WO2006093343A1

WO2006093343A1 - Electrophoresis-use barrier material and barrier structure and electrophoresis device

Info

Publication number: WO2006093343A1
Application number: PCT/JP2006/304661
Authority: WO
Inventors: Katsunori Aizawa
Original assignee: Kabushikikaisya Advance
Priority date: 2005-03-04
Filing date: 2006-03-03
Publication date: 2006-09-08
Also published as: JPWO2006093343A1; JP4814216B2

Abstract

An electrophoresis-use barrier material which is a barrier material used to prevent the effect of an electrolytic reaction caused in the vicinity of an electrode during electrophoresis, and which is characterized by comprising a compound capable of neutralizing acidic and (or) alkaline substances produced electrolytic reaction in the vicinity of an electrode, or a compound capable of eliminating an electrolyzed product strong in oxidizing power or reducing power; and an electrophoresis device and an sample analyzing method using it. The barrier material can prevent convection caused by gas produced by an electrolytic reaction caused during electrophoresis without using high voltage, a large amount of buffer solution, ion exchange membrane and ion exchange resin, neutralize acidic or alkaline substances produced during electrophoresis or eliminate a substance strong in oxidizing power or reducing power, and further supply constantly a support electrolyte to an electrophoresis portion.

Description

Electrophoretic barrier material, barrier structure, and electrophoresis apparatus

The present invention relates to an electrophoresis technique, and more particularly, to a technique for minimizing the influence of an electrolysis reaction generated in the vicinity of an electrode on an electrophoresis result in an electrophoresis apparatus. The present invention particularly relates to a barrier substance for electrophoresis, a barrier structure, and an electrophoresis apparatus. The present invention also relates to a method for separating and analyzing biological samples and other samples using such electrophoresis technology. Background art

Electrophoresis is, for example, a method in which a mixed sample of biomolecules (mainly DNA, RNA, protein, etc.) is separated according to the difference in molecular size and charge state of each molecule. For this reason, electrophoresis apparatuses that employ various methods are commercially available. Electrophoresis devices are used in fields such as life science, medicine, pharmacy, agriculture, and environmental science.

By the way, in an electrophoresis apparatus, an electrolysis reaction can occur at the electrode. Depending on the material of the electrode and the composition of the solution in contact with the electrode, various reactions occur at and around the electrode. Furthermore, in recent electrophoretic devices, because of their excellent durability and operability, platinum wires and materials with platinum attached are often used as electrode materials. Under the buffer solution used as the base, electrolysis generates oxonium ions, hydroxide ions, oxygen gas and hydrogen gas. Also, the type of solution used Depending on the type, chlorine gas, iodine, etc. are produced. In particular, when a solution containing a large amount of chloride ions is used for electrophoresis, hypochlorite (HC 1), which has a pH of 3 or more and part of the chlorine gas generated by this electrolysis, damages proteins and nucleic acids. ○) is converted into a strong oxidant such as In addition, various ionic substances may be adsorbed on the electrode and the electrical resistance around the electrode may change.

Many of these by-products produced during electrolysis have an adverse effect on the results of electrophoresis. For example, in the part close to the anode, the solution becomes acidic PH due to oxonium ions generated by electrolysis, and in the part close to the cathode, it becomes alkaline pH due to hydroxide ions.

By the way, many of the samples that are separated and analyzed by electrophoresis have many pKa values and isoelectric points that are unique to those samples. Unexpected changes in electrolyte pH will not result in the expected mobility. Also, if the conductivity (conductivity) changes, the potential gradient at that site changes, and in this case as well, the expected mobility is not brought about.

Furthermore, gases such as oxygen gas and hydrogen gas generated by electrolysis become bubbles in the buffer solution during electrophoresis and rise to the water surface of the buffer solution. This leads to the occurrence of convection. Such convection leads to the action of stirring the buffer solution, but it does not necessarily stir the solution uniformly, so it does not reach the action of keeping the pH at each site where electrophoresis is performed constant. Rather, depending on some conditions and the electrophoresis apparatus, the pH and conductivity of the solution in the vicinity of each electrode become non-uniform, and thus the pH and conductivity of the electrophoretic site also become non-uniform.

In addition, buffers, surfactants, and samples used for electrophoresis If a substance containing halide ions is used, chlorine gas and iodine are also generated. In particular, when chlorine gas is generated, part of this gas dissolves in the buffer solution, and in addition to hydrochloric acid, it produces harmful substances with strong oxidizing power such as hypochlorous acid. Naturally, this not only leads to instability of electrophoresis itself, but also may cause deterioration and denaturation of the sample to be separated. In fact, HC10 is known to denature certain proteins even at low concentrations of l O ppm.

Several methods have been proposed and put to practical use to reduce the effect of electrolysis on electrophoresis.

The first method used was to increase the distance between the electrode part where electrolysis occurs and the electrophoretic part, and it is relatively easy to design an electrophoretic apparatus that satisfies this condition. However, compared to an electrophoresis apparatus in which the distance between the electrode part and the electrophoresis part is short, it is necessary to increase the voltage in order to ensure a certain mobility. As a result, there are inconveniences such as excessive generation of Joule heat and increased risk of leakage due to high voltage. In addition, since some of the by-products of electrolysis reach the site where electrophoresis is performed in a short time, it is not suitable for long-time electrophoresis experiments.

Next, there is a method in which a large amount of buffer solution is used as an electrode solution in the vicinity of the electrode in order to neutralize the acid (oxonium) and alkali (hydroxide ion) generated by electrolysis during electrophoresis. However, in this method, oxygen gas generated by electrolysis, hydrogen gas, chlorine gas generation and iodine generation, adsorption of various ionic substances to the electrode, dissolution of metal ions from the electrode, etc. The effects cannot be excluded.

In addition, an ion exchanger layer such as an ion exchange membrane or ion exchange resin is formed between the electrode part and the electrophoresis part, and this is electrolyzed. A method has also been proposed in which the ions that are produced are bound, and at the same time, the ions that have been bound to these ion exchangers are supplied to the electrophoresis site. However, this method, which has been partially put into practical use, also has a drawback. For example, the ion exchange capacity is relatively small (often 0.3 mol ZL or less), that is, it is difficult to secure an electrolyte that can maintain long-term electrophoresis, and these ion exchange membranes and Typical drawbacks include the need to replace or regenerate the ion exchange resin.

The electrophoresis technology described above is, for example,

US Patent No. 5, 5 8 2, 7 0 2

US Patent No. 5, 8 6 8, 9 74

Special Table 1 1 1 5 0 5 3 2 5

J.Am.Chem.Soc., 4-5, 9 5

Disclosure of the invention described in

The object of the present invention is to solve the above-mentioned problems in electrophoresis technology.

That is, the object of the present invention is to prevent convection caused by a gas generated by an electrolysis reaction that occurs during electrophoresis without using a high voltage, a large amount of buffer solution, an ion exchange membrane or an ion exchange resin, Neutralizes acidic and alkaline substances generated during electrophoresis, and in some cases, removes products with strong oxidizing power and reducing power generated by electrolysis, and stabilizes the supporting electrolyte at the electrophoresis site. It is an object of the present invention to provide an electrophoretic device that can be supplied in a simple manner and that has a simple structure, and components that can be used advantageously in the configuration of the electrophoretic device.

Another object of the present invention is to separate various samples using electrophoresis. And providing a sample analysis method that can be carried out accurately and easily without adversely affecting the results of electrophoresis.

As a result of intensive studies to achieve these objects, the present inventor has barrier structures on the cathode side and the anode side of the portion where electrophoresis is performed (hereinafter also referred to as “electrophoresis portion”). By forming a site and reacting with or absorbing a buffering solution or electrolysis product there, or by stably presenting a concentrated solution of the supporting electrolyte for electrophoresis,

1) Neutralize acidic and alkaline substances generated during electrophoresis 2) React with or absorb electrolysis products as required

3) Furthermore, we found that we can provide a mechanism to stably supply the supporting electrolyte to the electrophoresis site,

At the same time, by devising the shape of the electrophoresis tank so that this barrier structure stays in the same site stably in the electrophoresis tank,

4) The inventors have found that convection caused by gas produced by electrolysis can be prevented at the same time, and have completed the present invention.

In one aspect, the present invention is a paria substance used to suppress the influence of an electrolysis reaction caused in the vicinity of an electrode during electrophoresis, and in many cases, several kinds of solutes and solvents. It is a mixture of This substance consists of a compound that can neutralize acidic and / or alkaline substances produced by the electrolysis reaction in the vicinity of the electrode, or reacts with or absorbs the electrolysis product, and the electrolysis product It is characterized by being a compound capable of reducing the amount of.

A single Paria substance provides a stable supply of supporting electrolyte to the electrophoresis site It is preferable to further have a function.

Further, it is preferable that the barrier material is used in the form of a caustic solution, glue or gel which can be used in any form.

In addition, a single substance may be used by itself, but an optional additive can be used in combination for further effect. Examples of suitable additives include substances that can stabilize the function of the barrier substance, and substances that can display a state in which the function of the barrier substance is stabilized.

In another aspect, the present invention provides an electrophoresis barrier used for preventing direct contact between the electrode and a buffer solution in an electrophoresis tank in an electrophoresis apparatus having an electrode composed of an anode and a cathode. A structure that mainly forms a physical barrier.

The barrier structure for electrophoresis according to the present invention includes two barrier substances for electrophoresis according to the present invention, each of which holds the one barrier substance in a state where any one of the electrodes and the barrier substance are in contact with each other. A barrier chamber and at least one bulkhead defining each barrier chamber;

It is characterized by comprising. In the barrier chamber, the barrier substance exposed from the partition is in contact with the buffer in the electrophoresis chamber.

The barrier structure can be configured in various forms and sizes within the scope of the present invention. For example, the partition wall defining the barrier chamber is preferably capable of preventing or suppressing convection of the buffer solution due to the gas generated in the electrolysis reaction caused in the vicinity of the electrode during electrophoresis. Therefore, it is preferable that the partition wall has a shape or the like that can induce such an action and is disposed at an appropriate position.

In particular, the partition wall is arranged in the form of a beam in the vicinity of the electrode in the vertical or near-vertical direction and has an opening through which the barrier material can move. A first partition wall (beam structure) provided in the upper part of the first partition wall and a vertical position in the form of a sill at a position farther from the electrode than the first partition wall so that the barrier substance and the buffer solution can come into contact with each other. Preferably comprises a second partition (sill structure) terminating in a buffer solution.

Further, in such a barrier structure, the first partition and the second partition may be arranged simply in combination, but an elastic packing having electrical conductivity is provided between the first partition and the second partition. Members may be further arranged.

In yet another aspect, the present invention provides an electrophoresis apparatus including an electrophoresis tank, electrodes each composed of an anode and a cathode disposed at predetermined positions of the swimming tank, and a buffer solution contained in the electrophoresis tank. Because

An electrophoretic apparatus is characterized by further comprising an electrophoresis barrier structure according to the present invention.

The electrophoresis apparatus of the present invention can be configured based on various electrophoresis techniques known in the technical field. Suitable electrophoresis techniques include, for example, vertical electrophoresis, horizontal electrophoresis, submarine electrophoresis, cylindrical electrophoresis, capillary electrophoresis, chip electrophoresis, and preparative electrophoresis. Examples thereof include electrophoresis, electrochromatography, pulse field electrophoresis, and isoelectric focusing.

For example, when the electrophoresis technique is based on the submarine type electrophoresis method, the electrophoresis apparatus is composed of a single electrophoresis tank, and a sample or a sample-containing medium is contained in a buffer solution in the electrophoresis tank. It is preferable to constitute so as to be immersed.

In addition, when the electrophoresis technology is based on vertical electrophoresis, columnar electrophoresis, or capillary type electrophoresis, etc., two electrophoresis cells with electrophoresis tanks spaced apart from each other are arranged. Each tank consists of a tank It is preferable that the tank contains a buffer solution and that the sample or the sample-containing medium is contained in a communication member that communicates the two migration tanks, such as a capillary tube or a hollow tube.

In yet another aspect, the present invention provides a method for separating or analyzing a sample by electrophoresis and further sorting the sample, characterized by using the electrophoresis apparatus according to the present invention. In the implementation of the method of the present invention in the method, the details of the kind of sample and analysis conditions are not particularly limited, and can be arbitrarily changed within the scope of the present invention. For example, the sample to be separated and analyzed is preferably a biological sample, and specific examples include biomolecules such as DNA, RNA, and protein. Brief Description of Drawings

Fig. 1 is a cross-sectional view schematically showing an electrophoresis tank applied to a submarine type electrophoresis apparatus.

FIG. 2 is a cross-sectional view schematically showing an electrophoresis tank applied to a vertical electrophoresis apparatus.

Fig. 3 is a cross-sectional view schematically showing an electrophoresis tank applied to a cylindrical or capillary type electrophoresis apparatus.

FIG. 4A is a top view showing the shape of an electrophoresis tank having a beam structure type partition wall and a sill structure type partition wall used in the examples.

FIG. 4B is a cross-sectional view of the electrophoresis tank shown in FIG. 4A as viewed from the side, and

FIG. 5 is a cross-sectional view showing the shape of an electrophoresis tank having a beam structure type partition wall and a sill structure type partition wall used in the examples. BEST MODE FOR CARRYING OUT THE INVENTION

The present invention can be advantageously implemented in various forms. Hereinafter, preferred embodiments of the present invention will be described with particular reference to several electrophoresis apparatuses, but the present invention is not limited to these embodiments.

The electrophoresis apparatus according to the present invention can have the same constituent elements as those of a conventional electrophoresis apparatus as the essential constituent elements. Ie

The electrophoresis apparatus according to the present invention includes one or more electrophoresis tanks and a pair of electrodes composed of an anode and a cathode arranged in the swimming tank or one of the anode and the cathode (an electrode is provided for each electrophoresis tank). And a buffer solution accommodated in the electrophoresis tank. In addition to these components, the electrophoresis apparatus of the present invention includes additional components such as a power source for pulse output or DC output, a buffer discharge port, an electrophoresis cover, a gel tray, and a comb. Can be provided. The main components will be described below.

Electrophoresis tank

The electrophoresis tank can have any shape, but is usually a box type, a cylindrical type, a column type, or the like. When handling, mounting of other components, and compactness are considered, it is recommended to use a box-type electrophoresis tank. The size of the electrophoresis tank is not particularly limited, but the electrophoresis tank of the present invention can be miniaturized due to its specific configuration. For example

An example of a compact electrophoresis apparatus is a box-type electrophoresis tank with a width of around 1300 mm, a length of around 12.0 mm, a depth of around 50 mm, and a buffer volume of around 2500 ml. Can be mentioned. In addition, the material of the electrophoresis tank is not particularly limited as long as it is resistant to an electrolytic solution and can be used stably over a long period of time. For example, polycarbonate, polystyrene, propylene copolymer It can be produced by molding plastic materials such as coalescence, polymethylpentene, borisulphone, polyphenylene oxide, and acrylic, and by adhering each member. In some cases, FRP may be used for the material.

One electrophoresis tank may be used, and two or more electrophoresis tanks may be connected as necessary. For example, it is common to use one electrophoresis tank for a submarine type electrophoresis apparatus, but two electrophoresis tanks for a vertical electrophoresis apparatus, a cylindrical type, a capillary type, etc. In general, these are used in combination. When two or more electrophoresis tanks are used, each electrophoresis tank can be connected with an arbitrary communication member to move the buffer solution. Examples of the communicating member include a capillary tube and a hollow tube. When a plurality of electrophoresis tanks are used, the shape and size of each electrophoresis tank may be the same or different.

Electrode

The electrode may be an electrode generally used in an electrophoresis apparatus, and the material, shape, size and the like are not particularly limited. For example, the anode can be formed in the form of a thin wire, a rod, a thin film, a thin plate, or the like from a conductive material resistant to a buffer solution, such as platinum, a platinum plating material, or carbon. Taking the case where an anode made of a thin wire (platinum) is attached to the box-type migration tank of the above size as an example, the size of the anode is about 122 mm in length and about 0.25 mm in diameter.

As with the anode, the material, shape, size, etc. of the cathode are not particularly limited. It can be formed of the same material and the same size as the anode.

Buffer The buffer solution used in the electrophoresis tank may be a buffer solution generally used in electrophoresis apparatuses. The buffer solution is usually composed of an electrolyte and a solvent in which the electrolyte is dissolved, and optional additives are used together as necessary. In particular, in the present invention, as will be described in detail below, a barrier substance is used in the vicinity of each electrode in order to prevent the buffer solution from coming into direct contact with the electrode.

〔Electrolytes〕

The electrolyte may be a compound commonly used in electrophoresis apparatus buffers. Suitable electrolytes include, but are not limited to, for example, the compounds listed below. In the following compound names, the letters or abbreviations enclosed in parentheses are common names of the corresponding compounds. The following compounds may be used singly or in combination of two or more compounds.

N-(2-acetamido) 1 2-aminoethanesulfonic acid (ACES), N- (2-acetamido) iminoniacetic acid (ADA), acetic acid, N- (2-aminoethyl) trimethylammonium chloride ( C HO L AM I NE), ascorbic acid, ammonia solution, barbituric acid, benzene quexacarboxylic acid (me 1 1 itic), benzempen carboxylic acid, benzene — 1, 2, 4, 4, 5 —tetrastrand Acid (pyromel 1 itic), benzene— 1, 2, 3 — tricarponic acid (hemime 1 1 itic), benzene— 1, 3, 5 — tricarboxylic acid (trimesic), benzoic acid, N, N—bis (2 — Hydroxetyl) 1 2 —Aminoethanesulfonic acid (BES), N 2, N—Bis (2 —Hydrochetil) Gilicin (BICINE), Bis (2—Hydroxychetyl) Iminotris (Hydroxymethyl) Methane BIS- TRIS), boric acid, Burushintetoraha Idrate, Butane-1,2,3,4-Tetracarboxylic acid, n-Butyric acid, Carbonic acid, Chenic acid, Cyclohexanediacetic acid, 3-Cyclohexylamino 1 Monopropanesulfonic acid (CAPS ), Cyclopentanediacetic acid, cyclopentanediacetic acid (3,3-tetramethylenedaltaric acid), cyclopentanetetra-1,2-, 3,4-carboxylic acid, ethylamine, dimethylamine, 2,2 — Dimethyldaluric acid, 3, 3 — Dimethyldaluric acid, dimethylmalonic acid, 2, 2-dimethylcono, succinic acid, 2, 2 — jetylsuccinic acid, 3, 6 _endomethylene 1, 2, 3, 3, 6 — tetrahydroff Evening acid (EMTA), ethanolamine, ethylamine, ethylenediammine tetraacetic acid (EDTA), formic acid, fumaric acid, glycine phosphate, glycine amide, glycine, N-darlicyl Lysine, hippuric acid, histidine, N—2—Hydrochetylpiperazine 1 N′—2—ethanesulfonic acid (HEPES), hydroxysylamine, 2—hydroxichetiliminotoris (hydroxymethyl) methane (MO NO) — TRIS), 4 (2 — Hydroxyl) _ 1 — Piperazinepropanesulfonic acid (EPPS), Imidazole, Itaconic acid, Lactic acid, Maleic acid, Lingoic acid, Malonic acid, Mandelic acid, Methylamine , 2 -methylpropane-1, 2, 3-triscarboxylic acid, 2 _ (N-morpholino) monoethanesulfonic acid (MES), 2- (N-morpholino) -propanesulfonic acid (MO PS) , Nitric acid, oxalic acid, propane 1, 2, 3 — tricarboxylic acid (tricarbal 1 y 1 1 ic), pyrophosphoric acid, phosphoric acid, phthalic acid, 1,4-piperazinebis (ethanesulfonic acid) (PIPES), propionic acid, pyridine, salicylic acid, succinic acid, sulfuric acid, tartaric acid, triethylamine, tris (hydroxymethyl) aminomethane (TRIS), N-tris (hydroxymethyl) methyl-2-amino Ethanesulfonic acid (TES), N-tris (hydroxymethyl) (TRICINE), N— 卜 ris (hydroxymethyl) methyl-2-aminopropanesulfonic acid (TAPS), trimethylamine, and others.

In these compounds, when the compound is an acid, in addition to use as an acid, lithium, sodium, potassium, magnesium, calcium, barium, zinc, molybdenum, manganese, iron, nickel, copper, tin Also included is use as a compound in combination with metal ions such as aluminum. Moreover, when it is a base, the use as a compound compounded with the following arbitrary acids is also included.

[Solvent]

The electrolyte is used by dissolving in an arbitrary solvent in order to prepare a buffer solution. Suitable solvents are not limited to those listed below, but include, for example, water, heavy water or water-soluble organic solvents such as monohydric alcohol, dihydric alcohol, trihydric alcohol or polyhydric alcohol, Primary alcohol, Secondary alcohol, Tertiary alcohol or higher alcohol, Acetonitrile, Acetone, Acetylacetone, Dimethylformamide, Ethylene glycol, N_ethylformamide, Dimethyl sulfoxide, 1, 4 Monodioxane, Ethylene dallicol , N-methyl amide, pyridine, tetrahydrofuran and the like. These solvents may be used alone or in combination of two or more.

[Barrier component]

In the electrophoresis apparatus of the present invention, it is essential that a barrier substance is interposed between the electrode and the buffer solution contained in the electrophoresis tank. The barrier substance neutralizes acidic substances and alkaline substances generated during electrophoresis, removes electrolysis products with strong oxidizing power and reducing power, and stably supplies a supporting electrolyte to the electrophoresis site. And even produced by electrolysis The main purpose is to prevent the convection caused by the generated gas. Usually, a barrier section containing a barrier substance or a barrier section on the cathode side and the anode side of the portion where electrophoresis is performed in the electrophoresis tank. Used in the form of a barrier chamber.

The barrier substance used in the practice of the present invention is not particularly limited as long as it exhibits the above-described effects and does not adversely affect the desired electrophoretic properties. Suitable components of the barrier are not limited to those listed below, but in addition to solutions that increase the specific gravity such as glycerol and heavy water, reducing substances such as concentrated buffer, sulfite and sodium sulfate. Or metal such as oxide or iron powder, chelate, etc. These barrier substances may be used alone or in combination of two or more.

The barrier substance is used in the form of isolating the electrode from the buffer solution in the vicinity of the electrode in the electrophoresis chamber. The paria substance can be used in various states, but is preferably used in the form of a solution barrier, a paste-like barrier or a gel-like barrier. Any state of the barrier can be easily prepared.

The solution barrier, paste-like barrier or gel-like barrier may be infiltrated with a buffer solution in contact therewith, or may be contained otherwise. The concentration of the buffer contained in these barriers can vary over a wide range, but it is defined by the electrolyte concentration and is usually about 1 η Μ to 1 表 as expressed by the mora concentration at the time of use. It is preferably about 1 mM to 500 mM. However, in solution barriers, paste-like barriers, or gel-like barriers, the electrolyte does not have to be completely dissociated, and depending on the conditions, there is a solid micelle state or complex that can be released into the electrolyte. You may do it.

In addition, the barrier on the cathode side includes the above It is necessary to have a composition that forms one anionic electrolyte, or sometimes two or more anionic electrolytes, or a composition that dissociates to form an anionic electrolyte. The type of electrolyte added to the cathode-side barrier is not limited unless it is a cation-type electrolyte that significantly changes the electrical resistance around the electrode by adsorption to the electrode.

On the other hand, the barrier on the anode side includes a composition that forms one of the above cation electrolytes, or sometimes two or more cation electrolytes, or dissociates to form a cation electrolyte. It is necessary to be. The type of electrolyte added to the anode side barrier is not limited unless it is an anionic electrolyte that significantly changes the electrical resistance around the electrode by adsorbing to the electrode.

Furthermore, in order to adjust the conductivity in the barrier or to form an electrically conductive phase different from the barrier single layer made of an aqueous solution at an arbitrary position in the vicinity of the electrode, it is added to the barrier material and the electrolyte or added separately, and is added to the ionicity. A solution can also be used. Suitable ionic liquids for adjusting the conductivity are, for example, imidazolium salt derivatives, pyridinium salt derivatives, phosphonium salts, tetraalkylammonium salts, and the like.

In the practice of the present invention, the equilibrium state of the electrolyte in the solution barrier, the paste-like barrier, or the gel-like barrier is moved, and the dissociation state is adjusted so that the portion of the barrier does not have too high conductivity. May be preferred. In such a case, the present inventors have found that it is preferable to further contain a water-soluble organic solvent in order to adjust the dissociation state in the barrier.

Suitable water-soluble organic solvents include, for example, monohydric alcohols, dihydric alcohols, trihydric alcohols or polyhydric alcohols, primary alcohols, secondary alcohols, tertiary alcohols or higher alcohols, acetonitrile. Acetone, acetylacetone, dimethylform Examples include muamide, ethylene glycol, N-ethylformamide, dimethyl sulfoxide, 1,4-dioxane, ethylene glycol, N-methylamide, pyridine, and tetrahydrofuran. These organic solvents may be used alone for the preparation of a solution, or two or more kinds of solvents may be used in combination. In the obtained barrier solution, the concentration of the barrier substance is not particularly limited, but the concentration at the time of use is about 0.1 to 99% by weight, preferably about 1 to 80% by weight. .

It is also desirable to include additional additives for different purposes in the preparation of solution barriers, paste-like parias or gel-like barriers, depending on the type of each barrier.

For example, various saccharides such as saccharose, polysaccharides such as starch, ethylene glycol, polyethylene glycol, polyvinyl alcohol, dextran, Ficoll (registered trademark), Percol (registered trademark) and other high specific gravity substances such as In addition to the ingredients, they are added to the solution barrier, or a substance that increases the viscosity is added to the solution barrier to form a paste-like barrier, thereby contacting these barriers and the barriers. It is desirable to have a function to prevent mixing with the buffer solution at the electrophoresis site. In the obtained solution-like barrier or paste-like barrier, the concentration of the barrier substance is not particularly limited, but the concentration at the time of use is about 2 to 99% by weight, preferably 5 to 80% by weight. %.

In the case of preparing a gel-like barrier, for example, it is not limited to those listed below, but polyacrylamide gel, agarose gel, composite gel, protein nucleic acid enzyme, Vol. 43, No. 15, page 2191 The elastic gel described in 1998, the elastomer gel described in Polymers for advanced Technologies vol. 11, p.481 (2000), etc. The electrolytes can be included in those gels that can be used. The composition of the electrolyte to be included may be the same as or different from the composition of the electrolyte at the electrophoresis site, or the electrolyte having another composition may be used.

In addition, solution barriers, glue-like barriers or gel-like barriers

It is also possible to use an additive component that makes it possible to visually observe the state of stabilization and the state of the PH value, in other words, an additive that can visually show the state. As such additives, for example, various pH indicators are effective. When pH indicator is added, part of the mixing situation of the barrier substance and the buffer solution at the electrophoresis site can be visualized by the color shift of pH indicator. Suitable pH indicators include, for example, Alizarin Red S, Alizarin Yellow R, Benzoperpurin 48, Brilliane Cresyl Blue, Brilliane Yellow

, Bromocresol Green, Promocresol Blue, Bromophenol Blue, Bromofu X Nord Red, Bromothymol Bull

― ク D DFenol Red, Clay Ton Yellow, 3 Ngolet

, Cresol red, o-cresolphthalein, m-cresol violet, curcumin, 2,4-dinitrophenol, 2, 6-di double-mouthed X-nor, Jerioku Pome Black, Ellis P-Sin, Ye

, ゝ,

Loulenn, indigo carmine, indigo sulfonic acid, lacmoide

, Maracay 卜 Green, Metallic Yellow, Methylolene, Methyl Red, Methyl Baylet 、, Methyl Ichiichi, Naphthol Benzene, One-Tall Red, One-Lamine, m-Two-Minute Phenolic,

P — 2-D phenol, phenolphthalein, phenol red

, ヽ,

, Kinnarun Reds, Resaz U N, Thiazol Luye Mouth G, Timor Bull I, Timor Fu Yurain, 1, 3, 5-Tri Nittoguchi Benzene

, 2, 4 6 — 卜 Linitro Toluene, tropeoline, etc. You can.

[Barrier structure]

In the electrophoresis apparatus of the present invention, the above-described barrier substance is preferably used in the form of a barrier structure in which the barrier substance is incorporated therein. The barrier structure is preferably a single substance for electrophoresis according to the present invention, a barrier chamber that holds the barrier substance in contact with either one of the anode and the negative electrode constituting the electrode, and the barrier chamber It is characterized by including at least one partition wall that defines In addition, it is usually advantageous to form the barrier chamber by combining the partition wall with the side wall and bottom wall of the electrophoresis tank.

The barrier structure prevents the convection generated by the gas generated by the electrolysis reaction from reaching the site where electrophoresis is performed, and at the same time allows the barrier substance of the present invention to exist stably. A typical example of a barrier structure is shown in FIGS. 1, 2 and 3 in the form of a migration tank containing it.

The electrophoresis tanks shown in Fig. 1, Fig. 2 and Fig. 3 can be applied to different electrophoresis methods. For example, the electrophoresis tank shown in Fig. 1 can be used for submarine electrophoresis, pulse field electrophoresis, and the like. The electrophoresis tank shown in Fig. 2 can be used for DNA sequencing, vertical electrophoresis, which is widely used for protein separation and analysis. In addition, the electrophoresis tank shown in Fig. 3 can be used for column-type and capillary-type electrophoresis methods. Hereinafter, each electrophoresis tank will be described in more detail.

The electrophoresis tank in Fig. 1 has a structure that can be used for submarine type electrophoresis and pulse field electrophoresis. Also, this structure is slightly modified, and a gel or other separation medium is sandwiched between glass plates, If the structure does not contain conductive buffer, it can be applied to horizontal electrophoresis. It becomes possible.

The electrophoresis tank 10 in FIG. 1 is a cross-sectional view of the electrophoresis tank of the submarine type electrophoresis apparatus. In the case of this electrophoresis tank 10, an anode 14 (1) made of a platinum wire and a negative electrode 14 (2) made of a platinum wire are disposed at the portion where the side wall and the bottom wall are in contact with each other. An output power source 19 is connected to the anode 14 (1) and the cathode 14 (2). Buffer 1 is accommodated in the electrophoresis tank 10 up to a level of 1 L.

Further, inside the migration tank 10, the first partition wall 11 having a beam structure (beam), the second partition wall 13 having a sill structure (sill), and the side wall and bottom of the migration tank 10. A barrier chamber is formed with the wall. Here, the first partition wall 11 extending downward from the upper surface of the swimming tank 10 opens at the lower part of the electrophoresis tank 10, and the second partition wall extends upward from the lower surface of the electrophoresis tank 10. 1 3 terminates in the middle of buffer 1. Further, in the formed barrier chamber, a solution or paste is formed so as to surround the anode 14 (1) and the cathode 14 (2) up to the upper end of the second partition wall 13, that is, up to the level 12. Barrier material 1 2 is retained. A platform 16 for placing a sample is provided in a substantially central portion of the electrophoresis tank 10. A separation medium 15 such as a gel, which is a sample, is placed on the platform 16. In the case of the illustrated example, the separation medium 15 has a rectangular shape that is long horizontally, but other forms may also be used. Further, the composition of the solution or paste-like barrier material may be the same or different on the anode side and the cathode side. Similarly, the other components of the electrophoresis tank 10 may be bilaterally symmetric or asymmetrical.

FIG. 2 is a cross-sectional view of the electrophoresis tank of the vertical electrophoresis apparatus. As shown in the figure, this electrophoresis tank is provided with two electrophoresis tanks 20 (1) and 20 (2) each having an anode 24 (1) and a cathode 24 (2) attached thereto. Yes. Both the anode 2 4 (1) and the cathode 2 4 (2) are formed of a platinum wire, and an output power source 29 is connected thereto. In the electrophoresis tanks 20 (1) and 20 (2), a buffer solution 1 is accommodated. In addition, the inside of the two migration tanks 20 (1) and 20 (2) has a first partition wall 21 having a beam structure (beam) and a sill structure (sill), respectively. A barrier chamber is formed by the second partition wall 23 and the side wall and bottom wall of the electrophoresis tank 20. In each electrophoresis tank, as in the electrophoresis tank 10 of FIG. 1, the first partition 21 opens at the lower part of the electrophoresis tank 10, and the second partition 23 has buffer 1 Terminates on the way. Further, in the formed barrier chamber, up to level 2 2 L, a solution-like or paste-like barrier substance 22 is held so as to surround the anode 24 (1) or the cathode 24 (2).

Further, in the illustrated migration tanks 20 (1) and 20 (2), the packing members 26 and 6 are sealed so as to close the spaces formed by the separated first partition wall 21 and second partition wall 23, respectively. Is inserted. The packing member 26 is effective in preventing unnecessary mixing of the solution barrier, the paste-like barrier 1 or the gel-like barrier 1 with the buffer solution at the electrophoresis site. As the packing member 26, polyacryl amide gel, agarose gel, composite gel, elasticized gel, elastomer gel and the like can be used. Also, various porous materials such as filter paper and sponges can be used as the packing member 26 only when the gas generated by the electrode is less likely to adhere to the inside or surface of the porous material. Can.

The electrophoresis tanks 20 (1) and 20 (2) arranged above and below are connected by a conduit formed by combining glass plates (not shown), and inside the conduit, A separation medium 25 such as a gel is filled. FIG. 3 is a cross-sectional view of an electrophoresis tank of a cylindrical electrophoresis apparatus. In this swimming tank, as shown in the figure, the communicating member filled with the separation medium 35 such as a gel is composed of a glass tube (may be a single tube, etc.), and the difference is that the mounting position is changed. Thus, it has the same configuration as the swimming tank shown in FIG.

In order to avoid duplication, the electrophoresis tank shown in Fig. 3 has two electrophoresis tanks 30 (1) and 3 with an anode 3 4 (1) and a cathode 3 4 (2), respectively. 0 (2) equipped with electrophoresis tank 30 (1

) And 30 (2) contain a buffer solution 1 respectively.

In addition, in the two migration tanks 30 (1) and 30 (2), the side walls and bottom of the first partition 31, the second partition 33, and the migration tank 30, respectively. A barrier ¾ is formed with the wall. In the barrier chamber, the anode 3

A solution or paste-like barrier material 3 2 is held so as to surround 4 (1) or the cathode 3 4 (2). Further, the packing member 3 so as to close the space formed by the first partition wall 31 and the second partition wall 33.

6 is fitted

The three types of electrophoresis tanks have been described above with reference to FIGS. 1 to 3. The combination of the components shown in the figure is a chip-type electrophoresis method, a preparative electrophoresis method, a gas chromatography method, and the like. And can be advantageously applied to isoelectric focusing.

The present invention also resides in a sample analysis method using electrophoresis. The sample analysis method of the present invention is characterized by using the electrophoretic device of the present invention having the above-described configuration. The sample to be analyzed (also referred to as analyte or test sample) is not particularly limited as long as it can be separated, analyzed and identified by electrophoresis. Examples of suitable samples include biological samples such as biomolecules such as DNA, RNA, and protein. For example, DNA Examples of such samples include complex of protein and protein, artificial and natural pigments, powder suspension samples, and force-bonn nanotubes. Specific examples of the sample analysis method of the present invention are not limited to those listed below, but DNA (plasmid and PCR product) size specification and fractionation, DNA sequencing, protein separation, analysis and Includes taking. Since these analysis methods can be carried out in accordance with conventional methods, for details of the analysis methods, refer to detailed descriptions in technical books, patent documents, and the like.

As can be easily understood from the matters described in detail above and specifically explained in the following examples, according to the present invention, barrier substances are provided on the cathode side and the anode side of the portion where electrophoresis is performed. A barrier structure containing a single chamber is placed, and a buffer solution or a concentrated solution of electrophoresis supporting electrolyte is stably present in the barrier structure. It is possible to provide a mechanism for neutralizing substances, removing electrolysis products with strong oxidizing power and reducing power, and supplying a stable supporting electrolyte to the electrophoretic site. At the same time, by contriving the configuration and shape of the electrophoresis tank so that one barrier substance stays stably at the same site, convection of the buffer solution caused by the gas generated in the electrolysis reaction can be prevented at the same time. Example

EXAMPLES Hereinafter, although this invention is demonstrated with reference to the Example and comparative example, this invention is not limited by the following Example.

In the following example, the submarine type electrophoresis apparatus schematically shown in FIG. 4A and FIG. 4B or FIG. 5 has the same configuration as the electrophoresis apparatus described above with reference to FIG. Was used after making changes as necessary. That is, the electrophoresis apparatus shown in the figure is basically as follows. It has such a configuration.

The electrophoretic apparatus shown in FIGS. 4A and 4B includes an electrophoretic tank 40, and an anode 4 4 (1) and a cathode 4 made of platinum wire are provided at both ends of the bottom of the electrophoretic tank 40, respectively. 4 (2) is installed. The anode 4 4 (1) and the cathode 4 4 (2) are arranged on the electrode installation part 48 that is slightly elevated from the bottom of the electrophoresis tank 40 so as to face each other in parallel in the vertical direction. An output power source (not shown) is connected to the anode 4 4 (1) and the cathode 4 4 (2).

In order to form the barrier chamber of the present invention inside the electrophoresis tank 40, a first partition 4 1 having a beam structure and a second partition 4 3 having a sill structure are provided. Is attached. A barrier chamber is formed by the side wall and bottom wall of the electrophoresis tank 10. A barrier substance (not shown) is added to the barrier chamber. Here, the first partition wall 11 extending downward from the upper surface of the electrophoresis tank 10 opens at the lower part of the electrophoresis tank 10, and a sample is provided at a substantially central portion of the electrophoresis tank 40. A platform 46 for placing the gel-like separation medium 45 is provided.

The electrophoresis apparatus shown in Fig. 5 has a structure similar to that of the apparatus shown in Figs. 4A and 4B and has no beam on the side of the force cathode.

Using the electrophoresis apparatus shown in the figure, the experiment was conducted at normal temperature and under a constant voltage (Example 1 to Example 5: 100 Port; Example 6 to Example 7: 42 Port). The gel 45 used in Examples 1-5 was 2% agarose, the gel thickness was 6 mm, and the length was 6 cm. Gel 45 was allowed to stand on platform 46 as shown.

In Example 6 and Example 7, a small gel piece containing about 0.2 g of a polyacrylamide gel was suspended in a dialysis tube 1 cm in diameter and 1 cm in length filled with an electrophoresis buffer. The medium 5 5 in the state was left on the platform 5 6. The buffer for electrophoresis was fixed by pouring to a volume where the liquid surface reached 1.1 cm above the platform 46. During the experiment, it was confirmed by a level that the electrophoresis apparatus was placed horizontally.

In Examples 1 to 5, 5 mm from both ends of the platform 46 to the anode 44 (1) and cathode 44 (2), respectively, at predetermined times before and after the start of the electrophoresis experiment. In Example 6 and Example 7 where 0.5 mL of the buffer solution (test solution) was separated from a distance of 5 mm from the liquid surface, in the center of Platform 5 6 5 mm deep from the surface, or 5 mm deep from the liquid level immediately above the second partition wall (sill) 53 on the anode side, or from the same anode side and cathode side positions as in Examples 1 to 5 A buffer solution with a volume of 0.5 mL was collected.

The test solution collected at each position was kept warm under the same conditions of 25 ° C, and its pH value and conductivity (m S / cm) were measured.

Example 1 (Comparative example)

In this example, for comparison, an electrophoresis experiment was performed by omitting the use of a barrier chamber and a barrier substance. That is, the electrophoretic device used in this example is one in which the partitions 41 and 43 are removed from the device shown in FIGS. 4A and 4B. The dimensions of the electrophoresis apparatus were as follows.

Length of electrophoresis tank 40: 1 3 6. 6 mm, length b: 1 5 2. 8 mm, 1st partition (beam structure) 4 1 height c: 45 mm, 1st facing Distance between partition walls 4 1 d: 1 2 7 mm, distance from first partition wall 41 from side wall of electrophoresis tank 40: e 2.6 mm, platform 4 6 height g: 2 4 mm and width f: 60 mm.

The buffer used in this example is commonly called “1 χ ΤΑ Ε” and its composition And Tris-acetate: 4 O mM and EDTA: 1 mM. When the experiment was repeated for 0 to 60 minutes, the experimental results shown in Table 1 below were obtained. In the case of this example, it was observed that the difference in pH and conductivity between the cathode side and the anode side became remarkable as the time required for electrophoresis increased.

Table 1

Example 2

In this example, electrophoresis experiments were performed using a barrier chamber and a barrier substance. That is, the electrophoresis apparatus used in this example is the apparatus itself shown in FIGS. 4A and 4B, and there is no change in size etc. The buffer used in this example is the same as that used in Example 1. The composition was tris-acetate: 40 mM and EDTA: I mM. The barrier substance placed in the barrier chamber was used in the form of a solution with the following composition: Tris-acetate: 9 3 2mM, EDTA: 23.3mM and glycerol: 53% (V / V) It was. 8 mL each of this solution barrier was poured into the barrier chambers on the anode side and cathode side.

When the experiment was repeated for 0 to 180 minutes, the experimental results described in Table 2 below were obtained. In this example, compared to Example 1, It was observed to be stable for longer and up to 1 hour, with little difference in PH and conductivity between the cathode and anode sides.

Table 2

Example 3

The procedure described in Example 2 was repeated. In this example, the composition of the barrier material was changed as follows.

The buffer used in this example was the same as that used in Example 1, and its composition was tris-acetate: 4 O mM and ED TA: I mM. The barrier substance placed in the barrier chamber is used in the form of a solution. The composition is tris-acetate: 7 7 O mM, ED TA: 1 9.2 mM, glycerol: 3 8.5% (v / v ) And 1,3-butanediol: 14.4% (V / V). 8 mL of this solution barrier was poured into each of the anode chambers on the anode side and the cathode side.

When the experiment was repeated for 0 to 180 minutes, the experimental results shown in Table 3 below were obtained. In the case of this example, it was observed that the difference between pH and conductivity on the cathode side and the anode side was small and stable for up to 2 hours compared to Example 1 and Example 2. Table 3

Example 4 (Comparative example)

In this example, for comparison, an electrophoresis experiment was performed by omitting the use of one barrier chamber and one barrier substance. That is, the electrophoretic device used in this example is one in which the partitions 41 and 43 are removed from the device shown in FIGS. 4A and 4B. The dimensions of the electrophoresis apparatus are as described in Example 1.

The buffer used in this example is commonly called “0.5 × TB E”, and its composition was tris-porate: 44.5 mM and EDTA: 1 mM. When the experiment was repeated for 0 to 180 minutes, the experimental results shown in Table 4 below were obtained. In the case of this example, it was observed that the difference in pH between the cathode side and the anode side became remarkable as the time required for electrophoresis increased. Table 4

Example 5

In this example, an electrophoresis experiment was performed using a barrier chamber and a barrier substance. That is, the electrophoresis apparatus used in this example is the apparatus itself shown in FIGS. 4A and 4B, and there is no change in size etc. The buffer used in this example is the same as that used in Example 4. The composition was tris-porate: 44.5 mM and EDTA: 1 mM. The barrier substance placed in the barrier chamber is used in the form of a solution, the composition of which is tris-porate: 1 78 mM, EDTA: 4 mM, glycerol: 40% (V / V) and 1, 3 — Butanediol: 15% (v / v). 8 mL of this solution barrier was poured into each barrier chamber on the anode side and cathode side.

When the experiment was repeated for 0 to 180 minutes, the experimental results described in Table 5 below were obtained. In the case of this example, it was observed that the difference in conductivity between the cathode side and the anode side was stable up to about 3 hours. No changes were observed at pH. Table 5

Example 6 (Comparative example)

In this example, for comparison, an electroelution experiment, which is a type of electrophoretic movement, was performed without using a barrier substance. In other words, this example shows the result when no barrier substance is added to the anode in FIG. The dimensions of the electrophoresis apparatus are as described in Example 1.

The buffer used in this example was tris-hydrochloric acid, and its composition was tris: 37. 5 mM, and titrated with hydrochloric acid so that the pH was 8.9. When the experiment was repeated for 0 to 60 minutes, the results shown in Table 6 below were obtained. In this example, as the time required for electroelution increased, an increase in hypochlorous acid with strong oxidizing power was observed at the center of the platform and the upper part of the partition wall (sil) on the anode side.

Table 6

Example 7

In this example, an electroelution experiment, which is a type of electrophoresis, was performed using a barrier substance. That is, this example is the result in FIG. 5 with the barrier material added to the anode. The dimensions of the electrophoresis apparatus are as described in Example 1.

The buffer used in this example was tris-hydrochloric acid, the composition of which was tris: 37. 5 mM, and titrated with hydrochloric acid so that the pH was 8.9. 8 ml injection of 75% (w / w) glycerol solution (prepared with distilled water) containing 1.2% 5% (w / w) sulfurous acid that can reduce hypochlorous acid in the anode barrier chamber Added. When the experiment was repeated for 0 to 60 minutes, the results shown in Table 7 below were obtained. In this example, no strong hypochlorous acid was observed in the center of the platform and the upper part of the anode-side partition (sil) for up to 1 hour. In addition, no sulfurous acid leak from the barrier chamber was observed on the platform where electroelution was performed. Table 7

Industrial applicability

In the electrophoretic method, a potential gradient is formed, and the observation target is separated and analyzed by the driving force. Therefore, the stable conductivity at the electrophoresis site retained by the present invention leads to the stability and reproducibility of the analysis results. In addition, by removing the stable PH environment or electrolysis products with strong oxidizing power and reducing power at the electrophoresis site held by the present invention, the charged state of the observation object can be made constant, and the stability of the observation object This also leads to stability and reproducibility of the analysis results. Because of such remarkable effects, the present invention can be used widely and advantageously from the separation and analysis of various samples to fractionation.

Claims

1. A barrier substance used to suppress the influence of the electrolysis reaction caused in the vicinity of the electrode during electrophoresis, and contains acidic and / or alkaline substances generated by the electrolysis reaction in the vicinity of the electrode. Whether it is composed of compatible compounds or reacts with electrolysis products, or

Consists of a compound that can absorb this and reduce the amount of electrolysis products.

2. The electrophoretic barrier substance according to claim 1, further having a function of stably supplying the supporting electrolyte to the electrophoretic site. Surrounding

3. The barrier material for electrophoresis according to claim 1 or 2, wherein the barrier material is in the form of a solution, glue or gel.

4. The substance according to any one of claims 1 to 3, further comprising a substance capable of stabilizing the function of the barrier substance and / or a substance capable of displaying a state in which the function of the barrier substance is stabilized. Or the barrier substance for electrophoresis according to 1 above.

5. An electrophoretic barrier structure used to prevent direct contact between the electrode and a buffer in the electrophoresis tank in an electrophoresis apparatus having an electrode composed of an anode and a cathode,

The barrier substance for electrophoresis according to any one of claims 1 to 4,

Each comprising two barrier chambers holding the barrier material in a state where either one of the electrodes is in contact with the barrier material, and at least one partition wall defining the barrier chamber. An electrophoretic barrier structure characterized by the above.

6. The barrier structure for electrophoresis according to claim 5, wherein in the barrier chamber, the one substance of the barrier exposed from the partition wall is in contact with a buffer solution in the electrophoresis tank.

7. The partition wall is disposed at a position for preventing or suppressing convection of the buffer solution by a gas generated by an electrolysis reaction caused in the vicinity of the electrode during electrophoresis. A barrier structure for electrophoresis as described in 1.

8. The partition is vertically arranged in the form of a beam at a position close to the electrode, and has an opening at a lower end portion through which the barrier material can move, and the partition is further away from the electrode than the first partition. 2. A second partition wall arranged vertically in the form of a sill and having an upper end that terminates in a buffer solution so that the barrier substance and the buffer solution can come into contact with each other. The barrier structure for electrophoresis according to any one of 5 to 7.

9. The electrophoretic barrier structure according to claim 8, further comprising an elastic packing member having electrical conductivity between the first partition and the second partition. .

10. An electrophoresis apparatus comprising: an electrophoresis tank; an electrode comprising an anode and a cathode respectively disposed at predetermined positions of the electrophoresis tank; and a buffer contained in the electrophoresis tank,

An electrophoresis apparatus, further comprising the barrier structure for electrophoresis according to any one of claims 5 to 9.

1 1. Vertical Electrophoresis, Horizontal Electrophoresis, Submarin Electrophoresis, Cylindrical Electrophoresis, Capillary Electrophoresis, Chip Electrophoresis, Preparative Electrophoresis, One Electrochromatography The electrophoresis apparatus according to claim 10, which is based on pulse field electrophoresis or isoelectric focusing.

1 2. Based on the submarine type electrophoresis method, The electrophoresis apparatus according to claim 10 or 11, wherein the electrophoresis apparatus comprises a single electrophoresis tank, and the sample or the sample-containing medium is immersed in a buffer solution in the electrophoresis tank.

1 3. Based on vertical electrophoresis, columnar electrophoresis, or capillary electrophoresis, the electrophoresis tank is composed of two electrophoresis tanks arranged vertically apart from each other. The electrophoresis apparatus according to claim 10 or 11, wherein a buffer solution is placed in the electrophoresis tank, and a sample or a sample-containing medium is contained in a communication member that communicates with the electrophoresis tank.

14. A method for analyzing or separating a sample by an electrophoresis method, wherein the electrophoresis apparatus according to any one of claims 10 to 13 is used.

15. The sample analysis method according to claim 14, wherein the sample is a powder selected from the group consisting of biomolecules, dyes, and carbon nanotubes.