Electrophoresis device

Abstract

The purpose of the present invention is to provide an electrophoresis device that has a high degree of freedom in selecting a laser device while limiting deterioration in analysis performance. To this

Description

Electrophoresis equipment

[0001] The present invention relates to an electrophoresis device.

[0002] In a multi-focusing capillary electrophoresis apparatus, a laser beam is irradiated from both ends of a capillary array consisting of multiple capillaries arranged in a row, thereby detecting a uniform signal intensity from all capillaries. In such a multi-focusing apparatus, reflected and transmitted light from the capillaries may return to the laser unit, which is the light source, resulting in unstable laser oscillation and adversely affecting analytical performance.

[0003] Patent Document 1 describes an electrophoresis device that suppresses reflected and transmitted light. For example, in FIG. 11 (Example 6) of Patent Document 1, a technique is disclosed in which a polarizer and a half-wave plate are used to suppress transmitted light while irradiating laser light from both ends of a capillary array perpendicular to the capillary axis. Furthermore, in FIG. 12 (Example 7) of Patent Document 1, a technique is disclosed in which laser light is irradiated at an angle shifted from perpendicular to the capillary axis in order to suppress reflected light.

[0004] US Patent Application Publication No. 2003 / 0226756

[0005] In the technology shown in FIG. 11 of Patent Document 1, if the polarization state of the laser light is disrupted when passing through the capillary array, variations in the polarization angle of the light after passing through the half-wave plate occur, and some components pass through the polarizer, making it difficult to completely block the transmitted return light. Furthermore, since this technology makes it difficult to suppress reflected return light, it is necessary to use a laser unit that is highly resistant to return light, which limits the flexibility in selecting a laser device. Furthermore, in the technology shown in FIG. 12 of Patent Document 1, the laser is irradiated at an angle relative to the capillary axis, which widens the irradiation area of ​​the laser light and may result in false signals due to spectral shift. The generation of false signals degrades the sample separation performance of the electrophoresis device.

[0006] An object of the present invention is to provide an electrophoresis apparatus that allows a high degree of freedom in selecting a laser device while suppressing deterioration of analytical performance.

[0007] In order to solve the above-mentioned problems, the present invention provides an electrophoresis device that irradiates laser light from both ends of a capillary array in which multiple capillaries are arranged, and detects light from the multiple capillaries, and an optical isolator is provided on the optical path from the light source of the laser light to the capillary array.

[0008] According to the present invention, since reflected light can be suppressed without irradiating the capillary with a laser beam tilted relative to the axis of the capillary, an electrophoresis device with high analytical performance can be provided. Furthermore, since transmitted light and reflected light can be reliably suppressed, the degree of freedom in selecting a laser device is increased.

[0009] 1 is a schematic diagram of an electrophoresis device according to the present embodiment; FIG. 2 is a diagram showing a state in which DNA bands separated by electrophoresis move within capillaries; and FIG. 3 is a graph showing an example of changes in emission spectrum. A diagram schematically showing the path of laser light in the optical irradiation system of the electrophoresis device according to Example 1; FIG. 3 is a diagram showing how laser light is incident on a polarization-dependent optical isolator in the forward direction; FIG. 4 is a diagram showing how returned light is incident on a polarization-dependent optical isolator in the reverse direction; and FIG. 5 is a conceptual diagram showing transmitted returned light and reflected returned light when a capillary array is irradiated with laser light. A diagram showing the linear polarization state of laser light that is incident on a polarization-dependent optical isolator and then irradiated onto the capillary array from below, and the transmitted returned light. A diagram showing the polarization state of laser light that is incident on a polarization-dependent optical isolator and then irradiated onto the capillary array from above, and the transmitted returned light. A diagram showing the optical path followed by laser light that is incident on a polarization-independent optical isolator in the forward direction, and the polarization direction at each position as viewed from the beam propagation direction. The polarization direction of the returned light is measured by the polarization-independent optical isolator, and the polarization direction at each position is shown in Fig. 1. The polarization direction of the returned light is measured by the polarization-independent optical isolator ...

[0010] The configuration of an electrophoresis apparatus according to an embodiment of the present invention will be described with reference to FIG. 1 . FIG. 1 is a schematic diagram of the electrophoresis apparatus according to this embodiment. As shown in FIG. 1 , the electrophoresis apparatus 101 includes a capillary array 117 composed of one or more capillaries 102, a thermostatic bath 118 that maintains the capillaries 102 at a constant temperature, a high-voltage power supply 104 that applies a voltage to the capillaries 102, a pump mechanism 103 that injects a polymer into the capillaries 102, and a transport mechanism 125. The transport mechanism 125 transports a buffer container 121, a washing container 122, a waste liquid container 123, and a sample container 124 to a capillary cathode end 127.

[0011] The capillary array 117 has a load header 129 provided at one end, a capillary head 112 provided at the other end, and a detection unit 116 formed between the load header 129 and the capillary head 112 for detecting samples undergoing electrophoresis in the capillaries 102. The capillary array 117 is composed of, for example, 24 capillaries 102, and is replaced with capillaries of a different length when the measurement method is changed. Also, if a capillary 102 is damaged or its quality deteriorates, it is replaced with a new capillary array 117.

[0012] The capillary 102 is formed from a glass tube with an inner diameter of 50 μm and an outer diameter of 320 μm, and its surface is coated with polyimide to improve its strength. However, the polyimide coating has been removed from the detection unit 116 of the capillary 102, where the laser light is irradiated, to allow the internal light emission to easily leak to the outside. The interior of the capillary 102 is filled with a separation medium, which creates a migration difference during electrophoresis, by a pump mechanism 103. In this embodiment, a polymer, which is a highly viscous solution, is used as the separation medium.

[0013] The capillary cathode ends 127 are fixed through respective metallic hollow electrodes 126, with the tips of the capillaries 102 protruding approximately 0.5 mm from the hollow electrodes 126. All of the hollow electrodes 126 provided for each capillary 102 are mounted together on a load header 129. All of the hollow electrodes 126 are electrically connected to the high-voltage power supply 104 mounted in the main body of the apparatus, and function as cathode electrodes when voltage is applied for electrophoresis, sample introduction, etc.

[0014] The capillary ends opposite the capillary cathode end 127 are bound together and glued by the capillary head 112. The capillary head 112 is connected to the block 107 in a pressure-tight and airtight manner. Then, new polymer is filled into the capillary 102 by the pump mechanism 103. The polymer in the capillary 102 is refilled for each measurement to improve the measurement performance.

[0015] The optical system is composed of a light irradiation mechanism 114 that irradiates the detection unit 116, an array holder 105 that holds the detection unit 116, a spectroscope 132 that separates the emitted light in the detection unit 116 into individual wavelengths, and a two-dimensional detector 115 that detects the separated light. When detecting a sample in the capillary 102 that has been separated by electrophoresis, the light irradiation mechanism 114 irradiates the detection unit 116, and the emitted light from the detection unit 116 is separated by the spectroscope 132 and then detected by the two-dimensional detector 115.

[0016] The thermostatic chamber 118 is covered with a heat insulating material, and its interior is controlled to a constant temperature by a heating and cooling mechanism 120. A fan 119 circulates and agitates the air within the thermostatic chamber 118, thereby maintaining the temperature of the capillary array 117 uniformly and constantly.

[0017] The pump mechanism 103 is composed of a plunger pump 106, a block 107, a check valve 108, an electric valve 113, a polymer container 109, and an anode buffer container 110. The block 107 is provided with a flow path that connects the plunger pump 106, the polymer container 109, the anode buffer container 110, and the capillary array 117. The flow path between the plunger pump 106 and the polymer container 109 is provided with a check valve 108 that prevents backflow of the polymer. The flow path between the block 107 and the anode buffer container 110 is provided with an electric valve 113. When the chamber 128 of the plunger pump 106 and the capillary array 117 are filled with polymer, the electric valve 113 closes to prevent the buffer solution from flowing in from the anode buffer container 110. When electrophoresis is performed, the electric valve 113 opens, and electricity is applied to the anode electrode 111 and the capillary cathode end 127.

[0018] The transport mechanism 125 is equipped with three electric motors and linear actuators (not shown), and is movable in three directions: up and down, left and right, and depth. One or more containers can be placed on the moving stage 130 of the transport mechanism 125. The moving stage 130 is also equipped with an electric grip 131, which can grasp and release each container. This allows the buffer container 121, washing container 122, waste liquid container 123, and sample container 124 to be transported to the load header 129 as needed. Unnecessary containers are stored in a designated storage area within the device.

[0019] Here, an overview of the false signal due to the spectral shift will be explained. FIG. 2 is a diagram showing the state in which a DNA band separated by electrophoresis moves within a capillary. A focused laser beam is irradiated onto the detection unit 116 of the capillary 102, and this laser beam has an irradiation area 702 of a predetermined width. Therefore, the signal obtained by the two-dimensional detector 115 changes from the time the DNA band 701 enters the irradiation area 702 of the laser beam until it exits it. Furthermore, since the electrophoretic direction is the same as the wavelength dispersion direction, the DNA band 701 moves in the wavelength dispersion direction while passing through the irradiation area 702 of the laser beam. Therefore, the emission spectrum 703 of the signal obtained by the two-dimensional detector 115 appears to change as shown in FIG. 3.

[0020] FIG. 3 is a graph showing an example of a change in the emission spectrum. The spectral shift shown in FIG. 3 has the same effect as the change in the wavelength of the emission spectrum 703 over time as the DNA band 701 passes through the laser light irradiation area 702. In electrophoresis, multiple fluorescent dyes are used, each corresponding to four types of bases. Therefore, if the emission spectrum 703 appears to change, it becomes impossible to completely match the observed emission spectrum 703 to each fluorescent dye or each base (by matrix conversion). In other words, when matrix conversion is performed on each component of the emission spectrum 703, false signals, which are unconvertible residual components, are generated. The false signals can reduce the discrimination ability of the fluorescent dyes and cause misdiagnosis. As the laser light irradiation area 702 becomes wider, the false signals due to the spectral shift become larger.

[0021] 4 is a diagram schematically illustrating the path of a laser beam in the optical irradiation system of the electrophoresis device according to Example 1. The light irradiation mechanism 114 in this example includes a laser unit 201, which is a light source that oscillates laser beam 202, a beam splitter 205 that splits the laser beam 202 into two beams, a reflecting mirror 203 that changes the path of the laser beam 202, and a condenser lens 206 that condenses the laser beam 202. A polarization-dependent optical isolator 204 is inserted on the optical path from the laser unit 201 to the beam splitter 205. One of the beams of laser beam 202 split by the beam splitter 205 is guided downward of the capillary array by the reflecting mirror 203, and the other is guided upward of the capillary array by the reflecting mirror 203. Furthermore, each laser beam 202 is focused by a focusing lens 206 and then incident on the upper or lower end of the capillary array, and the fluorescence emitted from the detection unit 116 of each capillary 102 is detected by a two-dimensional detector 115 .

[0022] 4 shows only five capillaries 102, but in this embodiment, the capillary array is made up of 24 capillaries 102, and the capillaries 102 are aligned and fixed to the reference base 207 in the detection unit 116. In this specification, the imaginary plane formed by the central axes (capillary axes) of the capillaries on the reference base 207 is called the capillary array plane, and the imaginary line on the capillary array plane that is perpendicular to each capillary axis is called the optical axis 208. Although the capillary array is made up of 24 capillaries 102 in this embodiment, the number of capillaries 102 is not limited to 24.

[0023] The light blocking principle of the polarization-dependent optical isolator 204 is shown using Figures 5A and 5B. Figure 5A shows the forward incidence of laser light on the polarization-dependent optical isolator, and Figure 5B shows the reverse incidence of returned light on the polarization-dependent optical isolator. The polarization-dependent optical isolator 204 is composed of a first polarizer 302a, a second polarizer 302b, and a Faraday rotator 301 positioned between the first polarizer 302a and the second polarizer 302b. Here, the second polarizer 302b is installed with its transmission axis tilted 45° relative to the first polarizer 302a. The Faraday rotator 301 rotates the polarization direction 303 by a fixed angle regardless of the incident direction or polarization direction. In this embodiment, a Faraday rotator 301 that rotates the polarization direction by 45° is used. However, the direction of rotation of the polarization direction 303 that the Faraday rotator 301 rotates for forward incident light is opposite to the direction of rotation of the polarization direction 303 that the Faraday rotator 301 rotates for backward incident light.

[0024] 5A, when laser light enters the polarization-dependent optical isolator 204 in the forward direction, the laser light passes through the first polarizer 302a, has its polarization direction rotated by 45° by the Faraday rotator 301, and then enters the second polarizer 302b. Since the transmission axis of the second polarizer 302b is tilted by 45°, the laser light passes through the second polarizer 302b.

[0025] 5B, when the returning light enters the polarization-dependent optical isolator 204 from the reverse direction, the returning light passes through the second polarizer 302b, and then the polarization direction of the returning light is rotated by 45° in the direction opposite to that of the forward direction by the Faraday rotator 301, and then enters the first polarizer 302a. In this case, the polarization direction of the returning light is perpendicular to the transmission axis of the first polarizer 302a, and the returning light is blocked by the first polarizer 302a.

[0026] Here, an overview of reflected return light and transmitted return light will be explained using Fig. 6. Fig. 6 is a conceptual diagram showing transmitted return light and reflected return light when a capillary array is irradiated with laser light. In Fig. 6, to make it easier to understand the transmitted light and reflected light from the capillary array, the incident light on the capillary array is shown slightly tilted from a direction perpendicular to the capillary axis for convenience.

[0027] When laser light 202 is incident on the capillary array, it is reflected at the interface between the air and the outer wall of the capillary and at the interface between the inner wall of the capillary and the gel. The former interface has a particularly large refractive index, resulting in a high intensity of reflected light. Since each capillary has two interfaces between the air and the outer wall of the capillary, a total of 48 reflections occur at the interfaces between the air and the outer wall of the capillary in a capillary array consisting of 24 capillaries. When this reflected return light 401 from the capillary array reaches the laser unit 201, which is the light source, the laser oscillation by the laser unit 201 becomes unstable.

[0028] Furthermore, the transmitted light that passes through the capillary array and is emitted from the opposite side to the incident side is transmitted return light 402. This transmitted return light 402 is attenuated compared to the incident light by the amount of reflected light. When this transmitted return light 402 reaches the laser unit 201, the laser oscillation by the laser unit 201 becomes unstable.

[0029] Figure 7A shows the linear polarization state of the laser light irradiating the capillary array from below after entering the polarization-dependent optical isolator, and the linear polarization state of the transmitted return light. The xy plane in Figure 7A is perpendicular to the direction of travel of the laser light, and shows the linear polarization state at each position (similar to Figures 7B, 9A, and 9B). Also, in Figure 7A, mirrors are not shown, and the beam splitter position is indicated as point A. The laser light emitted from the laser unit 201 enters the polarization-dependent optical isolator 204 in a linear polarization state in the x-axis direction. Since the laser light entering the polarization-dependent optical isolator 204 is forward incident, it passes through with its linear polarization rotated by 45°, as shown in Figure 5A. The laser light that travels straight through the beam splitter (point A) enters the capillary array 117 from below, maintaining a polarization angle of 45°. The transmitted return light that has passed through the capillary array 117 travels along the other laser path and then enters the polarization-dependent optical isolator 204 again, but because it is incident in the opposite direction, it is blocked by the first polarizer 302a as shown in Fig. 5B. Note that although not shown in Fig. 7A, the reflected return light travels along the path of the incident light and enters the polarization-dependent optical isolator 204 at the same polarization angle, so it is blocked in the same way as the transmitted return light.

[0030] Figure 7B shows the polarization state of the laser light that enters the polarization-dependent optical isolator and then irradiates the capillary array from above, as well as the polarization state of the transmitted return light. As in Figure 7A, the laser light emitted from the laser unit 201 enters the polarization-dependent optical isolator 204 in a linearly polarized state in the x-axis direction and passes through the polarization-dependent optical isolator 204 with its linear polarization rotated by 45°. The laser light reflected by the beam splitter (point A) enters the capillary array 117 from above, maintaining a polarization angle of 45°. The transmitted return light that passes through the capillary array 117 follows the other laser path and then re-enters the polarization-dependent optical isolator 204, but is blocked by the first polarizer 302a, as in Figure 7A. Although not shown in Figure 7B, the reflected return light enters the optical isolator at the same polarization angle while following the path of the incident light, and is therefore blocked in the same way as the transmitted return light.

[0031] In this embodiment, even if the polarization state of the laser light is disrupted during irradiation of the capillary array, resulting in variations in the polarization angle of the returned light, only linearly polarized light with a polarization angle of 45° passes through the second polarizer 302b and enters the Faraday rotator 301, as shown in FIG. 5B . Furthermore, the Faraday rotator 301 rotates the polarization direction of the returned light in the opposite direction to that in the forward direction, so the returned light is blocked by the first polarizer 302a. In other words, the polarization-dependent optical isolator of this embodiment reliably blocks the returned light even if the linearly polarized state is disrupted by irradiation of the capillary array with laser light. This allows the use of a laser unit with low resistance to returned light, thereby increasing the flexibility of laser device selection. Furthermore, because the optical axes of the laser light irradiated from both ends of the capillary array are coaxial and perpendicular to the capillary axis, the laser light irradiation area is narrowed, thereby reducing spurious signals due to spectral shift. As a result, the sample separation performance of the electrophoresis device is improved.

[0032] 8A to 9B, an optical irradiation system of an electrophoretic device according to Example 2 will be described. Unlike Example 1, the light irradiation mechanism 114 in this example has a polarization-independent optical isolator 801 as an optical isolator.

[0033] As shown in Figures 8A and 8B, the polarization-independent optical isolator 801 is composed of a first birefringent crystal 803a, a second birefringent crystal 803b, a Faraday rotator 301 located between the first birefringent crystal 803a and the second birefringent crystal 803b, a half-wave plate 802 located between the first birefringent crystal 803a and the second birefringent crystal 803b, and a pinhole plate 804.

[0034] Here, the Faraday rotator 301 used is the same as in the first embodiment, but in this embodiment, a half-wave plate 802 is also used. Like the Faraday rotator 301, the half-wave plate 802 also rotates the polarization direction by 45°. However, unlike the case of the Faraday rotator 301, the direction of rotation of the polarization direction that the half-wave plate 802 rotates for forward incident light and the direction of rotation of the polarization direction 303 that the half-wave plate 802 rotates for reverse incident light are the same.

[0035] 8A shows the optical path of a laser beam incident on a polarization-independent optical isolator from the forward direction and the polarization direction at each position as viewed from the beam propagation direction. Laser beam 202 traveling in the forward direction passes through a pinhole in pinhole plate 804 and enters first birefringent crystal 803a. Due to the difference in refractive index in each axial direction, first birefringent crystal 803a splits laser beam 202 into ordinary ray 806 and extraordinary ray 807, whose polarization directions differ by 90°. In this example, the polarization direction of ordinary ray 806 is perpendicular to the plane of FIG. 8A . Each beam is rotated 45° by Faraday rotator 301, then further rotated 45° in the same direction by half-wave plate 802, resulting in a total rotation of the polarization direction of the beam by 90°. The beams are then recombined by second birefringent crystal 803b.

[0036] 8B shows the optical path of the return light entering the polarization-independent optical isolator from the reverse direction and the polarization direction at each position as viewed from the beam propagation direction. When the return light 808 traveling in the reverse direction enters the second birefringent crystal 803b, it is separated into an ordinary ray 806 and an extraordinary ray 807. Each ray is rotated 45° by the half-wave plate 802 and then rotated 45° in the opposite direction by the Faraday rotator 301, so that it enters the first birefringent crystal 803a with its polarization direction still intact. However, because the polarization direction of each ray in the first birefringent crystal 803a remains the same as after passing through the second birefringent crystal 803b, the ray passes through the first birefringent crystal 803a without recombining and remains separated, and is blocked by the pinhole plate 804.

[0037] 9A shows the linear polarization state of laser light incident on a polarization-independent optical isolator and then irradiated onto a capillary array from below, as well as the linear polarization state of the transmitted return light. Since the laser light incident on the polarization-independent optical isolator 801 is incident in the forward direction, it passes through the polarization-independent optical isolator 801 as shown in FIG. 8A. The laser light traveling straight through the beam splitter (point A) is incident on the capillary array 117 from below. The transmitted return light that passes through the capillary array 117 continues along the other laser path and then re-enters the polarization-independent optical isolator 801. However, since it is incident in the reverse direction, it is blocked by the pinhole plate 804 as shown in FIG. 8B. Although not shown in FIG. 9A, the reflected return light re-enters the polarization-independent optical isolator 801 after retracing the path of the incident light, and is blocked in the same way as the transmitted return light.

[0038] 9B is a diagram showing the linear polarization state of the laser light incident on the polarization-independent optical isolator 801 and then irradiating the capillary array from above, and the transmitted return light. As in the case of FIG. 9A, the laser light incident on the polarization-independent optical isolator 801 passes through the polarization-independent optical isolator 801. The laser light then reflects off the beam splitter (point A) and enters the capillary array 117 from above. The transmitted return light that passes through the capillary array 117 travels along the other laser path and then re-enters the polarization-independent optical isolator 801, but is blocked by the pinhole plate 804. Although not shown in FIG. 9B, the reflected return light re-enters the polarization-independent optical isolator 801 after traveling along the path of the incident light, and is blocked in the same way as the transmitted return light.

[0039] In the case of the polarization-dependent optical isolator of Example 1, even if the return light varies, the polarizer blocks light other than that with a specific polarization angle, so the return light suppression effect is high. However, in the case of the polarization-independent optical isolator of this example, a certain degree of return light suppression effect can also be expected. In other words, this example also makes it possible to use a laser unit with low resistance to return light, thereby increasing the freedom of laser device selection. Furthermore, because the laser light irradiation area is narrow and false signals due to spectral shift can be reduced, the sample separation performance of the electrophoresis device is also improved.

[0040] The present invention is not limited to the above-described embodiments, and various modifications are possible. For example, in the above-described embodiments, an optical isolator is provided on the optical path from the light source to the beam splitter, thereby reducing the number of optical isolators to one. However, an optical isolator may be provided on each optical path after the beam splitter.

[0041] 101...electrophoresis apparatus, 102...capillary, 103...pump mechanism, 104...high voltage power supply, 105...array holder, 106...plunger pump, 107...block, 108...check valve, 109...polymer container, 110...anode buffer container, 111...anode electrode, 112...capillary head, 113...motorized valve, 114...light irradiation mechanism, 115...two-dimensional detector, 116...detection unit, 117...capillary array, 118...thermostatic bath, 119...fan, 120...heating and cooling mechanism, 121...buffer container, 122...washing container, 123...waste container, 124...sample container, 125...transport mechanism, 126...hollow electrode, 127...capillary cathode end, 128...chamber, 129...load head da, 130...moving stage, 131...grip, 201...laser unit, 202...laser light, 203...reflection mirror, 204...polarization-dependent optical isolator, 205...beam splitter, 206...condensing lens, 207...reference base, 208...optical axis, 301...Faraday rotator, 302a...first polarizer, 302b...second polarizer, 303...polarization direction, 401...reflected return light, 402...transmitted return light, 701...DNA band, 702...irradiation area, 703...emission spectrum, 801...polarization-independent optical isolator, 802...half-wave plate, 803a...first birefringent crystal, 803b...second birefringent crystal, 804...pinhole plate, 806...ordinary ray, 807...extraordinary ray, 808...return light

Claims

1. An electrophoresis device that irradiates a laser beam from both ends of a capillary array in which a plurality of capillaries are arranged, and detects the light from the plurality of capillaries, characterized in that an optical isolator is provided on the optical path from the light source of the laser beam to the capillary array.

2. An electrophoresis device according to claim 1, characterized in that the optical axes of the laser beams irradiated onto the capillary array from both ends are coaxial, perpendicular to the capillary axes and parallel to the arrangement plane of the capillary array.

3. An electrophoresis device according to claim 1, comprising: a beam splitter that splits the laser light emitted from the light source into two; and mirrors that guide the split laser light to one side and the other side of the capillary array, wherein the optical isolator is provided on the optical path from the light source to the beam splitter.

4. An electrophoretic device according to claim 1, characterized in that the optical isolator is a polarization-dependent optical isolator having a first polarizer, a second polarizer, and a Faraday rotator located between the first polarizer and the second polarizer.

5. An electrophoresis device according to claim 1, characterized in that the optical isolator is a polarization-independent optical isolator having a first birefringent crystal, a second birefringent crystal, a Faraday rotator positioned between the first birefringent crystal and the second birefringent crystal, and a half-wave plate positioned between the first birefringent crystal and the second birefringent crystal.

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