JPH0584861B2 - - Google Patents

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a method for producing a gradient gel membrane for electrophoresis for determining the base sequence of DNA or partially degraded DNA, and in particular, a method for producing a gradient gel membrane for electrophoresis. It relates to a coating head that applies a liquid to a web.

[Conventional technology]

Conventionally, in plate electrophoresis, acrylamide polymer concentration gradient gels that are not self-supporting are made by layering gels with different concentrations on one support or between two supports in the electrophoresis direction. It has been formed and used as a membrane.

However, in the method of forming a gel on a support, it is difficult to accidentally overfill the gel when forming the gel on the support, setting it in an electrophoresis tank, storing the gel, or adding an analytical sample. The gel could be broken or something other than the sample could be dropped onto the gel, damaging the gel, so careful handling and skill were required.

On the other hand, make a mold using two glass plates, etc.
In the vertical electrophoresis method, in which a gel is formed in the mold and electrophoretic analysis is performed while the mold is held vertically, it is difficult to make the mold thickness uniform, and the gel forming solution is This required a high level of skill to operate, as the gel solution had to be injected into a narrow mold before it turned into liquid.

Particularly in DNA sequencing operations, it is desirable to make long gels so that as many DNA fragments as possible can be analyzed in one gel.
Such gels were difficult to manufacture and handle. Furthermore, since a glass plate is used, there is a drawback that the glass plate is easily damaged.

In recent years, as a method for industrially producing gradient gel materials for electrophoresis, an aqueous solution or aqueous dispersion of a mixture of an acrylamide monomer and a crosslinking agent is prepared, and a free radical generating substance that absorbs light irradiation and initiates polymerization of the monomer is added. After addition, the monomer solution is formed into the desired gel product and the monomer solution or dispersion is polymerized and crosslinked by irradiating the formed solution with light to alter the porosity of the gel. Production of an electrophoretic gradient gel including a step of adjusting the applied light irradiation time (see JP-A No. 61-28512) Furthermore, it contains a monomer, a crosslinking agent, and a polymerization initiator, and has mutually different concentrations. Two types of aqueous solutions are introduced into a molding machine while being mixed while gradually changing the mixing ratio, and the polymerization of the monomer and crosslinking agent is completed in the molding machine, so that a concentration gradient of the polymer is created in the electrophoretic direction. Method for producing gradient gel for electrophoresis (Tokukosho
(See Publication No. 61-22903, Special Publication No. 61-39617)
etc. are disclosed.

Further, as a coating head for coating an electrophoretic separation medium liquid onto a continuously running web, a general coating head as shown in FIG. 6 has been used. That is, the electrophoretic separation medium liquid as the coating liquid 12 is pumped into the cavity 1 in the coating head 18 by the metering pump 22.
9. The cavity 19 is a liquid reservoir for supplying the coating liquid to the slot 23 communicating with the slot opening 24 and extending over the entire coating width. The coating liquid 12 condenses from the cavity 19 and flows into the slot 23.
The web 13 on the coating roller 20 is coated through the slot opening 24. At this time, a differential pressure chamber 21 is used to create back pressure behind the coating bead in order to stabilize coating.

[Problem that the invention seeks to solve]

However, the first production method has high costs due to poor light irradiation equipment and poor productivity, poor resolution due to the thick gel, and the reaction initiator remains polymerized in the presence of light even after production is completed. There are problems such as a cross-linking reaction occurs, making it impossible to obtain a gradient gel with stability and reproducibility. When the medium liquid is branched into the molding machine, it is difficult to branch it uniformly, and there are problems such as the inability to obtain a reproducible gradient gel.

Furthermore, the film thickness of the product coated with the conventional coating head in the width direction is not sufficiently uniform, and foreign matter may be generated due to long-term operation, making it necessary to disassemble and clean the coating head.

The purpose of the present invention is to solve the above problems, and to provide a gradient gel film for electrophoresis that has a uniform coating thickness over the entire coating width, does not generate foreign matter, and has a continuous concentration gradient in the running direction of the web. The objective is to provide a manufacturing method with high productivity, stability, and good reproducibility.

[Means for solving problems]

The object of the present invention is to provide an electrophoretic separation medium solution containing a polymerization reaction initiator and a monomer solution whose concentration changes over time by mixing two kinds of concentrated and light monomer solutions while continuously changing the flow rate ratio. A method for producing a gradient gel membrane for electrophoresis, in which a coating liquid is supplied to a coating head and the coating liquid is applied onto a running web to obtain a membrane having a continuous concentration gradient in the direction of electrophoresis, the method comprising: While the coating liquid reaches the slot opening from the supply port of the coating head, the width of the slot is continuously expanded in the width direction of the web, and the thickness of the slot is continuously decreased to reduce the cross-sectional area of the slot. The coating liquid is applied onto the web through the slot opening while minimizing sudden changes.

This is achieved by a method for producing a gradient gel membrane for electrophoresis, which is characterized by the following.

In the present invention, the monomer liquid whose concentration changes over time is produced by mixing two types of monomer liquids, one with a different concentration and the other with a different concentration, while continuously changing the flow rate ratio. And the change in the concentration of the monomer liquid is 3~
35W/V% is preferred.

The mixing method in the present invention may be carried out using a stirrer in a general tank, but mixing and preparation using a static mixer is most preferable.

A static mixer is one that produces a stirring action due to the movement of the fluid itself when the fluid is passed through the mixer. Examples of tubular types include spiral tubes, alternating left and right spirals, and propeller shapes with baffles. be.

The electrophoretic separation medium in the present invention may be anything as long as it can form an electrophoretic separation medium membrane, and typical examples include acrylamide gel, agarose gel, starch gel, agar gel,
A liquid that serves as a raw material for cellulose acetate porous membranes, filter paper, etc.

As a coating head for electrophoretic separation medium in the present invention, a coating head for slide bead coating, extrusion coating, hopper coating, terten coating, etc. is used. The thickness of the media membrane is selected depending on the purpose of separation, but is usually from 50 μm to about 1.0 mm, preferably from about 200 μm to about 0.5 mm. In the case of porous membranes and filter papers, the range is from about 70 μm to about 1 mm.

The running web in the present invention may be made of any material as long as it is sheet-like with good flatness, non-conductive, and substantially water-impermeable, such as polyethylene terephthalate, bisphenol A polyester, etc. Polyester like carbonate,
Vinyl polymers such as polymethyl methacrylate, polyethylene, polystyrene, and polyvinyl chloride, polyamides such as nylon, and copolymers thereof (eg, vinylideline chloride and vinyl chloride copolymers) are preferably used.

In the present invention, while the coating liquid reaches the slot opening from the supply port of the coating head, the slot width is continuously expanded in the width direction of the web, and the slot width is continuously decreased in the thickness direction. The coating liquid flow path from the mouth to the slot-shaped opening is changed continuously without abruptly changing the edge line of the coating liquid flow path.When increasing the width, the thickness is correspondingly reduced and the cut of the slot is changed. This refers to avoiding vortices and stagnation in the flow as a whole by regulating the angle of expansion and contraction to reduce sudden changes in area. As a result, the present invention eliminates the use of cavities conventionally used in coating heads.

Embodiments of the present invention will be described using figures.

FIG. 1 is a process diagram of the method for producing a gradient gel membrane for electrophoresis according to the present invention.

The electrophoresis medium liquid 12 is prepared by stirring and mixing a low concentration monomer liquid 1, a high concentration monomer liquid 2, and a polymerization reaction initiator liquid 3 in a static mixer 7.

FIG. 2a is an explanatory diagram of the relationship between the flow rate ratios of two kinds of concentrated and light monomer liquids of the present invention. Continuously changing the flow rate means, for example, initially high concentration monomer liquid 2 is 0 ml/min, low concentration monomer liquid 1 is 19 ml/min.
The flow rate starts at a flow rate of min, but if the product unit length of the gradient gel membrane for electrophoresis is l, the flow rate is changed as shown in the figure, as shown by the solid line for high concentration monomer solution 2 and the dotted line for low concentration monomer solution 1. , the total flow rate is 19
Although the rate is constant (ml/min), the concentration changes continuously. At the end of the product unit length l, the high concentration monomer liquid 2 is 19 ml/min and the low concentration monomer liquid 1 is 0 ml/min, making them highly concentrated membrane components. In order to repeatedly change the flow rate ratio as described above, in FIG.
is controlled by the controller 9a via the computer linkage adapter 10 in accordance with commands from the personal computer 11.

On the other hand, the polymerization reaction initiator liquid 3 is supplied by a constant flow liquid sending pump 6.
A fixed amount is sent to the static mixer 7 under the control of the controller 9b, and the low concentration monomer liquid 1, the high concentration monomer liquid 2, and the polymerization reaction initiator liquid 3 are stirred and mixed in the static mixer 7 for use in electrophoresis. A medium liquid 12 is produced. The electrophoresis medium 12 is supplied to the coating head 8 and is coated onto the web 13.

Figure 2b is a plan view of a gradient gel membrane for electrophoresis made with the concentration ratios shown in Figure 2a.
Figure c is a side view of Figure 2b.

The gap t between the product unit lengths l in FIG. 2a is punched out as a sample injection port, and is a portion that becomes the sample injection port with a cover sheet or the like.

Next, FIGS. 3a and 3b are side sectional views of one embodiment of the coating head of the present invention. The coating head 8 receives the electrophoretic separation medium liquid 12, which is the coating liquid from the static mixer 7, through the supply port 14,
6, the coating is applied onto the web 13 through the slot opening 17 on a coating roller 20.
In FIG. 3, a and b are different positions on the coating roller 20 at which the coating liquid 12 is applied onto the web 13, and either one may be used.

In the case of the present invention, the width and thickness of the coating liquid supplied from the supply port 14 to the slot 16 must be continuously expanded and contracted, and the angle of expansion and contraction must be relatively gentle. 16
The length is longer than the conventional one. Therefore, the position of the coating head 8 with respect to the coating roller 20 is as shown in FIGS. 3a and 3b.

In addition, in Fig. 3b, the angle of reduction α in the thickness direction
≦0.35° is preferred. FIG. 4 is a sectional view of the coating head of FIG. 3, viewed from the front, and FIG. 5 is a perspective view. In FIG. 4, the coating liquid 12 in the coating head 8 is
The slot 16 is continuously expanded in the width direction from the supply port 14 to the slot opening 17. Ideally, it is preferable to expand it into a ventilate shape as shown by the dotted line in the figure, but if you want to simplify the work by considering angle restrictions, the expansion angle θ
Straightening to ≦40° is commonly used.

The perspective view of FIG. 5 shows both the width and thickness.

[Effect]

In the present invention, a monomer liquid and a polymerization reaction initiator liquid having a concentration gradient are continuously mixed and supplied to a coating head as a medium liquid for electrophoretic separation. Since the liquid is sent to the coating head without mixing between the liquids before and after the liquid due to the self-feeding force, it is possible to stably produce a coating film with an unprecedented gradient of density. Moreover, it can be manufactured with high productivity.

Furthermore, the present invention continuously spreads the coating liquid in the width direction from the supply port to the slot-shaped opening, and concomitantly causes it to continuously contract in the thickness direction, thereby preventing rapid expansion and contraction of the liquid flow. Specifically, the edge line of the liquid flow path is expanded continuously (for example, like a ventilate) to avoid swirling (stagnation) occurring at the edge line of the passage. As the width is increased, the thickness is correspondingly reduced to reduce abrupt changes in the cross-sectional area of the slot, resulting in a uniform flow resistance across each channel. Regulate the angle of expansion and contraction. For example, the angle θ≦40° when the liquid spreads,
By setting the angle in the thickness direction to α≦0.35°, generation of vortices is prevented and partial retention of liquid is eliminated.

In particular, the reason why a cavity is not used is to prevent vortices generated in various parts of the contracted portion when the liquid flows out from the cavity to the slit, the resulting partial retention of the liquid, and the generation of foreign matter due to this.
By these methods, a uniform coating film can be obtained over the entire width of the coating.

〔Example〕

Examples of the present invention will be described.

The medium solution for electrophoretic separation is as a low concentration monomer - Agarose...65g Urea...4200g Acrylamide...549g 1,3,5-Triacryloyl-hexahydro-S-triazine...5.7g Deionized water up to 9000ml As a high concentration monomer - Agarose...40g Urea...4200g Acrylamide...1830g 1,3,5-triacryloyl-hexahydro-S-triazine...19g Deionized water Make up to 9000ml As a buffer - Trishydroxymethyl amine
…121.14g Boric acid …65.4g Ethylenediaminetetraacetic acid disodium salt …7.45g Deionized water Make up to 1000ml, buffer solution …750ml 2.9% polyvinylpyrrolidone aqueous solution, 800ml 25% N, N, N′, N'-Tetramethylethylenediamine solution...6.7ml of mixed solution, 9000ml of low concentration monomer, high concentration monomer
9000 ml of each, and make them low concentration monomer solution 1 and high concentration monomer solution 2, respectively.

Next, as polymerization reaction initiator solution 3, make 2% di-2 ethylhexyl sulfosuccinate solution...100ml 0.375% riboflavin phosphate ester sodium salt aqueous solution 150ml 3.75% ammonium peroxodisulfate aqueous solution...160ml, and add each of the above three solutions. Put it in the tank.

The sum of the flow rates of low concentration monomer liquid 1 and high concentration monomer liquid 2 is 19 ml/min using the flow rate gradient liquid sending pumps 4 and 5, and the flow rate of polymerization reaction initiator liquid 3 is 1.46 ml/min.
min amount is fed into the static mixer 7 by the constant flow liquid feeding pump 6.

In the static mixer 7, the three liquids are mixed and stirred by themselves to form a medium liquid 12 for electrophoretic separation.

The electrophoretic separation medium liquid 12 is applied onto the web 13 by the coating head 8 .

At that time, the flow rate ratio between the low concentration monomer liquid 1 and the high concentration monomer liquid 2 is as shown in Figure 2a, and the concentration of the applied gel film is as shown in Figures 2b and 2c. Become.

The electrophoretic separation medium solution prepared above is coated on a PET film with a coating width of 18 cm.
Applications were carried out at cc/m ² .

(Example 1) At this time, when the spread angle from the supply port in the width direction of the coating head to the slot was set to θ = 40°, and the thickness reduction angle α = 0.35°, the results showed that even after long-term operation, There were no malfunctions such as the occurrence of coating streaks, and a good quality coating with a uniform thickness over the entire width was obtained.

(Comparative Example 1) Coating was performed on PET film in the same manner as in Example 1 at the same coating solution concentration and temperature using a coating head with a spreading angle θ = 45° and a reduction angle α = 0.5°. The thickness of the coating was thinner at the edges, and the thickness of the coating was parabolic as a whole. Furthermore, the coating quality was somewhat streaky during long-term continuous operation.

[Effects of the Invention] The present invention continuously mixes a monomer solution with a concentration gradient and a polymerization reaction initiator solution, supplies it to a coating head as a medium solution for electrophoretic separation, and coats it onto a running web. A method for producing a gradient gel membrane for electrophoresis, which enables highly productive production of a gradient gel membrane for electrophoresis with accurate, stable, and reproducible concentration gradients by using a static mixer or the like before application. Ta. Furthermore, when supplying the coating liquid to the coating head, the coating liquid is continuously expanded in the width direction and continuously contracted in the thickness direction until it reaches the slot opening from the supply port, so that the coating liquid remains in the coating head. This eliminates the generation of foreign matter and enables uniform coating of ingredients in the width direction, further improving product quality.

[Brief explanation of the drawing]

Fig. 1 is a manufacturing process diagram of a medium liquid for electrophoretic separation according to the present invention, and Fig. 2 is a diagram showing the concentration gradient a of the medium liquid for electrophoretic separation according to the present invention, and a plan view b and a side view c of the coating film thereof. be. 3 is a side sectional view a, b of a coating head according to the present invention, FIG. 4 is a front sectional view of FIG. 3, FIG. 5 is a perspective view of FIG. 4, and FIG.
The figure shows a side sectional view of a conventional coating head. DESCRIPTION OF SYMBOLS 1...Low concentration monomer liquid, 2...High concentration monomer liquid, 3...Polymerization reaction initiator liquid, 4, 5...Flow rate gradient liquid feeding pump, 6...Constant flow liquid feeding pump, 7...
... Static mixer, 8 ... Application head, 9
a, 9b...controller, 10...computer linkage adapter, 11...personal computer, 12...medium liquid for electrophoretic separation, 1
3... Web, 14... Supply port, 16... Slot, 17... Slot opening, 18... Coating head, 19... Cavity, 20... Coating roller, 21... Differential pressure chamber, 22... Pump , 23...slot, 24...slot opening.

Claims (1)

[Claims] 1. An electrophoretic separation medium liquid, which is a mixture of a polymerization reaction initiator and a monomer liquid whose concentration changes over time by mixing two types of concentrated and light monomer liquids while continuously changing the flow rate ratio, is used as a coating liquid. A method for producing a gradient gel film for electrophoresis, in which the coating liquid is supplied to a coating head and applied onto a running web to obtain a film having a continuous concentration gradient in the direction of electrophoresis. While reaching the slot opening from the supply port of the coating head, the slot width is continuously widened in the width direction of the web, and the slot width is continuously reduced in the thickness direction to prevent a sudden change in the cross-sectional area of the slot. A method for producing a gradient gel membrane for electrophoresis, characterized in that the coating liquid is applied onto the web through the slot opening.