is installed so as to transmit the incident laser beam 43 and to block the transmitted light 122 and the reflected light 125. The slit 139 is installed so as to transmit the incident laser light 44 and to block the transmitted light 124 and the reflected light 126.

It is the schematic which shows the structure of the irradiation / detection system and the principal part in the Example of this invention. It is the schematic of the principal part of the electrophoresis apparatus in this invention. (A) is the principal part of the electrophoresis apparatus in the other Example of this invention, (b) is a top view of the irradiation and detection part of a capillary array, (c) is the pin attached to the laser main body exit. It is a top view of a hall plate. (A) is a schematic diagram of the fluorescence detection system in the present invention, and (b) is a schematic diagram showing the rotation angle of the capillary array, laser optical axis, diffraction grating and CCD of the detection system. (A), (b) is a top view of a pair of pinhole board used in this invention. (A) shows how to adjust the laser optical axis using a pinhole plate, (b) shows the change of the optical axis due to the combination of the pinhole plate and the condenser lens, and (c) shows two pinhole plates. The change of the optical axis when it replaces with the condensing lens of is shown. (A) And (b) is a top view which shows the shape of a pinhole board, (c) shows the light intensity on a pinhole board, (d) is light intensity distribution on the broken line of (c). is there. (A), (b) shows the method of adjusting so that a laser optical axis may become coaxial using a pair of pinhole board. (A), (b) is a figure explaining the adjustment method of the laser optical axis using one condensing lens. (A) thru | or (d) is a figure explaining the method to adjust a laser optical axis non-coaxially. (A), (b) is a top view which shows the structure of a pinhole board, (c) is a figure which shows the light intensity distribution on a pinhole board. (A) shows image formation on the CCD in a direction parallel to the grooves of the diffraction grating, and (b) schematically shows the light intensity distribution of the incident light laser on the capillary array. The adjustment method of the laser optical axis in another Example is shown. (A), (b) is a figure which shows the planar structure of the pinhole board in another Example, (c) is light intensity distribution on the surface of a pinhole board. (A), (b) is a figure explaining the adjustment method of the laser optical axis in another Example. (A), (b) is a figure which shows the planar structure of the pinhole board in another Example, (c) is light intensity distribution on the surface of a pinhole board. (A), (b) is a figure explaining the adjustment method of the laser optical axis in another Example. The signal intensity distribution in Example 1 of this invention is shown.

Explanation of symbols

  41 ... Half mirror, 42 ... Capillary array, 43, 44 ... Laser light, 45, 46 ... Condensing lens, 50 ... Pinhole plate.

Claims (11)

  Capillary array electrophoresis apparatus that irradiates one end or both ends of a capillary array composed of a plurality of capillaries arranged on a plane with laser light, propagates one after another to adjacent capillaries, and traverses the capillary array A capillary array electrophoresis apparatus characterized in that there is no overlap between the laser beam reflected by the capillary surface and the laser beam between the laser condensing means for condensing the laser beam on the capillary and the laser oscillator.   Capillaries that irradiate capillaries on both sides of a capillary array made up of a plurality of capillaries arranged on a plane, respectively, and each of the two laser beams propagate to adjacent capillaries one after another and traverse the capillary array 2. A capillary array electrophoresis apparatus, wherein one or both of the incident laser beams are not parallel to a plane formed by the capillary array.   Capillaries that irradiate capillaries on both sides of a capillary array made up of a plurality of capillaries arranged on a plane, respectively, and each of the two laser beams propagate to adjacent capillaries one after another and traverse the capillary array An array electrophoresis apparatus, wherein the orthogonal projections of the two incident laser beams with respect to a plane formed by the capillary array are not parallel to each other.   4. The capillary array electrophoresis apparatus according to claim 3, wherein the centers of the two opposed laser beams overlap each other in the vicinity of the center of the capillary array.   5. The capillary array electrophoresis apparatus according to claim 4, wherein a specific liquid having a refractive index of 1.41 is injected into all capillaries in the electrophoresis apparatus in which a diffraction grating is used in the fluorescence detection means. In the imaging after a specific Raman band from the liquid using each of the two incident laser beams facing each other as an excitation light source is dispersed by a diffraction grating, the Raman band from the capillary on the laser incident side A capillary array electrophoresis apparatus characterized in that an imaging position is on a shorter wavelength side than an imaging position of the Raman band from a capillary on an emission side.   6. The capillary array electrophoresis apparatus according to claim 1, wherein when the two condensing lenses for condensing the two incident laser beams facing each other are removed, the two laser beams facing each other are removed. A capillary array electrophoresis apparatus characterized by being substantially parallel and substantially perpendicular to a capillary axis.   6. The capillary array electrophoresis apparatus according to claim 5, further comprising a function of adjusting the position of the laser condenser lens.   6. A laser optical axis adjustment treatment comprising a set of two plates each having a hole having a size approximately equal to the diameter of the laser beam at each position where the two substantially parallel laser beams according to claim 5 pass. Ingredients.   6. The capillary array electrophoresis apparatus according to claim 1, wherein the fluorescence detecting means has wavelength dispersion means such as a diffraction grating or a prism, and passes through the capillary and the wavelength dispersion direction by the wavelength dispersion means. A capillary array electrophoresis apparatus characterized by being substantially perpendicular to a laser optical axis.   6. The capillary array electrophoresis apparatus according to claim 1, wherein a CCD (Charge Coupled Device) camera is used in the fluorescence detection means, and the CCD pixel lattice is substantially the same as the laser optical axis passing through the capillary. A capillary array electrophoresis apparatus characterized by being parallel. 6. The capillary array electrophoresis apparatus according to claim 1, further comprising a light-shielding plate installed to shield mainly light transmitted through the capillary or light reflected from the capillary.

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