JP3695631B2 - Electrophoresis device - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an electrophoresis apparatus used in the bio field, and more particularly to an improvement for increasing the speed and resolution of electrophoresis.
[0002]
[Prior art]
Conventionally, electrophoresis is well known as a method capable of performing gene structure analysis and structure analysis of proteins such as amino acids using an inexpensive and simple apparatus, and is widely used in the bio field.
[0003]
Electrophoresis includes disk electrophoresis using polyacrylamide, SDS (sodium dodecyl sulfate) polyacrylamide gel electrophoresis, isoelectric focusing, nucleic acid gel electrophoresis, There are electrophoresis methods utilizing the influence of interaction, two-dimensional electrophoresis methods, capillary electrophoresis methods and the like.
[0004]
FIG. 9 is a block diagram showing an example of a conventional electrophoretic measurement device. The electrophoretic measurement device is roughly composed of an electrophoretic device unit 10 and a signal processing device unit 20.
The electrophoresis apparatus unit 10 includes a migration unit 11 that performs electrophoresis, a first electrode 12 and a second electrode 13 for applying a voltage to the migration unit 11, and a support that holds the migration unit 11 and the electrodes 12 and 13. The plate 14, the electrophoretic power supply device 15 for applying a voltage to the electrode, the light source 16 for generating light for exciting the fluorescent material, the optical fiber 17 for guiding the light from the light source 16, and the fluorescent material It is composed of a photodetector 18 that condenses the fluorescence, selectively passes light of a specific wavelength by an optical filter, and converts it into an electrical signal.
[0005]
The signal processing unit 20 can receive an electrical signal from the photodetector 18 and perform appropriate processing, for example, pre-processing such as data conversion to digital data, addition averaging processing, and the like. . The output of the signal processing unit 20 is sent to a data processing device (not shown), and the sample is analyzed by analysis processing.
[0006]
In such an apparatus, a gel is injected into the electrophoresis unit 11, a sample of a DNA fragment labeled with a fluorescent substance is injected from above the gel, and then a voltage is applied to the first electrode 12 and the second electrode 13 by the power supply device 15. When is applied, electrophoresis starts. In each lane of the sample, the molecules contained in the sample gather for each molecular weight and form a band. The lighter the molecular weight, the faster the migration speed, and the longer the distance migrated in the same time.
[0007]
These bands are detected by irradiating the gel with light from the light source 16 (for example, laser light) to generate fluorescence in the fluorescent substance of the label gathered in the band in the gel, and this fluorescence is detected by the photodetector 18. To detect.
[0008]
That is, when the laser beam is irradiated, as shown in FIG. 10, the gel fluorescent substance existing on the optical path 31 is excited and emits fluorescence. This fluorescence is detected over time in the electrophoresis direction at a predetermined detection position for each lane. As a result, the fluorescence is detected when the band 32 of each lane passes the position on the optical path 31, and the pattern signal of the fluorescence intensity in one lane is obtained.
A data processor (not shown) can analyze each base sequence of DNA from the pattern signal, for example.
[0009]
[Problems to be solved by the invention]
However, such a conventional electrophoresis apparatus has the following problems.
(1) Measurement takes time.
(2) The resolution is not sufficient. Multiple separations require a large number of lanes. Since 2D is the limit, interrelated information of 3D or higher cannot be obtained.
(3) Large installation area. The area of the migration part is as large as 50 cm × 50 cm or 5 cm × 5 cm, for example.
(4) Position reproducibility is particularly poor in 2D. It is only necessary to put a marker in another lane and refer to it, but if the marker is put, the area increases.
[0010]
SUMMARY OF THE INVENTION An object of the present invention is to solve the above-mentioned problems, and an electrophoresis apparatus capable of obtaining a highly accurate electrophoresis pattern and a lot of correlated information in a short time with a compact migration part. Is to provide.
[0013]
[Means for Solving the Problems]
In order to achieve such an object, in the invention of claim 1,
An electrophoresis apparatus configured to perform electrophoresis on a sample labeled with a fluorescent dye in an electrophoresis section and read a fluorescent pattern that emits light,
A plurality of samples in which different fluorescent dyes are bound to various kinds of target substances such as proteins or DNAs are caused to flow in the same lane of the plate-like gel of the electrophoresis unit, and voltage, pH, density, An electrophoresis apparatus unit for performing electrophoresis with a physical gradient such as concentration;
A scanning or non-scanning confocal microscope or a two-photon excitation microscope configured to scan the sample of the migration section with excitation light and detect a fluorescence pattern of the sample emitted by irradiation with excitation light. And a three-dimensional position and concentration of the sample can be detected.
[0014]
According to such a configuration, the number of lanes is reduced and the migration section becomes compact, and voltage gradient nonuniformity and gel nonuniformity can be prevented and high-precision measurement is possible. In addition, simultaneous detection of multicolor fluorescence patterns and shortening of detection time are possible.In addition, physical gradients such as voltage, pH and density are provided in the thickness direction of the sample, and electrophoresis is performed simultaneously. A three-dimensional migration pattern can be detected.
[0015]
In this case, as described in claim 2, by carrying out simultaneous migration in the three axis directions by providing different physical gradients in the two axes in the plane direction of the gel and in the thickness direction perpendicular to the two axes , In addition, the interrelationship between the effects of the three axes can be measured.
[0016]
In addition, as described in claim 3, by mixing the sample and the marker in the same lane of the gel , the gel concentration, temperature difference, voltage distribution, and other conditions become the same, so a highly accurate and compact measurement is possible. It becomes possible.
[0017]
Further, when the confocal microscope opening is arranged so as to coincide with the position of each sample or inside the position of the sample as in claim 4 , the measurement of high S / N without the influence of the edge of the sample. Can be realized.
[0018]
Further, if the light condensing means is provided on the light source side of the opening as in claim 5 , the light source can be effectively used.
[0019]
Further, as in claim 6, the concentration distribution in the thickness direction can be realized by bringing a high-concentration solution into contact with only one side of the gel or by making a density difference in the thickness direction by centrifugation or multilayer lamination. it can.
[0020]
DETAILED DESCRIPTION OF THE INVENTION
The present invention will be described in detail below. FIG. 1 is a main part configuration diagram showing an embodiment of an electrophoresis apparatus according to the present invention. In the figure, 100 is a confocal microscope section, and 200 is a flat electrophoresis apparatus having a thin flat gel .
[0021]
A confocal microscope unit (also referred to as a confocal optical scanner) 100 can optically scan the carrier of the migration unit 201 and read the fluorescence migration pattern emitted from the carrier, and has the following configuration. Yes.
[0022]
Excitation light from the light source 101 (for example, blue laser light having a wavelength λ1) is converted into parallel light by the lens 102, reflected by the dichroic mirror 103, and then condensed on the slits of the slit array 105 via the lens 104. Then, the light passing through the slit is focused by the objective lens 106 and is incident on the carrier of the migration unit 201. The fluorescent substance in the migration part is excited by this light and emits fluorescence.
[0023]
This fluorescence returns again to the objective lens 106, the slit array 105, and the lens 104, passes through the dichroic mirror 103, and enters the next dichroic mirror 107.
The dichroic mirror 103 reflects light having a wavelength λ1 (for example, blue) and transmits light having a wavelength longer than the wavelength λ1. The dichroic mirror 107 reflects light having a wavelength λ2 (for example, green) and transmits light having a wavelength λ3 (for example, red). The wavelengths λ1, λ2, and λ3 have a relationship as shown in FIG.
[0024]
The light having the wavelength λ 2 reflected from the dichroic mirror 107 is collected on the light receiver 109 through the lens 108, and the light having the wavelength λ 3 transmitted through the dichroic mirror 107 is collected on the light receiver 111 through the lens 110.
If the slit array 105 is moved so that light from the light source scans the surface of the migration unit 201, the fluorescence migration pattern generated in the migration unit 201 is imaged on each of the light receivers 109 and 111.
[0025]
In this case, only the pattern that emits green light in the electrophoretic pattern forms an image on the light receiver 109, and only the pattern that emits red light in the electrophoretic pattern forms an image on the light receiver 111. The light receivers 109 and 111 convert the formed image into an electrical signal and output it.
[0026]
The electrophoresis apparatus unit 200 includes an electrophoresis unit 201 and a power source 202 that supplies a voltage for performing electrophoresis in the electrophoresis unit 201.
[0027]
In this way, it is possible to easily measure the multi-color fluorescence migration pattern generated in the migration unit 201 using a confocal scanner with high accuracy.
[0028]
By the way, the absolute value of the molecular weight cannot be obtained by electrophoresis. For this reason, as shown in FIG. 3, a marker molecule for reference is usually flowed to the adjacent lane. However, in this method, the measurement error is caused by the fact that it takes place and it is difficult to apply a voltage uniformly over the entire lane. There is a problem that arises.
[0029]
In the present invention, as shown in FIG. 4, a marker molecule for reference (hereinafter simply referred to as a marker) is also mixed and flowed together with the sample in the same lane. However, a dye having a different fluorescence wavelength is bound to each marker and the sample. By electrophoresing such a substance and optically scanning with a confocal scanner, a plurality of types of fluorophore patterns can be detected simultaneously.
[0030]
FIG. 5 shows another embodiment of the present invention. While two-dimensional electrophoresis is generally well known, FIG. 5 shows an example of three-dimensional electrophoresis in which one dimension is further added in the thickness direction (Z-axis direction).
[0031]
In this case, for example, the following voltage gradient and pH gradient are applied to the X-axis direction (vertical direction), the Y-axis direction (horizontal direction), and the Z-axis direction (thickness direction).
1) A high voltage is applied in the X-axis direction, a pH gradient is applied in the Y-axis direction, and a low voltage is applied in the Z-axis direction.
2) A voltage is applied in the X-axis direction, a pH gradient is applied in the Y-axis direction, and the gel concentration is changed in the Z-axis direction.
3) Affinity electrophoresis is performed by applying a voltage in the X-axis direction, a pH gradient in the Y-axis direction, and a voltage gradient in the Z-axis direction.
[0032]
In this case, for example, the objective lens 106 of the confocal scanner 100 is configured to be movable up and down so that the optical scanning surface of the migration unit 201 can move up and down in the optical axis direction (Z-axis direction). Then, three-dimensional information can be easily obtained by detecting multiple colors of the XY-axis direction fluorescence migration pattern while controlling the optical scanning plane in the Z-axis direction.
[0033]
The above description merely shows a specific preferred embodiment for the purpose of explanation and illustration of the present invention. Therefore, the present invention is not limited to the above-described embodiments, and includes many changes and modifications without departing from the essence thereof.
[0034]
For example, in the embodiment of FIG. 5, if only XZ is used as a lane as shown in FIG. 6, for example, it becomes more compact than ordinary two-dimensional electrophoresis.
Further, the concentration distribution in the thickness direction (Z-axis direction) can be realized by bringing a high-concentration solution into contact with only one surface, or by providing a density difference in the thickness direction by centrifugation. Alternatively, it can be realized by a method of laminating gels having different concentrations in multiple layers.
[0035]
Further, when the sample and the marker are separated and inserted in the thickness direction as shown in FIG. 7, the other conditions are the same as those in FIG. In this case, since the thickness direction can be separated and measured by the confocal method, the fluorescent colors may be the same.
Furthermore, as shown in FIG. 8, when electrophoresis is measured with a non-scanning confocal microscope, the confocal opening 61 may be set to coincide with or partially measure the position 62 of the sample. High S / N measurement without the influence of the edge of the sample can be realized.
[0036]
The light source may be either a single photon type or a two-photon type light source, and the same effect can be obtained.
[0037]
【The invention's effect】
As described above, the present invention has the following effects.
1) Compact and highly accurate multicolor electrophoresis can be easily realized.
2) Three-dimensional electrophoresis can be realized, the apparatus is compact, and a lot of information having correlation can be obtained in a short time.
[Brief description of the drawings]
FIG. 1 is a configuration diagram showing an embodiment of a multicolor electrophoresis apparatus according to the present invention.
FIG. 2 is an explanatory diagram showing wavelength distributions of excitation light and fluorescence.
FIG. 3 is an explanatory diagram regarding the arrangement of a sample and a marker.
FIG. 4 is an explanatory diagram when a sample and a marker are injected into the same lane.
FIG. 5 is an explanatory diagram of a migration unit when performing three-dimensional migration.
FIG. 6 is an explanatory diagram when one axial direction is cut off.
FIG. 7 is an explanatory diagram when markers are arranged in the thickness direction of a sample.
FIG. 8 is an explanatory diagram showing the relationship between the position of a sample and an opening.
FIG. 9 is a configuration diagram illustrating an example of a conventional electrophoresis apparatus.
FIG. 10 is an explanatory diagram of a migration pattern.
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 100 Confocal microscope part 101 Light source 102,104,108,110 Lens 103,107 Dichroic mirror 105 Slit array 106 Objective lens 109,111 Light receiver 200 Electrophoresis apparatus part 201 Electrophoretic part 202 Power supply

Claims (6)

An electrophoresis apparatus configured to perform electrophoresis on a sample labeled with a fluorescent dye in an electrophoresis section and read a fluorescent pattern that emits light,
A plurality of samples in which different fluorescent dyes are bound to various kinds of target substances such as proteins or DNA are caused to flow in the same lane of the plate-like gel of the electrophoresis section, and the two axes in the planar direction of the gel and the two axes A plate-shaped electrophoresis apparatus unit that performs electrophoresis by providing a physical gradient such as voltage, pH, density, concentration, etc. in a thickness direction perpendicular to the thickness direction;
A scanning or non-scanning confocal microscope or a two-photon excitation microscope configured to scan the sample of the migration section with excitation light and detect a fluorescence pattern of the sample emitted by irradiation with excitation light. An electrophoretic device characterized in that the three-dimensional position and concentration of a sample can be detected.   The electrophoresis apparatus section is configured to provide different physical gradients in two axes in the plane direction of the gel and in a thickness direction perpendicular to the two axes, and to separate the three axes at the same time. The electrophoresis apparatus according to claim 1.   The electrophoresis apparatus according to claim 1, wherein the electrophoresis apparatus unit mixes a sample and a marker in the same lane of the gel.   The non-scanning confocal microscope is arranged so that the opening coincides with the position of each sample or is located inside the position of each sample. Electrophoresis device.   The electrophoresis apparatus according to claim 4, wherein the confocal microscope has a condensing unit on a light source side of the confocal opening.   The concentration distribution in the thickness direction is realized by bringing a high-concentration solution into contact with only one side of the gel, or by making a density difference in the thickness direction by centrifugation or multilayer lamination. Electrophoresis device.

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