JP3506652B2 - Capillary array electrophoresis device - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a capillary array electrophoresis apparatus for separating and analyzing samples such as DNA and proteins.

[0002]

2. Description of the Related Art A capillary array electrophoresis apparatus disclosed in Japanese Patent Laid-Open No. 09-096623 is a pumping optical system including a capillary array in which a plurality of capillaries are arranged in a plane and a laser light source, and a signal light. It is composed of a light receiving optical system for detecting fluorescence. In this example, the capillary containing the fluorescent sample is irradiated with laser from the side, and the laser is focused by the lens action of the capillary, so that the laser is irradiated to all capillaries and the fluorescence from each capillary is received by the light receiving optical system. It is something to detect.

US Pat. No. 5,790,727 (Aug. 4,1)
Reference numeral 998) is a device for receiving fluorescence from a sample emitted in a direction perpendicular to the laser irradiation direction with an optical fiber. In this example, a light extractor made of an optical fiber is attached in the fluorescence detection direction so that the reflected light from the capillary surface does not enter the light extractor, and an attempt is made to reduce the background light.

[0004]

[Patent Document 1] Japanese Patent Application Laid-Open No. 09-0966
In the method disclosed in Japanese Patent No. 23, a part of the fluorescence from the sample due to laser irradiation is reflected on the capillary surface, which causes an increase in background light, resulting in a decrease in detection accuracy.

US Pat. No. 5,790,727 (Aug.
4, 1998), the capillary array is a consumable item, whereas the fluorescence detector consisting of an optical fiber is an equipment item. Therefore, every time the capillary array is replaced, the capillary central axis and the detection side light are detected. It was necessary to align with the central axis of the fiber, which was not convenient for the user.

An object of the present invention is to reduce SN by reducing background light.
(EN) Provided is a capillary array electrophoresis apparatus having an improved ratio and a high detection accuracy, and at the same time, simplifying the attachment of a capillary to this apparatus and the introduction of an excitation optical system, and providing a user-friendly capillary array electrophoresis apparatus. .

[0007]

An opening (window) through which signal light (fluorescence) passes is formed in a holding substrate for a capillary array, and a light blocking area for blocking reflected light from the capillary surface is provided between the capillaries. A V-groove for positioning the capillaries is formed in order to facilitate the alignment between the window and the capillaries, and a notch for the incident laser is provided on the side surface of the substrate.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be described in detail below with reference to the drawings.

(First Embodiment) FIG. 1 shows a first embodiment of the present invention.
FIG. 3 is an explanatory diagram of a wind unit that is an embodiment of FIG.
It is roughly divided into a capillary 1 (16 in this embodiment), a window substrate 2, and a glass substrate 3. 2 is an explanatory view of the window substrate 2 shown in FIG. 1 viewed from the back, and FIGS. 3A and 3B are cross-sectional views, respectively, and FIGS.
It corresponds to A-A 'and non-irradiated part BB'. First, the configuration will be described.

As shown in FIG. 1, in the window substrate 2, through windows 6 through which light passes are formed at the same pitch as the arrangement intervals of the capillaries 1, and each through window 6 has a V groove for positioning the capillaries 1. 8 is formed. These through windows 6 are partitioned by a light-shielding area 7 for each capillary.
Further, a cutout portion 12 is provided on the side surface of the window substrate 2 in order to facilitate incidence of a laser for excitation. The capillaries 1 are arranged so as to fit in the V grooves 8 of the window substrate 2,
From the opposite side, the glass substrate 3 fixes the capillaries with an adhesive so as to sandwich them. The capillary 1 comprises a fused silica tube 9 and a coating (polyimide resin) 10. The polyimide resin 10 is removed from the region corresponding to the through window 6 so as not to interfere with the excitation laser light and the fluorescence. The glass substrate 3 is to suppress the reflected light in this region, and the sliding glass surface 11.

Next, the effect of the light shielding area 7 will be described. The capillary 1 used in the experiment has an outer diameter of 0.36 mm. A urea solution was used as a measurement sample. Here, the urea solution is a substitute for a buffer solution used for electrophoresis, and the refractive indexes of both are almost equal. FIG. 11 shows a wavelength of 584n
The fluorescence intensity characteristic of m (fluorescence due to OH stretching vibration of water) is shown. The horizontal axis represents the array number of the capillaries 1 arranged in a plane, that is, the array position, and the vertical axis represents the relative fluorescence intensity normalized by the maximum fluorescence intensity. FIG. 5A shows a conventional fluorescence emission characteristic without the window substrate 2, the background light intensity is about 20% of the capillary fluorescence intensity, and there is noise between the capillaries. On the other hand, FIG. 6B shows the characteristics when the window substrate 2 is provided. By providing the light shielding region 7, the reflected light on the surface of the capillary is blocked, so that the background light intensity is reduced to about 10%. I was able to. Furthermore, the noise between the capillaries could be suppressed. As a result, the SN ratio is improved, and a signal with a smaller fluorescence intensity can be detected, and highly accurate measurement can be realized.

[0012] Next, a description will be given of an effect of the laser input Imad 12. The laser entrance Imad, as shown in FIG. 2, is formed in the direction perpendicular to the capillary array groove 8 on the side surface of the window substrate 2. FIG. 3A is a cross section of the laser incident part, and FIG. 3B is a cross section of the non-incident part. For comparison, an example in which the entrance window 12 is not provided is shown in FIG. If there is no entrance window 12, although the incidence angle of the laser 33 for to reach the capillary is severely limited, tolerance of the angle of incidence θ is widened by providing the laser input Imad 12. As a result, there is an advantage that the assembling property and the productivity are significantly improved.

As shown in FIG. 1, in the capillary 1, the polyimide resin 10 in the region of the window substrate 2 where the through window 6 is formed is removed.
The area where 0 is removed is not limited to this. Further, the coating material of the capillary 1 is not limited to the polyimide resin 10, and the same electric insulation property as that of the polyimide resin 10,
And a member having various other characteristics may be used.

(Second Embodiment) FIGS. 5 and 6 show a system in which a laser entrance window 12 is provided and an optical fiber 100 is inserted therein. By designing the size of the entrance window according to the diameter of the optical fiber, it is possible to perform precise alignment. This embodiment has an advantage that the laser light can be incident simply and surely as compared with the method in which the laser light is incident from a distant place.

Next, a method of processing the window board 2 will be described with reference to FIG. Here, a single crystal silicon substrate was used as the substrate material. First, the thermal oxide film 201 is formed on the silicon substrate 2.
Is formed (), a resist is applied, exposure and development are performed, and an oxide film is etched with an HF solution (). Next KO
Anisotropic etching with an alkaline solution such as H (
~), And finally the oxide film 201 is removed (). The use of single crystal silicon and anisotropic etching has a processing accuracy in the unit of μm, so that the arrangement of capillaries and the through windows 6,
This is because the dimensions of the laser incident window 12 or the light shielding area 7 can be easily defined by setting process conditions. Further, by using this etching technique, mass production is possible at low cost. Here, the etching mask material is not limited to the thermal oxide film 201, but may be a silicon nitride film or the like.

(Third Embodiment) FIG. 8 shows the configuration of a capillary array unit. Capillary 1, wind
A unit 29, a buffer solution inlet 30, an electrode plate 31 with a protective cover, a conductive fluorescent sample inlet 32 and the like,
As the window unit 29, the window unit shown in (first embodiment) is used. Practically, the capillaries are exchanged after being used several times to a dozen or more times, and are treated as consumables. Therefore, there is an advantage in that the capillaries can be easily attached to the device by being unitized in this way. Further, there is an effect that a user's mounting mistake can be prevented.

(Fourth Embodiment) FIG. 9 shows a fourth embodiment of the present invention.
2 is an explanatory view of the capillary array electrophoresis apparatus according to the embodiment of FIG. The capillary array electrophoresis apparatus is the capillary array unit, buffer container 17, fluorescent sample container 18, high voltage power source 19, laser light source 20, mirror 21, beam splitter 22, condensing lens 23 shown in the third embodiment. , A first lens 24, an optical filter and an image division prism 25, a second lens 26, a CCD camera 27, an arithmetic processing unit 28, and the like. Laser light source 20
The laser 33 generated by is split into two by the beam splitter 22, and the traveling direction is changed by the mirror 21. The laser 33 is condensed by the condenser lens 23 and irradiates the capillary 1. The inside of the capillary 1 is filled with a fluorescently labeled sample (fluorescent sample 34), and the laser 3
By irradiating the sample 34 with 3
5 occurs. This fluorescent light is made into parallel light by the first lens 24, image-divided by the optical filter and the image-division prism 25, and then imaged on the CCD camera 27 by the second lens 26. The processing arithmetic unit 28 analyzes the signal (fluorescence) captured as image data.

In FIG. 9, the laser 33 irradiates from both sides of the window unit 29, but it is also possible to irradiate only one side, and the number of the capillaries 1 of the capillary array unit is not limited to 16. Absent.

Next, the operation principle of the capillary array electrophoresis apparatus will be described. Buffer solution 3 contained in the buffer solution container 17
6 is injected into the capillary 1 through the injection port 30 of the capillary array unit. Next, the conductive sample injection port 32 is put into the sample container 18 filled with the fluorescent sample 34, and the fluorescent sample 34 is injected into the capillary 1. Then, the sample inlet 32 is put in a container (not shown) containing a buffer solution,
Electrophoresis is caused by applying a voltage 19 between the inlet 30 and the electrode portion 31. Since the migration speed of electrophoresis is proportional to the charge amount of the molecule and inversely proportional to the size of the molecule, the fluorescent sample 34 is separated. Continue applying high voltage,
The fluorescence 35 emitted at this time is continuously measured.

By providing the light shielding area 7 in the window unit, the reflected light reflected on the surface of the capillary 1 is shielded, so that the background light can be reduced and the SN ratio can be improved. Further, by providing the laser incident window 12 on the side surface of the window unit 29, there is an advantage that the allowable incident angle is increased and the arrangement margin of the optical system (20 to 23) is expanded.

(Fifth Embodiment) FIG. 10 is an explanatory diagram of a capillary array electrophoresis apparatus according to a fifth embodiment of the present invention. An optical fiber 100 and an optical branching device 101 are used as an optical system for incident light. In this embodiment, the optical fiber 10 is provided in the entrance windows 12 on both side surfaces of the window unit 29.
Since 0 is inserted, the optical system becomes simple. In particular, there is an advantage that the alignment (positioning) of the incident light and the capillary can be surely performed.

[0022]

EFFECTS OF THE INVENTION In a capillary array electrophoresis apparatus, a background light is reduced by forming a light emission window having a light-shielding region for each capillary and a window for excitation laser light incidence on a holding substrate for a capillary array. The SN ratio can be improved. Further, there is an effect that the laser beam can be easily incident and the arrangement of the optical system can be made to have a margin, so that the assembling can be simplified.

[Brief description of drawings]

FIG. 1 is a diagram showing a window unit according to a first embodiment of the present invention.

FIG. 2 is an explanatory view of the window board shown in FIG.

3 is a schematic cross-sectional view of FIG. 2, in which (a) is a laser irradiation part and (b) is a laser non-irradiation part.

FIG. 4 is a schematic cross-sectional view in the case where the window substrate has no laser incident portion.

5 is an explanatory view of another window board of FIG. 1. FIG.

FIG. 6 is a diagram showing a second embodiment in which a laser incident portion of a window substrate is used as an optical fiber fixing groove.

FIG. 7 is a diagram showing an example of a manufacturing process when silicon is used as a window substrate material.

FIG. 8 is a diagram showing an example of a capillary array unit (embodiment 3) including a window unit, capillaries, and electrodes.

FIG. 9 Capillary Array Electrophoresis Device (Embodiment 4)
FIG.

FIG. 10 is an explanatory diagram of a capillary array electrophoresis apparatus (embodiment 5).

FIG. 11 is an explanatory diagram of a fluorescence measurement result according to the first embodiment of the present invention.

[Explanation of symbols]

1 ... Capillary, 2 ... Window substrate, 3 ... Glass substrate,
4 ... Laser irradiation part, 5 ... Laser non-irradiation part, 6 ... Through window,
7 ... Shading frame, 8 ... V groove, 9 ... Fused quartz tube, 10 ... Polyimide resin, 11 ... Ground glass surface, 12 ... Irradiation window, 17
... buffer solution dissolver, 18 ... fluorescent sample container, 19 ... high-voltage power supply, 20 ... laser light source, 21 ... mirror, 22 ... beam splitter, 23 ... focusing lens, 24 ... first lens, 2
5 ... Optical filter and image division prism, 26 ... Second lens, 27 ... CCD camera, 28 ... Processing arithmetic unit, 29 ...
Window unit, 30 ... Buffer injection port, 31 ... Electrode plate with protective cover, 32 ... Conductive fluorescent sample injection port, 33
... laser, 34 ... fluorescent sample, 35 ... fluorescent, 36 ... buffer solution, 100 ... optical fiber, 101 ... optical demultiplexer, 201 ...
Silicon oxide film or silicon nitride film, 202 ... Etching groove.

─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Koji Shimizu 882 Ichige, Itamachi, Hitachinaka City, Ibaraki Prefecture Hitachi Co., Ltd. within the measuring instruments group (72) Toshiaki Kita 882, Ichige, Itamachi, Hitachinaka City, Ibaraki Hitachi, Ltd. Mfg. Co., Ltd. (72) Inventor Kiyoji Tsukada 882 Ichige, Image, Hitachinaka City, Ibaraki, Hitachi Ltd. Mfg. Co., Ltd. (56) Reference JP 9-96623 (JP, A) JP 9- 105738 (JP, A) JP 7-306181 (JP, A) JP 7-306180 (JP, A) JP 4-232840 (JP, A) JP 2000-97908 (JP, A) Actual Kaihei 5-79469 (JP, U) US Patent 5790727 (US, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) G01N 27/447 G01N 21/64

Claims (14)

(57) [Claims] 1. A window substrate, which holds a plurality of capillaries, is provided with a light-shielding region for blocking the signal light between the capillaries at an opening through which the signal light passes, and is provided with a notch for introducing excitation light. A window unit for a capillary array electrophoresis apparatus, which comprises: 2. The window unit of the capillary array electrophoresis apparatus according to claim 1, wherein a V groove for positioning the capillary is provided in the window substrate. 3. The window according to claim 1 or 2.
In the unit, a separate substrate is provided so as to face the window substrate, and the plurality of capillaries are sandwiched between the window substrate and the separate substrate, and a window unit of a capillary array electrophoresis apparatus. 4. The window unit according to claim 1 or 3, a sample inlet connected to one end of the capillary, and a buffer inlet connected to a different end. Characteristic capillary array unit. 5. The capillary array unit according to claim 4, an excitation optical system, and a light receiving optical system for detecting signal light generated from a sample filling the inside of the capillary, wherein beam-shaped excitation light is supplied to the window. A capillary array electrophoresis device, which is made incident from the outside of the substrate through the window substrate notch according to claim 1. 6. A capillary array electrophoresis apparatus, wherein an optical fiber is fixed to or supported by the cutout portion of the window substrate according to claim 1, and the optical fiber irradiates the capillary with excitation light. 7. A plurality of capillaries, an excitation optical system for irradiating the excitation light of the capillaries, a light receiving optical system for detecting the signal light generated from the sample in the capillary, and a plurality of capillaries for holding the signal light. And a sample window connected to one end of the capillary and a window unit having a light-shielding region for blocking the signal light between the capillaries and having an entrance window for introducing excitation light. An electrophoretic device comprising an inlet and a buffer solution inlet connected to one end different from the sample inlet. 8. The electrophoretic device according to claim 7, wherein the window unit includes a V groove for positioning a capillary. 9. The electrophoretic device according to claim 7, wherein the window unit has a window substrate and a separate substrate facing the window substrate, and the plurality of capillaries are separate from the window substrate. electrophoresis apparatus characterized by sandwiching a. 10. The electrophoretic device according to claim 7, wherein:
The electrophoresis apparatus, wherein the window unit includes the entrance window for introducing excitation light that fixes or supports an optical fiber, and irradiates the capillary with the excitation light by the optical fiber. 11. A plurality of capillaries and an opening for holding the plurality of capillaries, through which signal light passes, the opening having a light-shielding region for blocking signals between the capillaries and for introducing excitation light. A capillary array comprising: a window unit having an entrance window for the sample, a sample inlet connected to one end of the capillary, and a buffer inlet connected to one end different from the sample inlet. unit. 12. The capillary array unit according to claim 11, wherein the window unit is provided with a V groove for positioning the capillary. 13. The capillary array unit according to claim 11, wherein the window unit has a window substrate and a separate substrate facing the window substrate, and a plurality of capillaries are separated from the window substrate. capillary array unit, characterized in that for clamping the substrate. 14. The capillary array unit according to claim 11, wherein the window unit has an entrance window for introducing excitation light for fixing or supporting an optical fiber for irradiating the capillary with excitation light. Capillary array unit.