JPH10253592A - Sample plate and multi-capillary cataphoresis device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a sample plate that is suited for a multi-capillary cataphoresis device. SOLUTION: A base plate 101 is made of an insulation material and has a plane surface and a connector part 106 that is connected to it. A plurality of wells 102 are arranged on the surface of the base plate 101 with a constant gap horizontally and vertically, and an individual electrode pattern 104 reaching the connector part 106 is formed at its bottom in each well 102 through a base plate surface. The connector part 106 is connected to an external high-voltage application part.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a multi-capillary array electrophoresis section in which a plurality of capillary columns are arranged, a plurality of samples are injected into the capillary columns one by one, and all the capillary columns are electrophoresed simultaneously. A multi-capillary electrophoresis device equipped with an optical measurement unit that irradiates the capillary with light and measures the absorbance of the irradiated part by the sample and the fluorescence from the sample, and when introducing the sample into the multiple capillary columns It relates to a sample plate used for the above. Such a multi-capillary electrophoresis apparatus is used for protein separation and DNA base sequence determination. In a multi-capillary electrophoresis apparatus for DNA base sequence determination, a DNA fragment (fragment) sample in which a primer or a terminator is labeled with a fluorescent substance is electrophoresed using a Sanger reaction, and fluorescence from the DNA fragment sample is analyzed during electrophoresis. Detect and determine the base sequence.

[0002]

2. Description of the Related Art A DNA sequencer having high sensitivity, high speed, and large processing capacity is required for base sequence determination of DNA having a long base sequence such as a human genome. As one of the methods, there has been proposed a multi-capillary DNA sequencer in which a plurality of capillary columns filled with a gel are arranged instead of using a plate-shaped slab gel. Capillary columns, compared to slab gels,
Not only is it easy to handle and inject samples, but it can also be run at high speed and detected with high sensitivity. In other words, when a high voltage is applied to a slab gel, problems such as broadening of the band and the occurrence of a temperature gradient occur due to the influence of Joule heat.However, such a problem is small in a capillary column. Even if it does, the band spread is small and high-sensitivity detection can be performed.

[0003] In capillary electrophoresis, a sample is introduced into a capillary column by a method using pressure or an electrophoretic method by applying a voltage. Among them, the method of introducing a sample by electrophoresis is simple in device configuration,
It is widely and generally employed in terms of ease of operation and good controllability of parameters. When introducing a sample by electrophoresis, one end of the capillary column must be immersed in the prepared sample, the other end must be immersed in the buffer solution, and an electrode such as a platinum wire must be immersed in the sample near the end of the capillary column. is there.

[0004]

In the structure in which the electrodes are held near the end of each capillary column at the time of electrophoretic sample introduction, a multi-capillary electrophoresis system in which a plurality of capillary columns are collectively arranged in an array and electrophoresed simultaneously. In the case of the electrophoresis device, the electrode structure for introducing the sample becomes complicated. Also, after injecting the end of the capillary column into the sample in the sample injection container and injecting by applying voltage, etc., the end of the capillary column must be transferred to the reservoir containing the buffer solution for electrophoresis. It takes time and effort to perform the above operations, and it would be advantageous if automation could be performed.

A first object of the present invention is to provide a sample plate for introducing a sample having a simple electrode structure suitable for a multi-capillary electrophoresis apparatus. A second object of the present invention is to use such a sample plate,
An object of the present invention is to provide a multi-capillary electrophoresis apparatus that can automate the steps from sample injection to electrophoresis to a large number of capillary columns.

[0006]

In the sample plate of the present invention, a plurality of holes with bottoms are formed as wells on the surface of an insulating base plate having a planar surface and a connector portion connected to the surface. In the well, individual electrode patterns from the bottom to the connector portion via the base plate surface are formed.

A multi-capillary electrophoresis apparatus according to the present invention comprises a multi-capillary electrophoresis unit, in which a plurality of capillary columns are arranged, a plurality of samples are injected into the capillary columns one by one, and the electrophoresis is simultaneously performed on all the capillary columns. The capillary array electrophoresis unit irradiates light to the capillary, and has an optical measurement unit that measures the absorbance of the irradiated part by the sample and the fluorescence from the sample.The sample injection side of the multi-capillary array electrophoresis unit A sample plate in which wells containing samples are two-dimensionally arranged below the capillary column ends, corresponding to the arrangement of the capillary column ends, below the capillary column ends, and All capillaries containing buffer solution for electrophoresis A reservoir for electrophoresis in which a voltage is applied to the ram is arranged, and the sample plate has a plurality of bottomed holes formed as wells on the surface of an insulating base plate having a planar surface and a connector portion connected thereto. Each well has an individual electrode pattern formed from the bottom of the well to the connector via the base plate surface. One of the sample plate and the reservoir for electrophoresis is switched to switch the end of the capillary column on the sample input side. There is provided a moving mechanism for making the contact.

When injecting a sample into a capillary column,
Each sample is put into the well of the sample plate, and the sample plate is moved by the moving mechanism, and one end of each capillary column is immersed in the sample in the well. A voltage is applied between the connector portion of the sample plate and the buffer solution at the other end of the capillary column, and the sample is electrophoretically injected into the capillary. After the sample injection, the sample plate is removed from contact with one end of the capillary column by the moving mechanism, and one end of the capillary column is immersed in the buffer solution of the electrophoresis reservoir. Thereafter, electrophoresis is performed by applying a voltage between the buffer solutions of the reservoirs at both ends of the capillary column. Thereby, the operation from the sample injection to the capillary column to the electrophoresis is automatically performed.

[0009] The wells of the sample plate are used for PCR (Polymerase Chain Reac-
After processing the sample by the method, the capillary end can be inserted into the sample in the well to perform sample injection. The PCR method is a method in which only a desired portion of DNA is greatly amplified. PCR
In the method, a primer is added to the sample DNA, the temperature is raised to dissociate the double-stranded DNA into single strands, then the temperature is lowered to bind the primer to the DNA strand, and the temperature is raised slightly to synthesize the DNA, The temperature is further increased to form a single strand.
This is a method of amplifying and synthesizing a predetermined portion of DNA in large quantities by repeating the operation of changing the temperature up and down.

[0010]

1A and 1B show an embodiment of a sample plate, in which FIG. 1A is a schematic perspective view, and FIG. 1B is a sectional view taken along the line AA 'in FIG. The cross-sectional structure of one well is shown. The base plate 101 of the sample plate 100 is made of an insulating material, and has a planar surface and a connector 106 connected to the surface.
The material of the base plate 101 is polyether sulfone,
Heat-resistant engineering plastics such as polyetherimide, polysulfone, and liquid crystal polymer are suitable. A plurality of wells 102 are arranged on the surface of the base plate 101 at regular intervals in the vertical and horizontal directions. The well 102 is a hole having a bottom, and each well 102 is formed with an individual electrode pattern 104 extending from the bottom to the connector 106 via the base plate surface. The connector section 106 is a section connected to an external high voltage application section.

The electrode pattern 104 is formed as a three-dimensional wiring pattern from the bottom of the well 102 to the connector 106 via the inner wall surface of the well 102 and the surface of the base plate. Such a wiring pattern is formed by a MID (Molded Interco.) Forming a conductive wiring pattern on a resin molded product.
nnect Device) using the base plate 101
Can be formed by plating a conductive material such as copper or nickel on the molded product by electroless plating. In consideration of the adsorption of the sample and the influence of the metal ions on the sample, it is preferable that the electrode pattern 104 is finally plated with gold.

FIG. 2 schematically shows a multi-capillary electrophoresis apparatus for injecting a sample using this sample plate. The multi-capillary electrophoresis apparatus is a gel electrophoresis apparatus in which a gel such as polyacrylamide gel, linear acrylamide gel, polyethylene oxide (PEO) gel is filled as a separation medium in a capillary column, or free electrophoresis in a capillary column without filling the gel The sample plate of the present invention can be applied to any electrophoresis apparatus using a plurality of capillary columns.

A pair of reservoirs 110 and 120 are filled with buffer solutions 112 and 122, respectively, and electrodes 130 and 132 are provided in both buffer solutions, respectively. Each well 1 of the sample plate 100 shown in FIG.
02, a sample is put in, and a high-voltage wiring cable is connected to the connector unit 106.

Reservoir 110 and sample plate 100
Is connected in a switchable manner by a high-voltage switching unit 136 so that the wiring is switched, and a high-voltage power supply for electrophoresis is connected between the high-voltage switching unit 136 and the electrode 132 provided in the other reservoir 120, and the sample injection is performed. And electrophoresis voltages are applied.

At the time of sample injection, one end 2a of the capillary array 2 is inserted one by one into each well 102 of the sample plate 100. After the sample injection, the one end 2a is switched to the reservoir 110 and the one end 2a is immersed in the buffer solution 112.
The other end 2b of the capillary array 2 is connected to the other reservoir 1
20 is immersed in a buffer solution 122, and the other end side is provided with a detected part 2c which is irradiated with measurement light or excitation light from an optical measurement part 10 for detecting a sample by absorbance or fluorescence and measures absorbance or fluorescence. Have been.

The capillary array 2 has a two-dimensional arrangement corresponding to the arrangement of the wells 102 of the sample plate 100 at one end 2a, and the capillary columns are arranged in a line at the detection target 2c, and are perpendicular to the arrangement surface of the capillary columns. Measurement light or excitation light is emitted from the direction. The sample plate 100 and the reservoir 110 are switched so as to be selectively brought into contact with the capillary end 2a by a moving mechanism (not shown in FIG. 2).

At the time of sample injection, the sample plate 100
The capillary column ends 2a are immersed one by one in the sample in the well, and the other ends of the capillary columns are immersed in the buffer solution 122 of the reservoir 120 at a time. Then, all the wells 102 are simultaneously formed through the electrode pattern 104,
Alternatively, a high voltage is applied in order to inject the sample into the capillary column. When a high voltage is sequentially applied to the wells, a switching unit may be provided. If a plurality of high voltage applying means are prepared, the sample can be introduced under different introduction conditions depending on the sample.

After the injection of the sample, the application of the high voltage is temporarily stopped, and the sample plate 100 and the reservoir 110 are moved by the moving mechanism, whereby the capillary end 2 on the sample side is moved.
a is immersed in the buffer solution 112 of the reservoir 110. Thereafter, a high voltage is applied between the two reservoirs 110 and 120 to perform electrophoretic separation. The voltage for sample injection and the power supply voltage for migration are, for example, 30 kV, and the current capacity is 10 to 30 mA.
It is.

The moving mechanism moves the sample plate 100 and the reservoir 110 in a horizontal plane, and moves the capillary end 2a.
May be moved in the vertical direction. The number of wells in the sample plate 110 can be arbitrarily set according to the number of capillary arrays. Also,
The reservoir 110 also has a format in which a plurality of wells are filled with a buffer solution like the sample plate 100, and an individual electrode pattern is provided in each well so that an independent voltage can be applied to each well. The electrophoresis may be performed simultaneously.

[0020]

According to the sample plate of the present invention, a plurality of bottomed holes are formed as wells on the surface of an insulating base plate having a flat surface and a connector portion connected to the flat surface. Since individual electrode patterns from the bottom to the connector portion through the base plate surface are formed, using the sample plate enables electrode wiring to a plurality of wells without taking a complicated electrode wiring structure. In particular, it becomes easy to inject a sample into a capillary column in an electrophoresis apparatus using a multi-capillary array having a large number. The multi-capillary electrophoresis apparatus of the present invention uses this sample plate for sample injection, and has a moving mechanism for switching any one of the sample plate and the electrophoresis reservoir to contact the capillary column end. The operation of sample injection and migration can be performed automatically.

[Brief description of the drawings]

1A and 1B show an embodiment of a sample plate, wherein FIG. 1A is a schematic perspective view, and FIG.
It is sectional drawing in the A 'line position.

FIG. 2 is a schematic perspective view showing one embodiment of a multi-capillary electrophoresis apparatus for injecting a sample using the sample plate of one embodiment.

[Explanation of symbols]

 2 Capillary array 2a Capillary array end on sample side 2c Detected part 10 Optical measuring part 100 Sample plate 102 Well 104 Electrode pattern 106 Connector part 110,120 Reservoir 134 High voltage power supply

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Yoshihide Hayashizaki 3-1-1 Takanodai, Tsukuba, Ibaraki Pref. RIKEN Life Science Tsukuba Research Center (72) Inventor Shin Nakamura Shinnokyo Kuwabara, Nakagyo-ku, Kyoto, Kyoto, Japan No. 1 in the Sanjo Plant of Shimadzu Corporation

Claims (2)

[Claims] A plurality of bottomed holes are formed as wells on the surface of an insulating base plate having a planar surface and a connector portion connected thereto, and each well is formed from the bottom through the base plate surface. A sample plate, wherein individual electrode patterns reaching the connector portion are formed. 2. A method according to claim 1, wherein a plurality of capillary columns are arranged,
A plurality of samples are injected one by one into the capillary column, and a multi-capillary array electrophoresis section that is electrophoresed simultaneously in all capillary columns, and irradiates the capillary with the multi-capillary array electrophoresis section, In a multi-capillary electrophoresis apparatus provided with an optical measurement unit for measuring absorbance by a sample and fluorescence from the sample, the end of the capillary column is two-dimensionally arranged and arranged downward on the sample injection side of the multi-capillary array electrophoresis unit. Below the capillary column end, corresponding to the arrangement of the capillary column end, a sample plate in which wells containing samples are arranged two-dimensionally, and a buffer solution for electrophoresis are contained, and voltage is applied to all capillary columns. An applied electrophoresis reservoir is arranged, In addition, the sample plate has a plurality of wells with bottoms formed as wells on the surface of the insulating base plate having a planar surface and a connector part connected thereto, and each well has a base plate surface from the bottom. An individual electrode pattern is formed to reach the connector section via the connector section, and a moving mechanism for switching any one of the sample plate and the reservoir for electrophoresis to contact the end of the capillary column is provided. Multi-capillary electrophoresis apparatus.