JP2001264293A - Capillary array electrophoresis device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a window base board for a capillary array cutting off background light so as to facilitate incidence of an excited laser beam and to improve an S/N ratio of an electrophoresis device by using the window base board for the capillary array. SOLUTION: In a holding base board constituting a window unit for the capillary array, light shielding areas each spaced at a pitch equal to an arrangement interval of a capillary 1 are arranged in a through window 6 for passing emitted light, and a window 12 for incidence of an excited laser beam is arranged on the base board side face. In this way, background light is reduced because reflected light on the surface of the capillary 1 is cut off by means of the light shielding areas 7 in the holding base board, and the S/N ratio can be improved. In addition, incidence of a laser beam is facilitated and the degree of freedom of an optical system arrangement is increased, so that assembly can be carried out easily.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a capillary array electrophoresis apparatus for separating and analyzing samples such as DNA and protein.

[0002]

2. Description of the Related Art A capillary array electrophoresis apparatus described in Japanese Patent Application Laid-Open No. 09-096623 is an excitation optical system including a capillary array in which a plurality of capillaries are arranged in a plane, a laser light source and the like, and signal light. It consists of a light receiving optical system that detects fluorescence. In this example, the capillary containing the fluorescent sample is irradiated with laser from the side, and the laser is condensed by the lens function of the capillary, so that all the capillaries are irradiated with laser, and the fluorescence from each capillary is received by the receiving optical system. It is to detect.

US Pat. No. 5,790,727 (Aug. 4, 1)
998) is an apparatus for receiving fluorescence from a sample emitted in a direction perpendicular to the laser irradiation direction by an optical fiber. In this example, an attempt is made to reduce the background light by attaching a light extractor made of an optical fiber in the fluorescence detection direction so that light reflected from the capillary surface is not incident on the light extractor.

[0004]

Problems to be Solved by the Invention Japanese Patent Laid-Open No. 09-0966
In the method disclosed in Japanese Patent Publication No. 23, part of the fluorescence from the sample due to laser irradiation is reflected on the surface of the capillary, and this causes an increase in background light, leading to a decrease in detection accuracy.

Further, US Pat. No. 5,790,727 (Aug.
No. 4,1998), the capillary array is a consumable item, whereas the fluorescence detector made of optical fibers is equipment equipment. Therefore, every time the capillary array is replaced, the capillary central axis and the light on the detection side are changed. It was necessary to align the center axis of the fiber, and it was not convenient for the user.

[0006] It is an object of the present invention to reduce the background light to reduce the SN.
It is an object of the present invention to provide a capillary array electrophoresis apparatus having an improved ratio and high detection accuracy, simplifying installation of a capillary in the apparatus and introduction of an excitation optical system, and being easy to use. .

[0007]

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

[0008]

Embodiments of the present invention will be described below in detail with reference to the drawings.

(First Embodiment) FIG. 1 shows a first embodiment of the present invention.
It is explanatory drawing of the wind unit which is the embodiment.
It is roughly composed of 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 as viewed from the back, and FIGS. 3A and 3B are cross-sectional views. FIGS.
It corresponds to AA 'and the non-irradiated portion BB'. First, the configuration will be described.

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

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 used for electrophoresis, and the refractive indexes of the two are almost equal. FIG. 11 shows a wavelength of 584n.
5 shows a fluorescence intensity characteristic of m (fluorescence due to OH stretching vibration of water). The horizontal axis represents the sequence number of the capillary 1 arranged in a plane, that is, the arrangement position, and the vertical axis represents the relative fluorescence intensity normalized by the maximum fluorescence intensity. (A) shows the conventional fluorescence emission characteristics 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. 2B shows the characteristics when the window substrate 2 is provided. Since the light shielding region 7 is provided, the reflected light on the capillary surface is blocked, and the background light intensity is reduced to about 10%. I was able to. Furthermore, noise between capillaries could be suppressed. As a result, the SN ratio is improved, a signal having a smaller fluorescence intensity can be detected, and high-accuracy measurement can be realized.

Next, the effect of the laser irradiation window 12 will be described. As shown in FIG. 2, the laser irradiation window is formed on the side surface of the window substrate 2 in a direction orthogonal to the capillary array grooves 8. FIG. 3A is a cross section of a laser incident portion, and FIG. 3B is a cross section of a non-incident portion. For comparison, an example in which the incident window 12 is not provided is shown in FIG. When the incident window 12 is not provided, the incident angle of the laser 33 for reaching the capillary is strictly limited. However, the provision of the irradiation window 12 increases the tolerance of the incident angle θ. Thereby, there is an advantage that assemblability and productivity are greatly improved.

As shown in FIG. 1, in the capillary 1, the polyimide resin 10 in a region where the through window 6 is formed in the window substrate 2 is removed.
The region from which 0 is removed is not limited to this. Further, the coating material of the capillary 1 does not need to be limited to the polyimide resin 10, and has the same electrical insulation properties as the polyimide resin 10,
Alternatively, a member having other various characteristics may be used.

(Second Embodiment) FIGS. 5 and 6 show a system in which a laser incident window 12 is provided and an optical fiber 100 is inserted into the window. By designing the size of the entrance window according to the diameter of the optical fiber, precise positioning can be achieved. This embodiment has an advantage that the laser beam can be incident simply and reliably as compared with the system in which the laser beam is incident from a remote place.

Next, a method of processing the window substrate 2 will be described with reference to FIG. Here, a single crystal silicon substrate was used as a 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
Perform anisotropic etching with an alkaline solution such as H (
~), And finally, the oxide film 201 is removed (). Single crystal silicon and anisotropic etching are used because they have a processing accuracy of μm unit, so that the arrangement of the capillaries and the windows 6, 1
This is because the dimension of the light-shielding region 2 or the light-shielding region 7 can be easily specified only by setting the process conditions. By using this etching technique, mass production can be performed at low cost. Here, the etching mask material is a thermal oxide film 20.
It is not limited to one, 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 the first embodiment is used. Practically, the capillary is replaced after being used several to ten and several times, and is treated as a consumable. Therefore, there is an advantage that the unit can be easily replaced by being unitized in this way. In addition, there is an effect that a user's mounting error can be prevented beforehand.

(Fourth Embodiment) FIG. 9 shows a fourth embodiment of the present invention.
1 is an explanatory diagram of a capillary array electrophoresis apparatus according to an embodiment of the present invention. The capillary array electrophoresis apparatus includes the capillary array unit described in the third embodiment, a buffer solution container 17, a fluorescent sample container 18, a high voltage power supply 19, a laser light source 20, a mirror 21, a beam splitter 22, a condenser lens 23. , A first lens 24, an optical filter and an image splitting prism 25, a second lens 26, a CCD camera 27, an arithmetic processing unit 28, and the like. Laser light source 20
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 the sample 3, the fluorescent 3
5 occurs. The fluorescent light is converted into parallel light by a first lens 24, image-divided by an optical filter and an image splitting prism 25, and then imaged on a CCD camera 27 by a second lens 26. The signal (fluorescence) captured as image data is analyzed by the processing operation device 28.

In FIG. 9, the laser 33 is irradiated from both sides of the window unit 29. However, the structure may be such that only one side is irradiated. 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 in buffer solution container 17
6 is injected into the capillary 1 from the inlet 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. Thereafter, the sample inlet 32 is put into a container (not shown) containing a buffer solution,
Electrophoresis is caused by applying a voltage 19 between the inlet 30 and the electrode unit 31. Since the moving speed of the 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 region 7 in the window unit, the reflected light reflected on the surface of the capillary 1 is blocked, 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 allowance of the optical system (20 to 23) is increased.

(Fifth Embodiment) FIG. 10 is an explanatory view of a capillary array electrophoresis apparatus according to a fifth embodiment of the present invention. An optical fiber 100 and an optical splitter 101 are used as an optical system for incident light. In this embodiment, the optical fiber 10 is inserted into the entrance windows 12 on both sides of the window unit 29.
Since 0 is inserted, the optical system is simplified. In particular, there is an advantage that the alignment (positioning) between the incident light and the capillary can be reliably performed.

[0022]

As described above, in the capillary array electrophoresis apparatus, a light emitting window having a light-shielding area for each capillary and a window for exciting laser beam incidence are formed on a holding substrate for the capillary array, thereby reducing background light. However, the SN ratio can be improved. Further, there is an effect that the incidence of the laser beam is facilitated, the arrangement of the optical system is given a margin, and the assembly is simplified.

[Brief description of the drawings]

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

FIG. 2 is an explanatory diagram of the window substrate of FIG. 1;

FIGS. 3A and 3B are schematic cross-sectional views of FIG. 2, in which FIG. 3A shows a laser irradiation part, and FIG.

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

FIG. 5 is an explanatory diagram of another window substrate of FIG. 1;

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 illustrating 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, a capillary, and electrodes.

FIG. 9 shows a capillary array electrophoresis apparatus (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 result of fluorescence measurement in 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: penetrating window,
7: light shielding frame, 8: V groove, 9: fused quartz tube, 10: polyimide resin, 11: ground glass surface, 12: irradiation window, 17
... Buffer dissolver, 18 ... Fluorescent sample container, 19 ... High voltage power supply, 20 ... Laser light source, 21 ... Mirror, 22 ... Beam splitter, 23 ... Condenser lens, 24 ... First lens, 2
5 optical filter and image splitting prism, 26 second lens, 27 CCD camera, 28 processing unit, 29
Wind unit, 30: buffer solution inlet, 31: electrode plate with protective cover, 32: conductive fluorescent sample inlet, 33
... laser, 34 ... fluorescent sample, 35 ... fluorescence, 36 ... buffer solution, 100 ... optical fiber, 101 ... optical demultiplexer, 201 ...
Silicon oxide film or silicon nitride film, 202: etching groove.

Continued on the front page (72) Inventor Koji Shimizu 882 Ma, Ichiki, Hitachinaka-shi, Ibaraki Pref.In the measuring instrument group of Hitachi, Ltd. Within the measuring instrument group (72) Inventor Kiyoji Tsukada 882, Momo, Oaza, Hitachinaka-shi, Ibaraki Prefecture Within the measuring instrument group of Hitachi, Ltd.

Claims (6)

[Claims] 1. A capillary holding substrate that holds a plurality of capillaries and has an opening through which signal light passes, wherein the opening has a light-shielding region that blocks signal light between the capillaries, and a cut-out area for introducing excitation light. A window substrate, wherein a notch is provided, and a window unit, wherein a plurality of capillaries and the window substrate are supported or fixed. 2. The window substrate according to claim 1, wherein
A window substrate provided with a V-groove for capillary positioning, and a window unit using the same. 3. The window according to claim 1, wherein
In a unit, a separate substrate is provided facing the window substrate, and the plurality of capillaries are sandwiched between the window substrate and the different substrate. 4. A plurality of capillaries, a 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 one end of the capillary. A capillary array unit comprising: 5. A capillary array unit according to claim 4, comprising: an excitation optical system; and a light receiving optical system for detecting signal light generated from a sample filling the capillary, wherein the excitation light has a beam shape. Claim 1 from the outside of the window substrate
A capillary array electrophoresis apparatus characterized in that the light is passed through the cutout portion of the window substrate described above. 6. A capillary array electrophoresis apparatus, wherein an optical fiber is fixed or supported in the excitation light introducing notch of the window substrate, and the capillary is irradiated with the excitation light by the optical fiber. .