JPS6332362A - Production of material for electrophorectic analysis - Google Patents

Abstract

PURPOSE:To produce a medium for electrophoresis with high productivity by projecting flash light from a xenon flash lamp to an aq. soln. contg. an acrylamide monomer formed like a film and crosslinking agent to generate crosslinking polymn., thereby forming an aq. gel film. CONSTITUTION:The flash light from the xenon flash lamp light source is projected to the aq. soln. contg. the acrylamide monomer formed like the film and the crosslinking agent. The crosslinking polymn. is generated in the aq. liquid to form the aq. gel film. The light of high intensity can be applied to the gel forming liquid without increasing the quantity of the heat to be applied to the gel forming liquid too much by using the xenon flash lamp light source at the time of gelling the gel forming liquid in the above-mentioned manner. The moisture content in the resulted gel medium is, therefore, easily controllable and the high-performance medium for electrophoresis is produced with high productivity.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of the Invention] The present invention relates to a method for producing materials for electrophoretic analysis. The present invention particularly relates to a method for manufacturing a plate-shaped material for electrophoretic analysis in which a separation medium membrane used for electrophoretic analysis is sandwiched between two films, which is easy to handle and easy to carry out in autoradiography. The present invention provides a photopolymerization method for producing materials for electrophoretic analysis useful for electrophoretic analysis.

[Background of the Invention] Plate electrophoresis is a method of analysis that takes advantage of the fact that the mobility caused by an electric field in a flat medium film that can conduct electricity differs depending on the substance. In the medical field, it has become an indispensable means for separating and analyzing biological substances such as proteins and nucleic acids. In particular, in order to determine the base sequence of DNA, which is often carried out in genetic engineering or genetic disease research, four types of DNA fractions degraded in a base-specific manner or base-specific chain elongation reactions are required. Since it is necessary to compare the migration distances of DNA mixtures applied to each other, a plate-type electrophoretic analysis method is essential.

Conventionally, in plate electrophoresis, non-self-supporting polymer gels such as agarose and polyacrylamide are used to form a gel on one support or between two supports, forming a film-like material (layered material). It has been used as a columnar object filled inside a cylinder. However, in the method of forming a gel on a support, it is difficult to use the method when forming the gel on the support, when setting the support with a polymer gel membrane in an electrophoresis tank, during storage of the gel membrane, or during analysis. It is possible to break the gel film by mistake when adding a sample, or damage the gel film by dropping something other than the sample onto the gel film, so extreme care and skill are required in operation. Ta. On the other hand, in the vertical electrophoresis method, in which a mold is made from two glass plates, etc., a gel is generated in the mold, and electrophoretic analysis is performed while the mold is held vertically, the thickness of the mold is uniform. In addition, a high level of skill is required in operation, as the gel solution must be injected into a narrow mold before the gel forming solution gels. In particular, in DNA base sequencing operations,
Although it is desirable to make long gel membranes so that as many DNA fragments as possible can be analyzed in one gel membrane, such gel membranes are difficult to manufacture and handle.

Furthermore, since a glass plate is used, there is also a drawback that the glass plate is easily damaged.

In electrophoretic analysis, a target component in a test sample is often labeled with a radioisotope, electrophoretically separated, and then a separated image of the target component in the test sample is obtained by autoradiography. In this method, an electrophoretic separation medium membrane containing a radioisotope and a photographic film for recording radiation from the membrane are stacked and left in a dark place (exposure operation). To prevent the photographic film from getting wet,
A thin water-impermeable sheet, such as a plastic roughing sheet, must be placed over the gel membrane. In addition, in the case of a gel film made between two glass plates, remove one glass (this is because radiation from the isotope of the test sample in the gel film is absorbed as it passes through the glass). In order to prevent its strength from being significantly weakened, it must be covered with a thin sheet.

When removing this glass plate, some of the gel film may accidentally adhere to the upper glass and another part to the lower glass, damaging the gel film. It required skill to remove just the glass.

Furthermore, when covering with a thin sheet such as a plastic roughing sheet, bubbles often remain between the gel film and the sheet, or wrinkles form on the sheet, resulting in poor adhesion between the gel film and the photographic film. Another major drawback was that the autoradiograph obtained was blurred and the resolution was reduced.

A gel membrane sheet that has improved this problem is disclosed in, for example, Japanese Patent Laid-Open No. 126237/1983. This gel medium is N,N'-methylenebisacrylamide (
The most widely used reaction initiation of 0 polymerization obtained by polymerization and crosslinking of acrylamide with difunctional residue compounds such as BIS) is potassium or ammonium persulfate and the reaction accelerator N, N, N', N'
- This is a method in which tetramethylethylenediamine (TEMED) is used, and a photosensitizer such as riboflavin or lipoflavin phosphate sodium salt is added to photopolymerize, polymerize, and crosslink. Polymerization initiated by a photosensitizer has a variable rate of polymerization depending on the appropriate wavelength (absorption) and light intensity of the ultraviolet (UV) source. is known to receive In producing gel for electrophoresis, it is preferable to gel in a short time from the viewpoint of productivity, and it is desirable to use a strong light source.

However, according to research conducted by the present inventor, high heat is generally generated when a high-intensity light source (for example, a high-pressure mercury lamp) is used, and when the gel-forming liquid is directly irradiated with light from such a high-intensity light source, high heat is generated. It has been found that when added to the gel medium, the temperature of the gel medium rises and water in the medium evaporates, resulting in a serious drawback that it is difficult to stabilize the performance. For this reason, it is conceivable to use a cooling method in which a layer of water is placed in the middle to absorb the heat generated by the light source, but even this method cannot absorb heat sufficiently. If you want to irradiate strong light without applying heat, it is also known to use the cold mirror method, in which the light is reflected by a mirror and irradiated onto the polymerization system. If used, the distance between the light source and the polymerization system will increase, resulting in a decrease in irradiation intensity,
This is unfavorable in terms of production since it requires the installation of equipment involving an engineering department.

[Structure of the Invention] The present invention involves irradiating flash light from a xenon flash lamp light source to an aqueous liquid containing an acrylamide monomer and a crosslinking agent formed into a film to cause crosslinking polymerization within the aqueous liquid. , a method for producing a material for electrophoretic analysis, which comprises forming an aqueous gel membrane.

According to the present invention, by using a xenon flash light source during gelation of the gel-forming liquid, high intensity light can be applied to the gel-forming liquid without significantly increasing the amount of light applied to the gel-forming liquid. . Therefore, the water content in the gel medium obtained by the production method of the present invention can be easily adjusted, and a highly reliable electrophoresis medium can be obtained with high productivity.

[Detailed Description of the Invention] The present invention provides an aqueous liquid (gel-forming liquid) containing an acrylamide monomer and a crosslinking agent formed into a film as described above.
The object of the present invention is to provide an improved method for producing a material for electrophoretic analysis, which comprises irradiating light to cause cross-linking polymerization in the gel-forming solution to form an aqueous gel film.

The operation of forming a gel-forming liquid into a film is usually carried out by applying the gel-forming liquid onto a sheet-like object such as a plastic film or a glass sheet. This sheet-like material is preferably a sheet-like material that carries Ia as it is as a support for the gel membrane (support for the electrophoresis medium material).

The support for the electrophoresis medium material is a sheet-like material (including a film-like material) made of a plastic material or glass.As the plastic sheet, one made of any plastic material can be used. Examples of preferred plastic materials include polyethylene terephthalate, polycarbonate of bisphenol A, polyvinyl chloride, polymethyl methacrylate, polyethylene, polypropylene, polystyrene, cellulose acetate, and cellulose acetate propionate.

It is preferable that the support made of plastic material has its surface made hydrophilic by a known surface activation treatment. For the surface hydrophilic treatment, known methods such as ultraviolet irradiation, glow discharge treatment, corona discharge treatment, flame treatment, electron beam irradiation, chemical etching, electrolytic etching, etc. can be applied.

The support generally has a thickness of about 50 to 500 lm.
, preferably about 70 to 300 m.

Next, the gel forming liquid will be explained.

The gel-forming liquid used in the present invention is a gel-forming liquid obtained by dissolving or dispersing an acrylamide monomer and a crosslinking agent in water as an aqueous solution or an aqueous dispersion.

Examples of acrylamide monomers include acrylamide, N-methylacrylamide, N,N-dimethylacrylamide, N-(hydroxymethyl)acrylamide,
Examples include acrylamide compounds such as diacetone acrylamide and methacrylamide compounds such as methacrylamide, and these compounds can be used alone or in combination of two or more. Among these 7-acrylamide monomers, acrylamide is most preferred, and a combination of acrylamide and one or more of other acrylamide compounds and methacrylamide compounds is also preferred.

As a crosslinking agent, r Electrophoresis J
Known compounds (one type or a combination of two or more types) described in 19B1.2゜220-228 and the like can be used. Specific examples of crosslinking agents include N,N'-methylenebisacrylamide (BIS), N,N'-propylenebisacrylamide (PBA), di(acrylamide dimethyl)ether (DAE), and 1,2-diacrylamide ethylene glycol. (DEC), ethylene ureabisacrylamide (EUB); ethylene diacrylate (ED
A); N,N'-diallyltartardiamide (N,N'
-dialyltartardiaside: DA
TD); and N,N'-bisacrylylcystamine (
N, N'-bisacry171cySta+*ine
.

Examples include Ninomiya compounds such as BAC).

The amount of crosslinking agent is about 2 to 30 wt%, preferably about 3 to 1.5 wt%, based on the total weight of acrylamide monomer and ffM agent.
It is in the range of Owt%.

The gel Q degree is S, Hjerten: r A
rch.

Biochem-Biaphys-J 1 (5u
pp! ), 147 (1962), the amount of monomer and crosslinking agent is about 3 w with respect to the volume of the gel film consisting of monomer, crosslinking agent and water.
/v% to about 30 w/v%.

The gel-forming solution may optionally contain a denaturing agent such as urea, formamide, or an anionic surfactant.

The gel forming liquid can contain a water-soluble polymer.

As the water-soluble polymer, addition polymerization type or condensation polymerization type water-soluble polymer can be used.

Specific examples of addition polymers include nonionic water-soluble polymers such as polyvinyl alcohol, polyvinylpyrrolidone, and polyacrylamide. Specific examples of condensation polymers include nonionic water-soluble polyalkylene glycols such as polyethylene glycol and polypropylene glycol. The molecular weight of the water-soluble polymer is preferably in the range of about 10,000 to about 100,000. Among these water-soluble polymers, polyethylene glycol and polyacrylamide are particularly preferred.

The water-soluble polymer ranges from about 2 wt% to about 100 wt%, preferably about 5 wt% based on the total weight of monomer and crosslinker.
Amounts ranging from wt% to about 50wt% are used.

The gel forming solution can contain agarose.

The agarose is not particularly limited as long as it is a known agarose, and any of low electroosmotic, medium electroosmotic, and high electroosmotic agaroses can be used. Examples of acarose that can be used include JP-A-55-5730, JP-A-55-110946, and JP-A-57-502098.
There are agaroses and the like disclosed in publications such as No.

Acarose is about 0.2 w/v% to about 2 w/v%, based on the volume of the gel composition solution containing the monomer and the crosslinking agent.
It is preferably added at a rate of about 0.3 w/v% to about 1.2 w/v%.

The gel forming solution has an appropriate pH/H value depending on the substance to be electrophoresed.
A buffering agent may be included. As a buffering agent, please refer to ``Chemical Handbook Basic Edition'' edited by the Japan Chemical Society (Tokyo, Maruzen Co., Ltd., 196).
6th year) +312-1320 pages; Data for B
iochemical Re5search (R,M,
C, Dawson et a1 2nd edition, 0xfo
rd at the C1arendon Press
, 1969) pp. 476-508; Bioche
mistry 5, 467 (1966) ;Ana1
7tical Biochemistry 104
, 300-310 (1980) and the like. A specific example is tris(hydroxymethyl)aminomethane (Tris) [CA
s Registry Mo7? -86-1], N, N
-Bis(2-hydroxyethyl)glycine
e) , N-2-hydroxyethylpiperazine-N'
-2-hydroxypropane-3-sulfonic acid Na salt or salt, etc., N-2-hydroxyethylpiperazine-N
NaPA or salt of '-3-propanesulfonic acid, etc.
a salt of N-[)lis(hydroxymethyl)methyl-3-aminopropanesulfonic acid and
Acids, alkalis, salts, and the like can be used in combination with any of these if necessary. Examples of particularly preferred buffers include Tris, boric acid and EDTA 2N
There is a combination of a salts (pH 8,3).

It is preferable that the gel forming liquid contains a catalyst for radical crosslinking polymerization. Examples of radical crosslinking polymerization catalysts include Electrophoresis, 1981.2
゜213-219. 1981.2.220-228
, Aomizu and Nagai, eds., "Latest Electrophoresis Methods J" (published in 1973), etc., known low-temperature radical polymerization initiators can be appropriately selected and used. Specific examples of preferable radical polymerization initiators include: Examples include β-dimethylaminopropionitrile (DMAPN)-ammonium peroxonisulfate mixture, N,N,N',N'-tetramethylethylenediamine (TEMED)-ammonium peroxonisulfate mixture, etc.The content of the radical polymerization initiator is , from about 0.3 to % SgL based on the total weight of monomer and crosslinker, and preferably from about 0.5 to
It is in the range of 3% by weight.

In addition, it is desirable to add a photosensitizer to the gel-forming solution in addition to the above-mentioned radical crosslinking polymerization catalyst. Examples of photosensitizers include riboflavin, riboflavin phosphate sodium salt, etc. Derivatives can be mentioned. The content of photosensitizer ranges from about 0.3 to 5% by weight, and preferably from about 0.5 to 3% by weight, based on the total weight of monomer and crosslinker.

The gel-forming liquid may contain other additives such as an antioxidant, if necessary. As the antioxidant, various compounds known to be able to be incorporated into the gel film can be used. Specific examples of antioxidants include dithiothreitol and 2-mercaptoethanol.

Other additives include wetting agents, and the polyacrylamide gel film can also contain polyol compounds such as glycerin and ethylene glycol. The content of the polyol compound is about 5 to about 40% based on the volume of the gel membrane.
Selected from the range of w/v%. Among the polyol compounds, glycerin is particularly preferred. By adding a wetting agent, we can prevent the gel film from drying out due to extreme water evaporation during storage, and also improve the physical properties of the gel film, such as preventing brittleness caused by extreme dryness and preventing cracking. It has the advantage of being improved.

- The gel-forming liquid is applied in the form of a film onto the support directly or through another intermediate layer such as an adhesive layer, and then cross-linked and polymerized to form a gel film. .

When cross-polymerizing the gel-forming liquid on the surface of the support,
The gel-forming liquid coating layer can be further covered with a covering material such as a cover film (including a sheet or plate). The cover film used for this purpose may be made of the same material as the support and, like the support, has reduced oxygen permeation residue. The thickness of this coating material is 3007 tm or less, with a practically preferred thickness range of about 4 to 200 gm, and a particularly preferred range of about 4 gm to 11007 gm.

In the production method of the present invention, flash light from a xenon flash lamp light source is used for crosslinking polymerization of the gel-forming liquid. This irradiation is preferably carried out under a nitrogenous atmosphere.

A xenon flash lamp consists of a cathode and a
This is a lamp in which a high voltage is applied between the nodes and a trigger induces a plasma discharge between the cathode and the anode to produce high-intensity light. In this case, the light emission time is generally set to several microseconds to several tens of microseconds, and the light emission interval is set to several hertz to several hundred hertz.

xenon flash lamp. It can be placed above or below the gel-forming liquid membrane, or above and below at the same time. Furthermore, other ultraviolet lamps can also be used together.

There is no particular limit to the number of xenon flash lamps installed, and the heat generated from the xenon flash lamps can be easily removed by flowing air around the lamps. Therefore, the light source device becomes smaller, which is preferable in terms of production equipment.
It also has the advantage of being easy to maintain and manage.

The electrophoresis medium material obtained according to the present invention can be used in any of the Mizuto type and vertical plate electrophoresis methods, disk electrophoresis methods, etc. according to the known methods described in the above-mentioned documents. .

Below, the present invention will be explained in more detail with reference to Examples.

[Example 1-3] Acrylamide, BIS, urea,
Potassium belloxonisulfate, TEMED (N, N, N
', N'-tetramethylethylenediamine) and riboflavin phosphate ester sodium salt in the amounts listed in Table 1.

Margin below Table 1 Acrylamide 7.84gBIS
O, 16g urea
42.0g tris(hydroxymethyl) 1, 08g aminomethane boron #0.55gED
TA-2Na 93mg (add water to make it roomy) TEMED 33 chlorine salt 1.5m (5wt% aqueous solution) Riboflavin phosphate ester Nap 2. OmM(0,
25wt% aqueous solution) Note: BIS: N,N'-methylenebisacrylamide TEMED: N,N,N',N
' - Tetramethylethylenediamine The support wave coated with the above gel-forming solution in a film form is passed through a hardened casing that has been sufficiently purged with nitrogen, and at the same time, 200 W of xenon is applied from the top or bottom, or from both the top and bottom. Light was irradiated from a flash lamp.

In addition to the above-mentioned light irradiation, the temperature on the gel-forming liquid film and the time required for gelation were measured. The results are listed in Table 2.

[Comparative Example 1-3] In the same manner as in Example 1, a support wave coated with a gel-forming liquid in the form of a film was prepared.

Next, the gel-forming liquid film was irradiated with light in the same manner as in Example 1, except that the xenon flash lamp was replaced with a 200 W high-pressure mercury lamp.

In addition to the above-mentioned light irradiation, the temperature on the gel-forming liquid film and the time required for gelation were measured. The results are listed in Table 2.

[Comparative Example 4] In the same manner as in Example 1, a support wave coated with a gel-forming liquid in a film was manufactured by iA.

Next, the gel-forming liquid film was irradiated with light in the same manner as in Example 1 except that the light irradiation from the xenon flash lamp was omitted. Along with this light irradiation, the temperature on the gel-forming liquid film increases,
The time required for gelation was measured. The results are listed in Table 2.

Table 2 Irradiation Gelling light source Part Temperature Time Example 1 (Canon top 25℃ 35 seconds 2
Flash lower 25℃ 35 seconds 3 lamp) Upper/lower 26℃ 30 seconds Comparative example 1 (high pressure
Upper 50℃ 20 seconds 2 mercury lamp) Lower 6
0℃ 20 seconds 3 Up/Down 80℃ 15
sec 4 No irradiation 20℃ 600 sec In all cases where a xenon flash lamp is used, the temperature of the gel forming solution (gel film) hardly rises, whereas in the case where a high-pressure mercury lamp is used, the temperature rises significantly. It is clear that It can be seen that the gelation time shows almost the same polymerization rate in all cases.

Claims (1)

[Scope of Claims] 1. Aqueous liquid containing an acrylamide monomer and a crosslinking agent formed in a membrane or column shape is irradiated with flash light from a xenon flash lamp light source to cause crosslinking polymerization within the aqueous liquid. A method for producing a material for electrophoretic analysis, which comprises forming an aqueous gel film. 2. The method for producing a material for electrophoretic analysis according to claim 1, wherein the crosslinking polymerization is carried out in a nitrogen atmosphere. 3. The electricity according to claim 1, characterized in that crosslinking polymerization occurs in a state in which an aqueous liquid containing an acrylamide monomer and a crosslinking agent formed in a membrane is sandwiched between two films. Method for producing materials for electrophoretic analysis. 4. The method for producing a material for electrophoretic analysis according to claim 1, wherein the aqueous liquid further contains a radical crosslinking polymerization catalyst and a photosensitizer.