JPH01147355A - Molecular sieve unit - Google Patents

Abstract

PURPOSE:To shorten a migration time and to execute separation by providing a voltage applying means to a molecular sieve where has the gel concentration gradient consisting of a stepwise concentration gradient for a rough separation and a continuous concentration gradient for a fine separation. CONSTITUTION:The molecular sieve unit 1 consists of the molecular sieve 2, a gel mounting base 3, and the voltage applying means 4. A rough separation migration zone (a) consists of gel migration parts a1-a5, which are prepared having mutually different gel concentration and the stepwise concentration gradient increasing in gel concentration in the order from a1 to a5. Further, fine separation migration zones (b)-(f) connected laterally from the respective gel migration parts a1-a5 are prepared so as to increase continuously in gel concentration from the gel migration part connection parts to zone end parts b1-f1. Further, the gel concentration at the zone end part b1-f1 is equalized to the gel concentration of the gel migration parts connected to adjacent fine separation migration zones below them.

Description

DETAILED DESCRIPTION OF THE INVENTION (a) Industrial Application Field This invention relates to a molecular sieve unit. More specifically, the present invention relates to a molecular sieve unit suitable as a filtration medium in an electrophoresis device that utilizes the sieving effect of gel.

(b) Conventional technology Conventionally, when separating target substances by electrophoresis, plate-shaped polyacrylamide gels have been used for electrophoresis because of the unique advantages of treating electroosmotic flow as zero and not having to worry about thermal convection. It is used as a medium.

This separation utilizes the sieving effect of the network structure (bore) of the gel, and the size of the bore is adjusted by adjusting the gel concentration. When performing separation and analysis using such a flat polyacrylamide gel, it is necessary to use a polyacrylamide gel with a predetermined width and a predetermined length (usually width: 5
A flat gel having a length of 150 mm (mm, length: 150 mm) is used, and is configured to have a concentration gradient in which the gel concentration can continuously increase along its longitudinal direction.

(c) Problems to be Solved by the Invention Separation by electrophoresis is determined by the potential gradient and migration distance. In terms of separation ability, the larger the potential gradient, the better the QL resistance, and the longer the electrophoresis distance, the better. However, in separation using the flat polyacrylamide gel described above, when a constant voltage is applied and the electrophoresis distance is set long in order to increase the separation ability, the potential gradient becomes small. If it is desired to obtain a potential gradient in one direction or the like, it is necessary to increase the applied voltage, but increasing the voltage causes the gel itself to generate heat. However, since electrophoresis must be performed while maintaining a constant temperature, a cooling device with a cooling capacity to eliminate the above-mentioned heat generation is required, and in the end, the separation performance is increased by increasing the electrophoresis distance and the applied voltage. In order to increase the power consumption, a larger cooling device and a power source with a larger output are required, which increases the size of the device and increases the cost.

Furthermore, as the migration distance increases, the migration time also increases in proportion. For example, the average electrophoresis time is currently 8 to 12 hours, which is a cause of impeding its use in the field of clinical medicine.

The present invention was made in view of this situation, and it is an object of the present invention to provide a molecular sieve unit that can shorten the electrophoresis time without impairing separation performance.

(d) Means for Solving the Problems Thus, according to the present invention, there is provided a belt-shaped main migration path having a stepped gel concentration gradient in which a plurality of gel migration sections each having a different gel concentration are connected in one direction; A plurality of belt-like sub-containers each consisting of a gel band connected to the side surface of each gel migration section, and having a gel concentration gradient that continuously slopes from the gel concentration gradient of each gel migration section to the gel concentration of the adjacent gel migration section. and a voltage applying means capable of applying a voltage between the gel migration sections of the strip-shaped main migration channel and between the gel migration section of any of the strip-shaped secondary migration channels and the ends thereof. A molecular sieve unit is provided.

The molecular sieve unit of the present invention first roughly separates a sample in a strip-shaped main migration path in which a stepped concentration gradient is set, and then continuously separates each coarsely separated component in a direction substantially perpendicular to the direction of the coarse separation. The present invention is characterized in that it is configured such that fine separation can be performed in each strip-shaped sub-phoresis path (hereinafter referred to as migration zone) in which a concentration gradient is set to slope.

As the molecular sieve used in the unit of the present invention, a plate-like gel formed by arranging a plurality of substantially identical flat gels having a predetermined width and a predetermined length in parallel is suitable. Each of the flat gels arranged in parallel has a gel migration section prepared at a single gel concentration at one end in the long plane direction, and a concentration gradient (electrophoresis) that continuously slopes from this gel migration section to the other end. zone).

However, when these are arranged in parallel, the gel concentration in the gel migration area of the other flat gel adjacent to one flat gel is the same gel concentration as the concentration prepared at the other end of the former flat gel. It is prepared as follows. By adjusting the gel concentration as described above, a gel is formed in a parallel arrangement.On one side of a flat gel, a gel migration section consisting of a plurality of gel migration sections each having a different gel concentration is arranged in a row, and a stepped concentration gradient is formed. will be set, and a plurality of migration zones will be set in parallel from the above-mentioned - side.

The parallel arrangement as described above is made on the gel mounting table. At this time, it is preferable that the respective gel migration parts in each flat gel are arranged in contact with each other, and the migration zone parts in the -direction flat gel are preferably separated and set with gaps between them.

The flat gels arranged in parallel as described above are preferably prepared to have the same shape and the same volume, and the size of one gel is, for example, 5 x 30.
Examples include XI (unit: mm).

The above-mentioned flat gel may be any gel as long as it is hydrophilic, absorbs and retains buffer solutions, etc., and has a molecular sieve effect, but from the viewpoint of electroosmotic flow, thermal convection, etc., polyacrylamide Preferably, it is composed of gel.

As described above, in the flat gel formed by the parallel arrangement of flat gels, the sample injection part is set in the gel migration part where the gel concentration is lowest.

In the unit of the present invention, the voltage application means is configured to be able to apply a predetermined voltage between the gel migration sections of the strip-shaped main migration path and to the migration zones formed in each of the flat gels. The voltage application means set in the strip-shaped main migration path may be configured to apply voltage to both ends of the migration path at once, and may be configured to apply voltage between any gel migration sections. It may be configured. As an example between the above arbitrary gel migration parts,
Examples include spaces between adjacent gel migration sections. Further, the means for applying voltage to the migration zone may be configured to apply voltage to all migration zones at once, or may be configured to apply voltage to only an arbitrary migration zone. good.

The electrode used in the voltage applying means is one that is configured to generate a negative electric field between intended predetermined portions of the gel. For the second configuration example, refer to the description of the embodiment described later.

The gel mounting table used in the unit of the present invention is made of a material that has thermal conductivity necessary for cooling the flat gel constituting the above-mentioned molecular sieve on which it is mounted, and an insulating property capable of preventing electric leakage. suitable, e.g. ceramic,
Examples include products coated with Teflon resin. A gel cooling means is provided at the bottom of the gel mounting table. Examples of the cooling means include a thermo module.

(E) Effect According to the present invention, a step-like concentration gradient is set by arranging a plurality of gel migration sections each having a different gel concentration, and after a sample is injected into the lowest gel concentration section of the strip-shaped main migration path, the strip-shaped main migration path is When a voltage is applied between the main migration paths, the injected sample is classified into corresponding gel migration areas by a stepwise sieving effect set by the stepped concentration gradient. After this, between each strip-shaped sub-phoresis path set up by connecting to the side of each gel migration section,
When a predetermined voltage is applied, the sample components classified in each gel migration section at the end of each strip-shaped sub-phoresis channel are finely separated according to the gradient continuously formed in the sub-phoresis channel. .

The present invention will be described in detail below with reference to Examples, but the present invention is not limited thereby.

(f) Example FIG. 1 is an explanatory diagram of the configuration of one embodiment of the molecular sieve unit of the present invention, FIG. 2 is a plan view of an example of the molecular sieve used in the unit of FIG. 1, and FIG. 3 is the unit of FIG. 1. FIGS. 4 and 5 are explanatory diagrams showing an example of the configuration of the electrodes shown in FIG. 4. FIGS.

In FIG. 1, the molecular sieve unit (1) is a molecular sieve (2).
, a gel mounting table (3) on which the molecular sieve (2) is placed, and a voltage application means (4) configured to apply a predetermined voltage to a predetermined portion of the molecular sieve (2). .

The molecular sieve (2) is arranged vertically (p-q
It is composed of a flat gel (21) with a width of 25 mm (direction), a width (x-y direction) of 30 mm, and a thickness of 0.5 mm.

The flat gel (21) has a width (f) in the longitudinal (p-q) direction.
f,) A main migration path (a) (hereinafter referred to as the coarse separation migration zone) spanning 5 mm is formed, while +A (x-y direction)
In the direction from the main migration path (a), there are five sub-paths of the same shape (hereinafter referred to as fine separation migration zones) (bXc) (d) (
e) (f) is set as a fraction. The size of the above one electrophoresis zone for fine separation is 5 mm in width (Q2) and 5 mm in length (
Q.) It is set to 25mm. Furthermore, regarding the fraction settings of (b), (c), (d), (e), and (r) of the five electrophoresis seams described above, the zone gap (1) is set to 1 mm.

The rough separation migration zone (a) has five gel migration sections (a).
It is composed of a row of υ(a=Xa-Xa,Xa-), and each gel migration area is prepared with a single gel concentration, but these five are prepared with different concentrations, and These are (al) → (a, ) → (a3) → (a4)
It is set to form a step-like concentration gradient in which the gel concentration increases in the order of "-" (a5). Also, each sub-separation section is connected in the lateral (x-"y) direction from each gel migration section. The electrophoresis zone is prepared so that the gel concentration continuously increases from the connection part of the gel electrophoresis section to the end of the zone, and the gel concentration at the end of the zone is lower than that of the adjacent area below. The a-modulation is made to match the gel concentration of the gel migration section connected to the separation migration zone. As an example of each gel concentration gtJ showing this, each gel migration part (a,) (a=Xa3) (a,)
The gel concentration of (a5) was 10%, 11%,
12%, 13%.

14%, and a gradient concentration of 10 to 11% from the gel migration part (aυ connection part to the zone end (bl)) for the fine separation migration zone (C).
) for its gel migration section (a,) from the connection point to its zone end (cl) at a gradient concentration of 11-12%, and for its separation migration zone (d) its gel migration section (a3)
A gradient concentration of 12 to 13% from the connection part to the end of the zone (dυ), and a gradient concentration of 13 to 14% from the connection part to the end of the zone (eυ) of the gel migration zone (a4) for the separation migration zone (e) % gradient concentration, a fine separation migration zone (f
), from the gel migration part (a,) connection part to its zone end (rυ) to a gradient concentration of 14 to 15%. Of the five gel migration sections that make up the zone, the sample injection section (a) should be set in the part with the lowest gel concentration.Therefore, in the above gel concentration preparation example, the sample injection section (a) should be set in the gel migration section (al). It is provided.

As shown in FIG.
a ceramic plate (31) on which the molecular sieve (2) is mounted;
2) and a cooling device (32) capable of cooling the air conditioner. The material of the ceramic plate (31) is S.
Examples include iC and AIN. Further, a thermo module is used in the cooling device (32).

The voltage applying means (4) includes an electrode (not shown) connected to a predetermined position of the flat gel (21) constituting the molecular sieve (2), and a power source (4) capable of applying a predetermined voltage between the electrodes.
1).

As an example of the structure of the electrode used in the voltage applying means (4), as shown in FIG. 43), a water-absorbing polymer (44) filled inside the exterior body (43), and a platinum electrode (45) inserted into the water-absorbing polymer (44). Be used. Note that the water-absorbing polymer (44) is used containing a buffer solution. Examples of the material for the porous polymer body (42) include polypropylene, such as Sunfine AQ, and examples of the water-absorbing polymer include vinyl alcohol-acrylic acid copolymer, such as Sumikagel. The above electrode is used with the size (length A) of the electrode surface changed depending on the part to which voltage is intended to be applied.

FIG. 4 shows an example of setting electrodes on a molecular sieve and an example of the shape of the electrodes. The reading is based on the planar gel (21) constituting the molecular sieve, and for the rough separation migration zone (a), both ends of the zone (p l) ( A pair of electrodes tffi having an electrode surface (A=5 mm) of a size corresponding to the zone width at qυ
(5×6) is set. In addition, in order to apply a voltage to the five electrophoresis zones for fine separation at the same time, an electrode surface (A = 25 mm) are set up.

As another example of electrode setting, if voltage is to be applied only between the intended adjacent gel migration zones for rough separation migration zone (a), it is recommended to set the electrodes as shown in Figure 5 (in this case, A = 5 mm), electrophoresis zone for fine separation (bXc) (dXe
) (f) When voltage is applied only to individual migration zones, the settings may be made as shown in FIG. 6 (A=5n+m in this case).

Next, the operation of the molecular sieve unit (1) configured as described above will be explained.

For example, in a molecular sieve unit (1) in which the gel concentration of the plate-shaped gel (21) is configured as in the above preparation example, and the electrodes are arranged as shown in FIG.
After sampling the sample into the sample injection part (a) provided in the gel migration part (al) of A predetermined voltage is applied to the positive side) so that an electric field of, for example, 50 V/cm is applied to the zone.

At this time, the sampled sample group is in the zone (a
) Gel concentration of each of the five gel migration sections arranged in a row, 10%
Mesh set as -11% = 12% → 13% → 14% (
Coarse separation is performed in order of bore size.

In other words, those that can pass through the 11% bore are transferred to the gel migration section (a,), and those with a larger molecular weight are transferred to the 11% bore.
% of the bore and remains in the gel migration section (a,). Next, the sample group that has passed through the 11% bore is further migrated according to the electric field, and those that can pass through the 12% bore are transferred to the gel migration section (a), and those that have a molecular weight that cannot pass through the bore remains in the gel migration section (a,). Thereafter, the sampling samples are similarly sieved into each of the five gel migration sections (a, Xa2Xa3) (a, Xa5).

At the point where the above rough separation is completed, the voltage application in the p-'q force direction is finished, and then the voltage is applied in the X - + y direction (X is the negative side, y
is the positive side). At this time, each of the gel migration sections (a, Xa
=Xa, Xa,) For example, 1
00 V/c++') A predetermined voltage is applied so that an N field is applied. In this manual, each gel migration section (aυ(
Each sample group roughly separated into (a, ) (a3) (a4) (a5) is simultaneously divided into zones (b) (cXd) (eXf) connected corresponding to each Kel migration section, respectively. X -1-
The gel concentration increases continuously in the y-direction through a bore set up, which allows each zone (bXc) (dX
eXf), each of the coarsely separated sample groups is finely separated.

Note that the voltage application described above is changed depending on the electrode arrangement set in the molecular sieve unit. For example, if the electrode arrangement in the p-"q direction is as shown in FIG. 5, and the electrode arrangement in the X-4y direction is as shown in FIG.
, Xa=) - After rough separation into these gel migration areas (al) and (a,) by applying a voltage so that a predetermined electric field is applied between - By applying a predetermined voltage, only the sample group coarsely separated in the gel migration section (aυ) is finely separated into the zone (b). After rough separation by applying a predetermined voltage to only the fine separation zone (c), a predetermined voltage in the X − + y direction is applied only to the fine separation zone (c), and the coarsely separated sample group is transferred to the gel migration section (a,). the zone (C)
subdivide into By repeating the same procedure, the sample will finally be separated into the fine separation zone (bXcXd) (eXf) in the same way as the sample obtained by the above method.

According to the separation method using the above molecular sieve unit, for example, compared to a conventional gel (width 5 mm, length 150 mm) that has the same gel concentration gradient (10 to 15%) as this molecular sieve continuously only in one dimension, , the total running time is 175~l/10
It can be shortened to a certain extent.

(G) Effects of the Invention According to the present invention, a voltage is applied in two dimensions to a molecular sieve having a two-dimensional gel concentration gradient consisting of a stepped IT gradient for coarse separation and a continuous concentration gradient for fine separation. is possible
74. Since the electrophoresis device is equipped with a pressure applying means, it is possible to shorten the electrophoresis distance while maintaining a high potential gradient, and it is possible to perform separation by shortening the electrophoresis time.

[Brief explanation of the drawing]

Fig. 1 is an explanatory diagram of the configuration of one embodiment of the molecular sieve unit of the present invention, Fig. 2 is a plan view of an example of the molecular sieve used in the unit shown in Fig. FIG. 4 is an explanatory diagram showing an example of the configuration of an electrode on a molecular sieve, and FIGS. It is a partial configuration explanatory diagram showing an example. (2)...Molecular sieve, (3)...
Gel mounting table, (4)...voltage application means, (5)
(6)・ Pair of electrodes, (7×8)...Pair of electrodes, (21)... Flat gel, (31)... Ceramic plate, (32)...
...cooling device, (41) ...power supply,
(42)...Porous polymer body, (43)...
... Exterior body, (44) ... Water absorbing polymer, (45) ... Platinum electrode, (a) ... Rough separation migration zone, (b) (c )(
dXeXf)......Migration zone for fine separation, (al
)(a2)(a3)(a4)(a,)...Gel migration section.

Claims (1)

[Scope of Claims] 1. A belt-shaped main migration path having a stepped gel concentration gradient in which a plurality of gel migration sections each having a different gel concentration are connected in one direction; and a plurality of belt-shaped secondary migration channels each having a gel concentration gradient that continuously slopes from the gel concentration of each migration section to the gel concentration of an adjacent gel migration section; 1. A molecular sieve unit comprising a voltage applying means capable of applying a voltage between the gel migration sections of the channel and between the gel migration section of the optional strip-shaped sub-transfer channel and its end.