JP2003121413A - Sampling structure of capillary array - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a DNA sequencer dispensing with a sample tray. SOLUTION: Electrode end structure of this capillary array is formed as structure wherein three members, namely, an electrode, a capillary, and a bonding agent for bonding the electrode to the capillary, are positioned on the same plane, and a sample is directly attached to the electrode end of the capillary array. The electrode end of the capillary array is subjected to hydrophilization treatment. Thus, the sample tray becomes unnecessary, and the sample quantity to be used can be reduced up to one tenth of the former quantity.

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 a sample such as DNA or protein, and more particularly to a capillary array.

[0002]

2. Description of the Related Art A technique for constructing an array by combining a plurality of capillaries, supplying an electrophoretic medium and a sample to be analyzed or separated to each capillary and causing the sample to migrate, and utilizing the target sample for separation and analysis is known. well known.

Further, such a technique for supplying a sample such as DNA or protein labeled with a fluorescent substance to a capillary is disclosed in US Pat. Nos. 5,366,608, 5,529,679, 5,516,409, 5,730,850 and 5,790,72.
7, 5558705, 5439578, 5274
240, etc.

From the viewpoint of the throughput of separation and analysis, the method using a multi-capillary has many advantages over the electrophoresis method using a slab gel.

Japanese Unexamined Patent Publication No. 9-96623 discloses a capillary array electrophoresis apparatus. FIG. 2 is a diagram for explaining the capillary array, and FIG. 3 is a schematic diagram showing the electrophoresis system. The outer diameter of the capillary 1 is 0.1 to 0.
It has a diameter of 7 mm and an inner diameter of 0.02 to 0.5 mm, and the outer cover is coated with resin such as polyimide. The capillaries themselves are quartz pipes, and a plurality of capillaries (generally several to several tens) are arranged to form an array.

Fluorescently labeled 10 to several 10 μl of DNA
A laser light source 6 to a mirror 7 or a mirror 7 to a load header 4 that captures DNA into a capillary by electrophoresis from a sample tray 3 that incorporates a large number of sample containers 2 containing samples and a detector 5 that fixes and arranges the capillaries in the order of sample numbers of the load header. A beam splitter 8, an excitation optical system for irradiating excitation light with a condenser lens 9, a detection lens system 11 for detecting fluorescence 10 as signal light, and a CCD camera 12 are provided. In this example, laser is irradiated from both sides of a capillary array containing DNA or protein to be electrophoresed, and all capillaries are irradiated with excitation light by focusing the laser by the lens action of the capillaries, and fluorescence from each capillary is emitted. Is detected by the detection optical system. On the side opposite to the load header of the capillary array, there is a capillary head 17 in which a plurality of capillaries are bundled and bonded and attached to a buffer solution container 14 containing a buffer solution 13 in a pressure-tight manner. A high voltage of about 15 kV is applied from the high voltage power supply 15 to the buffer solution container and the load header, and the sample in the sample container is electrophoresed by the buffer solution contained in the capillary from the buffer solution container to separate and analyze the sample. The purpose of the load header is to sample the sample and also to serve as an electrode 20 on the sample container side for applying a high voltage between the sample and the buffer solution container. FIG. 4 shows the structure of the load header. Each of the 16 capillaries 1 is inserted into an electrode of a thin pipe made of stainless steel (hereinafter referred to as a SUS symbol), and the tip on the sample side is slightly protruded from the electrode. It is fixed and sealed with an epoxy adhesive 27. The 16 electrodes are electrically connected in advance by a connecting plate 23 as shown in the drawing and aligned with the holder 25 with a small tolerance. The reason for using the SUS pipe electrode is that the samples and reagents used for the separation analysis are corrosive.

The electrodes are integrally molded in a plastic holder, and after incorporating the connection plate, the lid 26 is ultrasonically bonded to the holder to complete the process.

The capillaries are sealed with an adhesive 27 on the lid so as to prevent the capillaries from coming off and to prevent leakage of high voltage. A part of the connecting plate is bent 90 ° so that a high-voltage contact (not shown in the figure) can be connected through an opening provided in the holder. Further, FIG. 5 shows a state in which the electrode end of the load header is inserted into the sample 28 of the sample container of the sample tray.

A gel 29 for giving a migration speed difference at the time of electrophoresis and a buffer 30 as a medium for electrophoresis are enclosed in the inner diameter portion of the capillary. A DNA sample, which is cut into a large number of appropriate lengths and has fluorescent markings on its head for identifying four kinds of nucleoside base molecules of adenine, guanine, cytosine, and thymine, which compose DNA, is negatively charged.

When a positive potential is applied to the gel and a negative potential is applied to the electrode of the SUS pipe in this state, an electric field is generated in the direction indicated by the two arrows in the figure, and the marked DNA sample enters the gel of the positive potential. Go. After a certain amount of the sample is sampled in the gel, the electrode is taken out of the sample container, put in a buffer containing no sample, and electrophoresis is continued again.

The inside of the gel filled in the entire area of the capillary is D
Since the passage resistance varies depending on the size of the sample while the NA sample passes, the sample reaches the detection unit earlier in the ascending order. In the detector, the capillary is irradiated with a laser to detect fluorescence corresponding to four types of nucleoside bases of adenine, guanine, cytosine, and thymine from the fluorescence-marked DNA sample.

Reference numeral 16 in FIG. 2 is a separator for arranging a plurality of capillaries, and reference numeral 21 in FIG.
Is a signal processing arithmetic unit for arithmetically processing the detected signal.

The capillary array is a consumable item that is discarded for a few months or when the resolution decreases due to electrophoresis for several hundred times.

The load header described above has the following problems. (1) Since the sample is put in the sample container of the sample tray and the electrode end of the load header is inserted into the sample container for sampling, the sample amount used is as large as several tens of μl. (2) Manual work for dispensing a small amount of sample of several tens of μl is a difficult work that requires skill and patience. (3) The use of the sample tray requires contamination of the samples and mixing between the samples (called carryover), so high quality cleaning is required inside and outside the sample tray. (4) The length of the electrode is 20 to 40 mm when using the tray
It is necessary to increase the length, and a long electrode may bend and may not be sampled.

It should be noted that since the amount of sample used for DNA analysis is very small, the same DNA is used when analyzing with such a device.
Sequence samples are expanded by replication. This task is a difficult task created by biochemical operation of 10 steps or more.

[0016]

The first aspect of the present invention relates to an electrode portion structure for electrophoresis without using a sample tray, and it is a problem to be solved that the sample is directly attached to the electrode end and electrophoresis is performed.

The second aspect of the present invention is that the sample required for electrophoresis is several μm.
The problem to be solved is to make it 1 or less.

[0018]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention relates to a capillary array electrophoresis apparatus for separating and analyzing a sample such as DNA or protein, and in particular, the sample is directly attached to the electrode end and the electrode end is not directed downward. Characterize. Hereinafter, the present invention will be described in detail.

FIG. 1 is a diagram showing the present invention. FIG. 1 shows the tip of the electrode, which is the same as the conventional one in that a capillary is put inside the SUS pipe which is the electrode and the tip is attached with an adhesive. As described in the subject of the invention, in order to attach the sample to the end of the electrode as shown, the electrode, the capillary,
Also, it is characterized by cutting so that the surface of the adhesive becomes flat. By doing so, the sample can be directly attached to the electrode end by the nozzle. FIG. 6 shows another embodiment. The ends of the electrodes are flared as shown in the figure, so that the amount of sample adhered can be increased. The advantage of this structure is that you can control the amount of sample adhered by changing the diameter of the flare part while keeping the same dimensions of the original electrode and capillary, and the capillary can always sample from the center of the sample for efficient and uniform electrophoresis. It is possible.

To obtain such an electrode end, as shown in FIG. 7, a capillary is passed through a flared electrode pipe, and the tip is put out on the pipe and filled between the pipe and the capillary with an adhesive such as epoxy. To do. After the adhesive has hardened, it is cut with a high speed cutter as shown by the dotted line so as to obtain a desired diameter. The cutting may be slanted according to the use posture of the electrode pipe.

It is also effective to perform hydrophilic treatment 31 on the end face in order to improve the adhesion of the sample to the electrode end. The hydrophilic treatment also has the following effects.

After the electrophoresis is completed, the sample attached to the electrode is removed, and the gel in the capillary is replaced with a new gel for use. Before and after these operations, washing is performed to prevent carryover, but if hydrophilic treatment is performed, the washing effect is great and accurate DNA analysis can be performed.

FIG. 8 shows a load header using the present invention. It is a load header equipped with seven sampling electrodes. Since the electrodes are short and do not bend, the work of attaching the sample to the electrodes can be automated by the dispenser 32 and a robot (not shown).

Electrophoresis may be performed in the state shown in the figure, and it is not always necessary that the sample is under the end portion of the electrode.
This is the second invention. Furthermore, the electrode ends change their orientation before and after the application of the electrophoretic voltage, and are cleaned → sampled →
Sample uptake → Buffering → Analytical electrophoresis → Washing Take the necessary orientation according to the process. By doing so, a device with good operability is selected.

The amount of the sample used for the analysis is 0.26 to 2.2 with the sample attachment shape being a hemisphere and the electrode end diameter being 1 to 2 mm.
It becomes 1 μl, which can be reduced to 1/10 of the conventional one.

[0026]

According to the present invention, the sample tray is not required and the amount of sample used can be reduced to 1/10 of the conventional amount.

[Brief description of drawings]

FIG. 1 is a diagram showing the present invention.

FIG. 2 is a diagram showing a conventional device.

FIG. 3 is a diagram illustrating a separation analysis system.

FIG. 4 is a diagram illustrating a conventional device.

FIG. 5 is a diagram illustrating a main part of a conventional device.

FIG. 6 is a diagram illustrating an example.

FIG. 7 is a diagram illustrating a processing method of the present invention.

FIG. 8 is a diagram illustrating another invention.

[Explanation of symbols]

1 ... Capillary, 2 ... Sample container, 3 ... Sample tray,
4 ... Road header, 5 ... Detection part, 6 ... Laser light source, 7 ...
Mirror, 8 ... Beam splitter, 9 ... Condensing lens, 10
… Fluorescence, 11… Detection lens system, 12… CCD camera, 1
3 ... buffer solution, 14 ... buffer solution container, 15 ... high voltage power source, 1
6 ... Separator, 17 ... Capillary head, 20 ... Electrode, 21 ... Signal processing arithmetic unit, 23 ... Connection plate, 25 ... Holder, 26 ... Lid, 27 ... Adhesive, 28 ... Sample, 29
... gel, 30 ... buffer, 31 ... hydrophilic treatment part, 32 ...
Despencer.

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Akihiro Suzuki             882 Ichige, Ichima, Hitachinaka City, Ibaraki Prefecture             Ceremony company Hitachi High Technologies Design and manufacturing             Headquarters Naka Operations (72) Inventor Tomonari Morioka             882 Ichige, Ichima, Hitachinaka City, Ibaraki Prefecture             Ceremony company Hitachi High Technologies Design and manufacturing             Headquarters Naka Operations F-term (reference) 4B024 AA11 CA01 HA19                 4B029 AA23 BB20 CC13

Claims (6)

[Claims] 1. A plurality of capillaries for DNA analysis, and a plurality of capillaries for injecting a sample at one end of the capillaries, each having a window unit having an opening for aligning and holding the capillaries and irradiating ultraviolet rays in the middle of the capillaries. In a capillary array equipped with a load header having an inlet and a capillary head for injecting a buffer solution at the other end, the sample injection port of the load header passes the capillary through the electrode, and the sample is attached to the electrode end by surface tension. Capillary array characterized by. 2. The capillary array according to claim 1, wherein the electrode and the capillary are fixed with an adhesive, and the electrode, the capillary, and the adhesive form the same plane at the electrode end. 3. The capillary array according to claim 2, wherein an end surface of the electrode to which the sample is attached is hydrophilically treated. 4. A plurality of capillaries for DNA analysis, and a plurality of capillaries for injecting a sample at one end, the cap having a window unit having an opening for aligning and holding the capillaries and irradiating ultraviolet rays in the middle of the capillaries. In a capillary array with a load header having an inlet and a capillary head for injecting a buffer solution at the other end, the sample injection port of the load header passes the capillary through the electrode, and the sample is attached to the electrode end by surface tension, and the sample is attached to the electrode. A capillary array characterized in that the extreme is electrophoresed in a posture other than downward. 5. The capillary array according to claim 4, wherein the orientation of the electrode end face changes before and after the application of the electrophoretic voltage. 6. The capillary array according to claim 1 or 4, wherein the outer diameter of the electrode end surface is φ0.7 to φ2.0.