JP6113549B2 - Electrophoresis device - Google Patents

Description

  The present invention relates to a technique for separating and analyzing nucleic acids, proteins and the like by electrophoresis, for example, a capillary electrophoresis apparatus.

  For the purpose of determining the base sequence and base length of DNA, an electrophoresis method using a capillary is used. In a capillary electrophoresis apparatus, it is necessary to irradiate a plurality of capillaries with excitation light. One of the light irradiation methods for a plurality of capillaries is described in Japanese Patent Laid-Open No. 2001-324472 (Patent Document 1). There is a multi-focus method.

  FIG. 14 is a schematic diagram of the multi-focus method. In this system, a sample containing DNA labeled with a fluorescent dye is introduced into a capillary and irradiated with laser light 102 so as to propagate through a plurality of capillaries (capillary array 101) arranged in a row. Laser light 102 is irradiated to both ends of a capillary array 101 composed of a plurality of capillaries arranged on a flat substrate. The laser beam 102 propagates one after another to adjacent capillaries and traverses the capillary array 101. The fluorescence-labeled DNA emits fluorescence by the laser beam irradiated to the capillary. The light emitted from the capillary array 101 is detected by a photodetector. By measuring the fluorescence from each capillary, DNA analysis of the sample introduced into each capillary can be performed. The same applies to the analysis of proteins and the like.

  In the multi-focus method, when laser is applied to both ends of the capillary array, each capillary is irradiated with laser light substantially uniformly. Since each capillary is uniformly irradiated with laser light, the detected signal intensity is approximately the same. On the other hand, when the laser is irradiated only on one end of the capillary array, the laser beam attenuates every time it passes through the capillary. Since the laser beam applied to the capillary is attenuated, the detected signal intensity varies. Thus, in one-side irradiation, the signal intensity differs for each capillary. That is, since a difference occurs in detection accuracy for each sample introduced into the capillary, double-sided irradiation is used in the multifocus method.

  In the multi-focus method, laser instability occurs due to reflection and return light. Specifically, when laser light is irradiated from one end of the capillary array, there is a problem that reflected light from the surface of the capillary array returns to the laser oscillator and makes the oscillation of the laser unstable. 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 order to solve these problems, Patent Document 1 uses a method of providing an angle to the laser light introduced from both ends of the capillary array. Japanese Patent Application Laid-Open No. 2009-257804 (Patent Document 2) uses a technique in which a λ / 4 plate and a polarizer are arranged on a laser optical axis to block transmitted return light and reflected return light.

JP 2001-324472 A JP 2009-257804 A

  In the multi-focus method, as in Patent Document 1, in order to prevent transmitted return light from the capillary, the laser optical axis from both ends of the capillary is angled, and the laser position is aligned near the center where the irradiation intensities from both sides are almost equal. In this case, the laser position from both sides is shifted near both ends of the capillary array, so that the laser beam diameter is apparently thick. As a result, the spread of the intensity distribution of the laser beam is increased, causing a spectral shift in the measured data and generating a pseudo signal. In order to prevent the generation of the pseudo signal, a λ / 4 plate and a polarizer are required on the laser optical axis as in Patent Document 2.

  In the present invention, with respect to the multi-focus method, laser light irradiation to the capillary array is performed from one side, and an antireflection material or the like is provided on the transmission side to suppress the reflected light of the laser light.

  In other words, the electrophoresis apparatus of the present invention has a laser light source, a capillary array composed of a plurality of capillaries, a detection unit in which the capillary arrays are arranged in a plane, and a laser beam emitted from the laser light source on the detection unit. Irradiates from one side of a capillary array aligned in a line, propagates the laser light one after another through adjacent capillaries, and detects light emitted from the capillary array by laser light irradiation The irradiation system irradiates the laser beam at an angle with respect to the normal to the capillary surface, and includes a light absorber on the light transmission side of the capillary array and transmits the laser beam transmitted through the capillary array. It is incident on the light absorbing material and absorbed.

  In addition, the nonuniformity of the signal intensity from each capillary in the capillary array due to one-side incidence can be determined by using a variable density filter having a different optical density for each region through which light emitted from each capillary passes, or by changing the signal intensity to software. Uniform by correcting above.

By irradiating the capillary on one side, the irradiated laser beam diameter becomes smaller than on both sides, and the transmitted return light can be fundamentally eliminated. Thereby, the pseudo signal generated in the both-side irradiation can be suppressed.
Problems, configurations, and effects other than those described above will be clarified by the following description of embodiments.

Schematic of an electrophoresis apparatus. 1 is a schematic diagram of an irradiation system of Example 1. FIG. 1 is a schematic diagram of an irradiation system of Example 1. FIG. 1 is a schematic diagram of a detection system of Example 1. FIG. Schematic of the image obtained by the light-receiving surface of a two-dimensional image sensor. Schematic of signal intensity distribution in single-sided irradiation. The figure which shows an example of the performance of a variable density filter. Explanatory drawing of the effect by using a variable density filter. FIG. 3 is a schematic diagram of an irradiation system of Example 2. FIG. 6 is an explanatory diagram of signal equalization according to the third embodiment. FIG. 6 is an explanatory diagram of signal equalization according to the third embodiment. FIG. 6 is an explanatory diagram of signal equalization according to the third embodiment. FIG. 6 is an explanatory diagram of signal equalization according to the third embodiment. Schematic diagram of the multi-focus method.

  The above and other novel features and effects of the present invention will be described below with reference to the drawings. However, the drawings are for explanation only, and do not limit the scope of the present invention.

  FIG. 1 is a schematic diagram of an electrophoresis apparatus. This apparatus includes an irradiation system 201 for irradiating a sample with excitation light, a detection system 202 for optically detecting the sample, a thermostat 203 for keeping the capillary at a constant temperature, and a high voltage application for applying a high voltage to the capillary. A section 204, a capillary array 205 constituted by one or a plurality of capillaries, and a pump mechanism 208 for injecting the buffer solution 206 and the separation medium 207 into the capillaries.

  The capillary array 205 is an exchange member including one or a plurality of capillaries (eight in this example), and includes a load header 209, a detection unit 210, and a capillary head 221. The capillary array 205 is replaced with a new capillary array when the measurement method is changed, when the replacement time comes due to a predetermined number of times of use, or when the capillary is damaged or deteriorated in quality.

  The capillary is composed of a glass tube having an inner diameter of several tens to several hundreds of microns and an outer diameter of several hundreds of microns, and the surface is coated with polyimide to improve the strength. However, the irradiated portion irradiated with the laser light has a structure in which the polyimide coating is removed so that internal light emission easily leaks to the outside. The inside of the capillary is filled with a separation medium 207 for providing a moving speed difference during electrophoresis. The separation medium 207 has both fluidity and non-fluidity, but in this embodiment, a fluid polymer is used.

  The optical system of the capillary electrophoresis apparatus includes an irradiation system 201 including a light source, a detection unit 210, and a detection system 202 that detects fluorescence generated from the capillary. The light source emits laser light 211 that is coherent light. As the light source, an Ar gas laser, a solid-state laser, a semiconductor laser, or the like is used. In the detection unit 210, a plurality of capillaries constituting the capillary array are arranged in a plane and arranged, and the laser light 211 is irradiated from the side of the capillary array so as to penetrate the plurality of capillaries. The laser light excites the sample migrating in the capillary, and light emission such as fluorescence is emitted from the sample. This fluorescence can be detected by a detection system 202 including a two-dimensional CCD, and information depending on the test sample such as a DNA molecule sequence can be acquired.

[Example 1]
As described above, the capillary electrophoresis apparatus includes an irradiation system and a detection system.
2 and 3 are schematic views showing the configuration of the irradiation system in the first embodiment. 2 is a schematic diagram in which the migration direction of the capillary array is the front and back direction of the paper surface, and FIG. 3 is a schematic diagram in which the migration direction of the capillary array is the left-right direction of the paper surface.

  The irradiation system includes a semiconductor laser 301 as a light source, a reflection mirror 303, a condenser lens 304, and a light absorbing material 307. The laser beam 302 emitted from the semiconductor laser 301 is changed in the traveling direction by the reflection mirror 303, and is arranged in a plane on the detector with an angle θ (see FIG. 3) to prevent reflected return light. The capillary array 305 is irradiated obliquely with respect to the capillary surface from one side, that is, not perpendicularly incident on the capillary surface, but with an angle with respect to a perpendicular standing on the capillary surface. Since the beam diameter of the laser light applied to the capillary array 305 is about 0.8 mm, the incident angle θ needs to be at least about 0.58 ° so that the reflected light 308 on the capillary array surface does not return to the semiconductor laser 301. It becomes.

  The laser beam 302 incident at an angle θ is condensed by a condenser lens 304 and irradiated from one side to an irradiation part in the capillary array. The irradiated laser beam 302 propagates one after another in the direction intersecting the migration direction through the adjacent capillaries, traverses the plurality of capillaries, and passes outside the detection unit. The transmitted laser light 306 that has passed through the capillary array enters the light absorbing material 307 and is absorbed.

As the light absorbing material 307, for example, a light absorbing film sheet (Metal Velvet ^™ ) having an absorption rate of 99.99% is used. By providing this on the transmission side, it is possible to fundamentally eliminate the transmitted return light generated by the both-side irradiation. Furthermore, scattered light and background light to the capillary can be suppressed.

  Next, a detection system for detecting the fluorescence of the sample excited in the capillary will be described. FIG. 4 shows the configuration of the detection system. The detection system includes an optical filter 401, a fluorescent condensing lens 402, a transmissive diffraction grating 403, a focus lens 404, a variable density filter 405, and a two-dimensional image sensor 406 such as a CCD.

  The fluorescence emitted from the sample in the capillary passes through the optical filter 401, and after the excitation light is removed, it becomes parallel light, for example, by the fluorescence condensing lens 402 of F = 1.4. Spectroscopy by The fluorescence is dispersed for each wavelength, the signal for each capillary is made uniform by the variable density filter 405, and imaged on the two-dimensional image sensor 406 by the focus lens 404. FIG. 5 shows an image on the two-dimensional image sensor. Eight capillaries are arranged in the Y-axis direction, and fluorescence from each capillary is dispersed in the X-axis direction. The X-axis direction corresponds to the fluorescence wavelength.

  Next, signal equalization by the variable density filter 405 will be described. When the variable density filter is not installed, the signal intensity of the fluorescence of the same wavelength formed on the two-dimensional image sensor is attenuated every time it passes through the capillary as shown in FIG. In the diagram showing the signal intensity, the horizontal axis is the Y-axis (capillary position), the vertical axis is the signal intensity, and it can be seen that the signal intensity decreases as the irradiation position from the semiconductor laser 301 increases.

  The signal that attenuates in this way is made uniform by the variable density filter 405. FIG. 7 shows an example of the performance of the variable density filter. The variable density filter is a filter having different optical density and light transmittance for each region, and the optical density changes linearly with respect to the region as shown in FIG. By making the fluorescence having different intensities for each capillary by one-sided irradiation and the detection signal uniform through the variable density filter 405, a signal that changes depending on the capillary position as shown in FIG. Regardless of this, a uniform signal can be obtained.

As described above, in this embodiment, the transmitted light caused by the one-side irradiation is absorbed by the light absorbing material to prevent reflection, and the transmitted return light generated in the both-side irradiation is eliminated. Thereby, stabilization of laser oscillation and generation | occurrence | production of a pseudo signal can be suppressed. In this embodiment, a light absorbing film sheet (Metal Velvet ^™ ) is used as the light absorbing material, but an alumite-treated metal may be used, for example. Even if it is another material, a material will not be limited if it absorbs light.

[Example 2]
Unlike the first embodiment, this embodiment includes a mirror having an angle on the capillary transmission side, thereby eliminating transmitted light. FIG. 9 is a schematic view of an irradiation system according to the present embodiment.

  The transmitted laser light 706 that propagates through the capillary array 705 by one-side irradiation and passes through the capillary array changes its traveling direction by the mirror 708 and is incident on the light absorbing material 707 to be absorbed. The material of the light absorbing material 707 is not limited as long as it absorbs light as in the first embodiment.

  In this embodiment, the transmitted laser beam 706 can be absorbed more reliably. For example, when the light absorbing material 707 having a light absorption rate of 99% is provided on the transmission side as in the first embodiment, 99% of the transmitted light propagated through the capillary is absorbed, but the remaining 1% is reflected, and the capillary array And may return to the semiconductor laser 701. In order to prevent such reflected light from returning to the semiconductor laser 701, the traveling direction of the transmitted laser light 706 is changed by the mirror 708, and the transmitted light is absorbed by the angled light absorber 707. According to this arrangement, the probability that the transmitted laser light 706 transmitted through the capillary array returns to the semiconductor laser 701 can be further reduced.

[Example 3]
In the first and second embodiments, the signal intensity of the one-side irradiation is uniformized by the variable density filter, but in the present embodiment, it is performed by software. Details will be described below.

  The irradiation system may be any of the first and second embodiments. If the transmitted return light is eliminated, the irradiation system is not limited. Unlike the first and second embodiments, the detection system does not include a variable density filter. Therefore, as described with reference to FIG. 6, as described with reference to FIG.

  If the signal attenuated by the one-side irradiation is used as it is, a difference in detection accuracy occurs as described above. Therefore, the attenuated signal is corrected at the time of analysis as shown in FIG. The correction method will be described below.

  10 to 13 schematically show the correction method. As shown in FIG. 10, a signal obtained for each capillary in one-side irradiation is detected by a two-dimensional image sensor 801. The signal intensity is detected on the X axis for each capillary. The X-axis direction of the two-dimensional image sensor 801 corresponds to the fluorescence wavelength, and the Y-axis direction corresponds to the capillary position. The signal intensity gradually decreases from the capillary on the excitation light incident side of the capillary array toward the capillary on the excitation light emission side.

FIG. 11 shows an example of signal intensities of the first capillary (Ch1) and the eighth capillary (Ch8) for simplification. The signal strength is calculated by the following formula.
Signal strength = Peak-Offset
Peak is the highest signal, and Offset is the minimum baseline value. The Peak described here is the signal of the fluorescent dye, and the baseline is the Raman scattered light of the polymer as the separation medium.

Thus, the signal intensity is calculated for each capillary, and the signal intensity is plotted on the vertical axis with the horizontal axis as the capillary position as shown in FIG. Usually, the capillary having the highest signal intensity is Ch1, so that the signal intensity of each capillary is corrected to Ch1. That is, when Ch8 is corrected, the correction value is as follows.
Correction value (Ch8) = Signal intensity (Ch1) / Signal intensity (Ch8)
This correction value differs for each capillary. By multiplying the correction value by the signal intensity at the time of detection, the signals of the capillaries can be made pseudo uniform as shown in FIG.

  The correction value, that is, variation in signal intensity in one-sided irradiation, differs depending on the apparatus because it depends on the optical axis, the performance of the two-dimensional imaging device, the performance of the transmission diffraction grating, and the like. Therefore, a correction value is calculated for each apparatus and stored in the memory. When replacement of the light source, the optical component, the two-dimensional image sensor, etc. occurs, the correction value is calculated and stored again.

  As described above, in this embodiment, the emission intensity is uniformized by correcting the attenuation of the emission intensity due to the one-side irradiation by software.

  In addition, this invention is not limited to an above-described Example, Various modifications are included. For example, the above-described embodiments have been described in detail for easy understanding of the present invention, and are not necessarily limited to those having all the configurations described. Further, a part of the configuration of one embodiment can be replaced with the configuration of another embodiment, and the configuration of another embodiment can be added to the configuration of one embodiment. Further, it is possible to add, delete, and replace other configurations for a part of the configuration of each embodiment.

101, 205, 305, 705 Capillary arrays 102, 211, 302, 702 Laser light 201 Irradiation system 202 Detection system 203 Constant temperature bath 204 High voltage application unit 206 Buffer solution
207 Separation medium 208 Pump mechanism 209 Load header 210 Capillary array detector 221 Capillary heads 301 and 701 Semiconductor lasers 303 and 703 Incident light reflecting mirrors 304 and 704 Condensing lenses 306 and 706 Transmitted laser beams 307 and 707 Light absorber 308 Reflection Light 401 Optical filter 402 Fluorescence condensing lens 403 Transmission diffraction grating 404 Focus lens 405 Variable density filter 406 Two-dimensional image sensor 708 Transmitted light reflection mirror 801 Two-dimensional image sensor

Claims (3)

A laser light source;
A capillary array comprising a plurality of capillaries;
A detection unit in which the capillary array is aligned in a plane;
Laser light emitted from the laser light source is irradiated from one side of the capillary array aligned in a plane on the detection unit, and the laser light is propagated one after another through the plurality of capillaries to traverse the plurality of capillaries. An irradiation system
A detection system for detecting luminescence generated from the capillary array by the laser light irradiation,
The irradiation system irradiates laser light at an angle with respect to a normal to the capillary surface, and includes a mirror and a light absorbing material on the light transmission side of the capillary array, and transmits the laser light transmitted through the capillary array to the mirror The electrophoretic apparatus is characterized in that the light is reflected and incident on the light absorbing material at an angle . 2. The electrophoresis apparatus according to claim 1, wherein the optical density of the detection system is linear with respect to a region where light emitted from each capillary is transmitted in order to equalize the signal intensity from each capillary of the capillary array. An electrophoresis apparatus comprising a variable density filter that changes .   2. The electrophoresis apparatus according to claim 1, wherein the detection system corrects the signal intensity obtained from each capillary of the capillary array and makes the signal intensity between the capillaries uniform.

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