JP2004069430A - Electrophoresis chip, method for producing the same, and method for separating substances - Google Patents

Abstract

An electrophoresis chip which is inexpensive and easy to manufacture, a method for manufacturing the same, and a method for analyzing a substance are provided.
The electrophoresis chip includes a plate-like cover member, a plate-like intermediate member, and a plate-like base member, and the plate-like intermediate member is provided at a narrow groove-shaped through hole and at both ends thereof. And the flat cover member forms a liquid pool portion together with the liquid pool portion forming through hole provided in the flat intermediate member. It has through-holes 4a and 4b for forming a liquid pool, and each opening of the narrow groove-like through-hole is closed by a flat cover member and a flat base member to form a capillary. In the manufacturing method, the flat cover member, the flat intermediate member, and the flat base member are joined. The analysis method uses the electrophoresis chip.
[Selection diagram] Fig. 1

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to, for example, an electrophoresis chip that can be used for isoelectric focusing and a method for producing the same, an electrophoresis apparatus including the chip, and a method for separating substances.
[0002]
[Prior art]
The electrophoresis method is a method of separating a substance to be analyzed based on differences in charge, molecular weight, and the like, and is currently widely used for separation and analysis of biopolymers such as proteins and nucleic acids. Among them, the method called isoelectric focusing is a technique of separating and analyzing various charged substances by converging in a direction where the effective charge becomes zero. That is, it is an electroconvergent method. In the case of a mixture of substances having various kinds of dissociating groups, their effective charges move to a zero point and stop, respectively, and are called isoelectric focusing electrophoresis.
[0003]
In isoelectric focusing, in order to form a pH gradient in a separation medium, a mixture of a large number of compounds having slightly different pKa values from an acidic side to a basic side has conventionally been used as a wide-area buffer.
[0004]
In various electrophoresis, in order to suppress the influence of thermal convection generated by energization and to adjust resistance to molecular movement, various gel supports that are easily solidified, for example, agar, starch, or acrylamide are used. Have been. In conventional electrophoresis, it was difficult to perform detection (analysis) in real time while performing electrophoresis. Therefore, it was necessary to maintain a separated state until analysis was performed using a gel support.
[0005]
Subsequently, electrophoresis using a glass capillary having an inner diameter of 100 μm or less as an electrophoresis tank was developed. In this method, since a thin tubular capillary is used, the surface area is increased in comparison with the volume of the electrophoresis tank, thereby increasing the cooling efficiency, and without using a solid support, thermal convection even in a free solution. Can be suppressed, and a higher voltage can be applied.
[0006]
Also, the need to repeatedly use the same capillary has led to the use of additives that form sols that only provide high viscosity.
[0007]
In recent years, with the development of microchip chemistry, glass chips have started to be used instead of glass capillaries, which are smaller and easy to design electrophoresis vessels. For example, JP-A-2001-157855 discloses a flat substrate made of a polymer material [for example, polydimethylsiloxane (PDMS)] and a flat surface plate disposed on one surface of the substrate. A microchip for capillary gel electrophoresis having a two-layer structure is disclosed. On one surface of the substrate, a narrow groove forming a flow path of a predetermined shape is provided, and the narrow groove is sealed by the surface plate to form a capillary. The surface plate is provided with a pair of through holes as a liquid reservoir for introducing the electrophoresis medium and the sample into the capillary.
[0008]
The glass capillary is usually a tubular fused silica capillary having an inner diameter of 25 μm to 100 μm and a length of 10 cm or more, and its cross-sectional shape is circular, while the microchip easily changes its cross-sectional shape. can do. For example, the shape can be a quadrangle (including trapezoid), a triangle, a circle, or an ellipse. In particular, when the shape has a longer optical path length than a circle (for example, a deep rectangle), the measurement sensitivity can be improved. .
[0009]
Further, as compared with a glass capillary, a microchip has an advantage that the wall surface of the flow path can be easily processed. That is, since the fused silica capillary has only two open ends, internal processing is performed by, for example, a method of repeating etching and cleaning (Japanese Patent Application Laid-Open No. 7-151729). On the other hand, in the case of a microchip, when a fine groove is formed on a substrate, one surface is open, so that it is easy to perform surface processing inside the flow channel by laser processing or the like.
[0010]
In addition to these advantages, microchips have the advantage that electrophoresis can be completed in a short time due to the short flow path length, and it is easy to make multiple capillaries with multiple flow paths and high throughput. Is possible. Fused silica capillaries have low mechanical strength and are difficult to handle, so they are coated with a polyimide protective film, whereas microchips are easy to handle because microcapillaries are formed inside the substrate. Furthermore, in the case of a glass capillary, it is necessary to perform a washing operation and a filling of a separation medium for each analysis in order to use the glass capillary repeatedly, but a plastic microchip is inexpensive and therefore disposable. You can also.
[0011]
Here, Japanese Patent Application Laid-Open No. 2001-157855 describes that the manufacturing cost can be reduced because the microchip has a two-layer structure. However, in the microchip, since it is necessary to form a narrow groove on the surface of the substrate according to the shape of the capillary, a microchip with high reproducibility is manufactured with respect to the capillary shape that has the greatest influence on separation or analysis accuracy. This is not always advantageous in terms of manufacturing cost. Furthermore, since PDMS having high hydrophobicity is used, hydrophilic treatment such as plasma treatment is required.
[0012]
[Problems to be solved by the invention]
Therefore, an object of the present invention is to provide a microchip having high reproducibility with respect to the shape of a flow channel as a separation field (that is, a microchip having an accurate shape and size of a flow channel as designed), but at a very low cost. Another object of the present invention is to provide a disposable electrophoresis chip which is easy to manufacture and suitable for mass production.
[0013]
[Means for Solving the Problems]
The problem is solved by the present invention.
A flat base member, a flat intermediate member provided on one surface thereof, and a flat plate provided on the opposite surface of the flat intermediate member where the flat intermediate member comes into contact with the flat base member Including a cover member,
The plate-shaped intermediate member is provided at at least one narrow groove-shaped through hole penetrating in the thickness direction thereof, and a liquid provided at both ends of the narrow groove-shaped through hole, and penetrating in the thickness direction of the plate-shaped intermediate member. And a through hole for forming a reservoir,
A liquid reservoir penetrating in the thickness direction of the flat cover member, wherein the flat cover member forms a liquid pool portion together with the liquid reservoir forming through hole provided in the flat intermediate member. Having a through hole for forming a part,
The narrow groove-shaped through hole and the liquid reservoir forming through hole provided in the plate-shaped intermediate member are in contact with the plate-shaped base member, one opening of each of which is sealed,
The other opening of the narrow groove-shaped through hole provided in the flat intermediate member is sealed by contact with the flat cover member.
The above problem can be solved by the above-mentioned electrophoresis chip.
The present invention also provides (1) at least one narrow groove-shaped through hole penetrating in the thickness direction, and a liquid pool portion formed at both ends of the narrow groove-shaped through hole and penetrating in the thickness direction. (2) a liquid that penetrates in the thickness direction and that can form a liquid reservoir together with the liquid reservoir forming through-hole provided in the planar intermediate member. A plate-shaped cover member having a through hole for forming a reservoir portion, and (3) a plate-shaped base member,
The flat plate-shaped cover member is in contact with one surface of the flat-shaped intermediate member, and the flat-plate base member is joined so as to be in contact with the other surface of the flat-shaped intermediate member. And a method for producing the electrophoresis chip.
The present invention also relates to an electrophoresis device including the electrophoresis chip.
Furthermore, the present invention relates to a method for separating a substance, characterized by using the electrophoresis chip or the electrophoresis apparatus.
[0014]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention will be described in detail with reference to the accompanying drawings.
FIG. 1 shows one embodiment of the electrophoresis chip 11 according to the present invention in a state where it is separated and separated into respective members, that is, a flat cover member 1, a flat intermediate member 2, and a flat base member 3. It is an exploded perspective view. FIG. 2 is a cross-sectional view of one embodiment of the electrophoresis chip 11 according to the present invention shown in FIG. In this specification, a flat cover member, a flat intermediate member, a flat base member, a reinforcing flat member, and the like may be collectively referred to as a flat member.
[0015]
The electrophoresis chip 11 of the present invention includes, for example, as shown in FIGS. 1 and 2, a flat cover member 1, a flat intermediate member 2, and a flat base member 3 stacked in three layers.
The flat cover member 1 is provided with a pair of through-holes 4a, 4b for forming a liquid reservoir portion penetrating in the thickness direction, and the inner walls of the through-holes 4a, 4b for forming the liquid reservoir portion have: Electrodes 7a and 7b are provided, respectively. Electric circuits 8a and 8b are further connected to the electrodes 7a and 7b.
The plate-like intermediate member 2 has a pair of through-holes 5a and 5b for forming a liquid reservoir portion penetrating in the thickness direction, and the through-holes 5a and 5b for penetrating in the thickness direction and forming the liquid reservoir portion. A narrow groove-shaped through hole 6 to be connected is provided.
[0016]
In the electrophoresis chip 11 of the present invention, the plate-like cover member 1, the plate-like intermediate member 2, and the plate-like base member 3 are stacked in three layers to form a through-hole for forming a liquid reservoir portion provided in the plate-like cover member. One (for example, 4a), one of the through holes for forming a liquid reservoir portion provided in the plate-like intermediate member (for example, 5a), the narrow groove-like through hole 6, and the through hole for forming a liquid reservoir portion provided in the plate-like intermediate member. A continuous open (non-closed) space consisting of one of the remaining holes (for example, 5b) and the other of the through holes for forming a liquid reservoir formed in the flat cover member (for example, 4b) is formed. Perform capillary electrophoresis using. More specifically, the narrow groove-shaped through-holes are formed on one surface of the flat cover member (that is, the surface in contact with the flat intermediate member) and one surface of the flat base member (that is, the flat plate member). Together with the intermediate member) to form a flow path, and a through hole for forming a liquid reservoir formed in the flat cover member and the flat intermediate member is formed by a flat base member. Together with one of the surfaces (i.e., the surface in contact with the flat intermediate member) to form a liquid reservoir.
[0017]
The material of the plate member in the electrophoresis chip of the present invention is appropriately selected from transparent, translucent, or opaque materials in consideration of a detection method after electrophoresis, for example, detection by absorption or fluorescence. can do.
From the viewpoint of improving reproducibility, a material that can be molded in a mold, for example, a castable glass, a thermosetting resin, or a thermoplastic resin, in that a flat member having the same shape can be easily and inexpensively manufactured. It is preferable to use a resin or the like. It is more preferable to use a resin material because of its insulating property, freedom of molding, and the ability to keep the electroosmotic flow low. In particular, a thermoplastic resin material is effective from the viewpoint of productivity, for example, acrylic resin (for example, polymethyl methacrylate or polyacrylate), styrene resin (for example, polystyrene or styrene copolymer), polycarbonate, polyamide resin A resin (for example, nylon 6 or nylon 66), a polyester resin (for example, polyethylene terephthalate), an epoxy resin (for example, bisphenol A type epoxy resin), or a polyvinyl chloride resin is preferable.
[0018]
Above all, in terms of transparency and fluorescent characteristics, an acrylic resin or a styrene resin is more preferable, and polymethyl methacrylate or polystyrene is more preferable.
Also, there is no need for mechanical processing equipment.Since a mold is made of silicon resin and the like, and a liquid epoxy resin is injected and cured, chips can be easily manufactured. Epoxy resins are preferred from the standpoint of easy basic examination. In the case of an epoxy-based resin, for example, by mixing a pigment or a fluorescent substance with the liquid epoxy resin before curing, a flat member that does not transmit visible light and a flat member that emits fluorescence can be manufactured. It is also preferable in that it is possible.
[0019]
Further, since the chip for electrophoresis of the present invention is intended to be used as a disposable product, it is preferable to use a biodegradable plastic from such a viewpoint. Examples of biodegradable plastics include starch-based polymers, cellulose ester-based polymers, aliphatic polyester-based polymers, polylactic acid-based polymers, and microbial polyesters (eg, polyhydroxybutyrate / valerate). it can.
[0020]
In the electrophoresis chip of the present invention, the plate-like intermediate member can be formed from a material through which light (for example, visible light or ultraviolet (UV)) does not pass.
For example, when analyzing a substance to be separated based on absorption of visible light, the plate-shaped intermediate member can be formed from an opaque material having a light-shielding effect. In the case where a substance to be separated is analyzed based on UV absorption, the plate-shaped intermediate member can be formed from a material having a UV absorption effect (for example, epoxy resin).
When the plate-like intermediate member is formed of a material that does not transmit light, the narrow groove-shaped through-hole can have a function as a slit. Therefore, the effect of stray light from the surroundings is reduced, and the detection sensitivity of the target substance is increased. be able to.
[0021]
In a known microchip for electrophoresis having a two-layer structure (for example, a microchip described in JP-A-2001-157855), in order to impart such a stray light blocking effect, for example, a migration tank portion Other than the above, a light shielding means such as coloring or covering with a cover can be considered, but all of them require fine processing technology with high precision. On the other hand, in the electrophoresis chip of the present invention, by forming only the plate-shaped intermediate member from a material that does not transmit light, a stray light blocking effect can be easily imparted. That is, since the electrophoresis tank (flow path) and the electrode tank (liquid pool) are formed as through holes in the plate-shaped intermediate member, it is easy to use a member having a light-shielding effect only in the plate-shaped intermediate member. In addition, it is possible to impart a function as a slit to the electrophoresis tank.
[0022]
Further, in the electrophoresis chip of the present invention, one of the flat cover member and the flat base member can be formed from a material containing a substance that emits light by ultraviolet irradiation. Examples of the substance that emits light by irradiation with ultraviolet light include a substance that emits fluorescence by irradiation with ultraviolet light and a substance that emits phosphorescence by irradiation with ultraviolet light.
Examples of the substance that emits fluorescence by ultraviolet irradiation include a coumarin fluorescent dye, a pyridine fluorescent dye, and a rhodamine fluorescent dye.
Examples of the substance that emits phosphorescence by ultraviolet irradiation include CaS: Bi (purple blue light emission), CaSrS: Bi (blue light emission), ZnS: Cu (green light emission), and ZnCds: Cu (yellow to orange light emission). Can be mentioned. Further, a lanthanoid metal (eg, europium, samarium, terbium, or the like), and an alkaline earth metal aluminate activated with a lanthanoid metal (eg, europium, samarium, terbium, or the like), and the like can be given. .
[0023]
For example, in the case where an ultraviolet light source is provided on the flat cover member side and ultraviolet light is irradiated from the flat cover member side, the flat base member is formed from a material containing a substance that emits light by ultraviolet radiation. The absorbing material can be analyzed (detected or measured). Further, when the ultraviolet light source is provided on the flat base member side and the ultraviolet light is irradiated from the flat base member side, the flat cover member is formed from a material containing a substance which emits light by ultraviolet irradiation. The absorbent can be analyzed.
[0024]
The shape of the flat member is not particularly limited as long as it is flat, and can be appropriately determined depending on the electrophoresis apparatus to be used. For example, a square (for example, a rectangle or a square) or a circle Can be mentioned.
The size of the flat member is, for example, easy to handle, or, depending on the electrophoresis apparatus to be used, can be appropriately determined, for example, in the case of a square shape, so as to be easy to handle with one hand, The size is preferably about 10 mm square to 150 mm square, more preferably 10 mm square to 100 mm square, and further preferably 20 mm square to 50 mm square from the viewpoint of miniaturization of the electrophoresis apparatus.
[0025]
Among the flat members, the shape of the flat cover member is such that when the flat cover member and the flat intermediate member are joined, the flow path forms the opening of the narrow groove-shaped through hole provided in the flat intermediate member. The shape is not particularly limited as long as the shape can be sealed as described above, and examples thereof include a plate shape and a film shape.
Although the thickness of the flat cover member is not particularly limited, for example, it is preferably about 0.05 mm to 5 mm, and more preferably 0.05 mm to 1 mm, from the viewpoint of moldability or handleability.
[0026]
Among the flat members, the shape of the flat intermediate member is not particularly limited, and examples thereof include a plate shape and a film shape. From the viewpoint of accuracy, a plate having a certain thickness is preferable, preferably 5 to 1000 μm, more preferably 5 to 500 μm, and still more preferably 10 μm to 100 μm.
[0027]
Further, among the flat members, the shape of the flat base member is not particularly limited, and may be, for example, a plate or a film. Although the thickness of the flat base member is not particularly limited, for example, from the viewpoint of moldability or handleability, it is preferably about 0.05 mm to 5 mm, more preferably 0.05 mm to 1 mm.
[0028]
As a molding method of the flat member, for example, a method of molding by injection molding, injection molding, or press molding using a mold, or a method of molding by laser processing or mechanical processing, or a method of forming into a film shape (For example, inflation molding, calender molding, die extrusion molding, etc.) and the like, and a method of forming a film is preferable. In the case of molding into a plate shape, a method using a mold is preferable because a material having high reproducibility in both dimensions and shape can be obtained. Further, laser processing or mechanical processing is preferable from an economic viewpoint.
In addition, a commercially available material formed into a film can be used as the flat base member, or a film with an adhesive can be used when precision is not particularly required.
[0029]
The through-holes 4a and 4b for forming a liquid pool formed in the flat cover member 1 or the through-holes 5a and 5b for forming a liquid pool formed in the flat intermediate member 2 are, for example, buffer buffers for electrophoresis. And / or liquid reservoirs 9a and 9b for supplying or discharging a separation medium containing a polymer for molecular sieving or a sample liquid containing a substance to be separated, and an electrode tank for electrophoresis. It is necessary that two or more holes are formed so as to penetrate in the plate thickness direction. Further, it is necessary to provide the through-hole for forming the liquid pool portion at a position that does not reach the periphery of the plate-shaped cover member or the plate-shaped intermediate member.
The size of the through-hole for forming the liquid reservoir portion provided in the flat cover member or the flat intermediate member is not particularly limited as long as the separation medium and the sample liquid can be injected and discharged. From the viewpoint of the injection operation, the inner diameter is preferably set in the range of 0.5 to 10 mm, more preferably 1 to 5 mm.
[0030]
Also, as for the shape, the through-hole for forming the liquid pool portion provided in the flat cover member and the through-hole for forming the liquid pool portion provided in the flat intermediate member can form one continuous liquid pool portion. Moreover, the shape is not particularly limited as long as it has a shape capable of injecting and discharging the separation medium and the sample solution. For example, a columnar shape (including an elliptical columnar shape) or a rectangular columnar shape having a constant cross-sectional shape may be used. A truncated cone (including an elliptical truncated cone) or a truncated pyramid in which the area of the cross-sectional shape continuously increases or decreases, or a columnar shape in which the area of the cross-sectional shape discontinuously decreases and / or increases is given. it can.
In addition, the cross-sectional shape and the size of the through-hole for forming the liquid pool portion provided in the flat cover member and the through-hole for forming the liquid pool portion provided in the flat intermediate member may be the same, or one of the through holes may be one. Either or both of the cross-sectional shape and size may be different as long as a continuous liquid reservoir can be formed. It is preferable that the cross-sectional shape and the size are the same in that the injection and the discharge of the separation medium or the sample solution are facilitated.
[0031]
The narrow groove-shaped through-hole 6 which is a narrow groove formed in the plate-shaped intermediate member 2 is, for example, a narrow groove serving as a flow path for introducing or separating a separation medium or a sample liquid. Thus, it is necessary that the through holes 5a and 5b formed in the plate-shaped intermediate member 2 are formed so as to connect the through holes 5a and 5b. The narrow groove-shaped through-holes need to be provided at positions that do not reach the periphery of the plate-shaped intermediate member, and the narrow groove-shaped through-holes do not intersect.
The shape of the narrow groove-shaped through-hole can be arbitrarily designed depending on, for example, the accuracy or form of the analysis, and may be, for example, a linear shape, a loop shape, a V shape, or a complicatedly bent shape, or a combination thereof. can do. The cross-sectional shape is not particularly limited, and may be, for example, a triangle, a quadrangle (for example, a rectangle, a trapezoid, or a square), a circle, a U-shape, or a V-shape.
[0032]
The size of the narrow groove-shaped through-hole is not particularly limited as long as the substance to be separated can be separated, and can be arbitrarily designed. For example, from the viewpoint of handleability, moldability, or miniaturization of the electrophoresis apparatus, the width of the narrow groove-shaped through-hole is preferably from 10 to 2000 μm, more preferably from 20 to 1000 μm, and from 30 μm to More preferably, it is 500 μm. The depth of the narrow groove-shaped through-hole (that is, the thickness of the middle flat plate) is preferably 5 to 1000 μm, more preferably 5 to 500 μm, and still more preferably 10 μm to 100 μm. The length of the narrow groove-shaped through-hole is preferably about 5 mm to 150 mm, more preferably 5 mm to 20 mm, and still more preferably 5 to 10 mm.
Note that, depending on the shape and size of the narrow groove-shaped through-hole and the through-hole for forming the liquid reservoir, the through-hole for forming the narrow groove and the through-hole for forming the liquid reservoir may be unified, and the boundary may not be clear. Yes, such an embodiment is also included in the scope of the present invention. As such an embodiment, for example, there is a case where the shape of the through-hole for forming the liquid pool portion is a prismatic shape, and the inner diameter of the through-hole for forming the liquid pool portion matches the width of the narrow groove-shaped through hole. Can be.
[0033]
In the electrophoresis chip of the present invention, the number of narrow groove-shaped through holes provided in the flat intermediate member is not particularly limited, and may be one as shown in FIGS. 1 and 2, As shown in FIGS. 6 to 8, the number may be two or more. When a plurality of narrow groove-shaped through holes are provided, it is possible to simultaneously separate a plurality of substances to be separated. When a plurality of narrow groove-shaped through holes are provided, it is preferable that they are formed in parallel and arranged substantially in parallel from the viewpoint of analysis sensitivity.
[0034]
Also, the number of through-holes for forming a liquid pool portion provided in the flat intermediate member and the flat cover member is different from the number of through-holes for forming the liquid pool portion provided in the flat intermediate member and the number of through-holes formed in the flat cover member. There is no particular limitation as long as the number of through-holes is the same, and each is two or more. In the electrophoresis chip of the present invention, each of the liquid reservoir forming through holes provided in the plate-shaped intermediate member is provided in at least one other plate-shaped intermediate member through at least one narrow groove-shaped through hole. Is connected to the through hole for forming the liquid reservoir. As a combination of the through-hole for forming the liquid reservoir portion and the through-hole having a narrow groove provided in the plate-like intermediate member, various modes can be given as shown in FIG. 1 or FIGS.
[0035]
For example,
As shown in FIG. 1, a combination of one narrow groove-shaped through hole 6 and two through holes 5a and 5b for forming a liquid reservoir connected to both ends thereof;
As shown in FIG. 6, a plurality of (for example, three in FIG. 6) narrow groove-shaped through-holes 61a, 61b, and 61c, and a plurality of (for example, FIG. (In total, six) through holes 51a, 51b, 51c, 51d, 51e, 51f in combination with the liquid reservoir forming holes;
As shown in FIG. 7, two pairs of through-holes 52a and 52b for forming a liquid reservoir and a plurality of (for example, three in FIG. 7) narrow groove-like through-holes 62a, 62b and 62c connecting the through-holes are formed. combination;
As shown in FIG. 8, a plurality of (for example, three in FIG. 8) narrow groove-shaped through holes 63a, 63b, and 63c, and a plurality (for example, four in FIG. 8 in total) of through-holes for forming a liquid reservoir portion. And one end of each of the narrow groove-shaped through holes is connected to one and the same through hole 53a for forming a liquid pool portion, and the other end portion is formed of a separate liquid pool. Combination to connect with the through holes 53b, 53c, 53d for forming the part
And the like.
[0036]
When a plurality of different sample liquids are simultaneously separated, it is preferable that the liquid reservoirs for supplying and discharging the sample liquid are separately prepared for each sample liquid. It is preferable that two or more narrow groove-shaped through-holes that also serve as a liquid reservoir are formed with independent through-holes for forming a liquid reservoir portion at both ends (for example, an embodiment shown in FIG. 6).
[0037]
The method for forming the through hole for forming the liquid reservoir and the through hole having a narrow groove shape is not particularly limited, and they may be formed separately (in this case, either may be formed first). Although it is possible, a method of forming both at the same time by one molding is simple and preferable because the number of steps is reduced. For example, a method of performing injection molding, injection molding, or press molding using a metal mold is preferable because a product having high productivity and having high reproducibility in both dimensions and shape can be obtained.
[0038]
The electrodes 7a and 7b formed on the flat cover member 1 are used to separate a sample by moving a flow path formed by the narrow groove-shaped through-holes 6 by applying a voltage and moving a sample to be separated by a potential difference. The liquid reservoirs at both ends of the introduction flow path and the separation flow path of the sample liquid or the separation medium, that is, the inner wall of the through hole for forming the liquid reservoir formed in the flat cover member, or the inner wall and the vicinity thereof Can be formed.
In the electrophoresis chip of the present invention, when mechanical strength (for example, rigidity) is required, the surface of the flat cover member 1 opposite to the surface in contact with the flat intermediate member, or A reinforcing flat member can be provided on one or both surfaces of the flat base member 3 opposite to the surface in contact with the flat intermediate member. In the case where the reinforcing flat member is provided on the flat cover member side, the through holes corresponding to each of the through holes for forming the liquid pool portion provided on the flat cover member (that is, the liquid pool provided on the flat cover member). It is necessary to provide a through-hole capable of forming a liquid pool together with the through-hole for forming a portion) also in the reinforcing plate-shaped member. When the reinforcing flat member is provided on the flat cover member side, an electrode can be provided on the surface of the reinforcing flat member.
[0039]
In the electrophoresis chip of the present invention, from the viewpoint of moldability or handleability, the electrode is formed as a through hole for forming a liquid pool portion of the flat cover member (when a reinforcing flat member is provided on the flat cover member side). Is preferably formed on the inner wall of the through-hole provided in the reinforcing flat member and its peripheral portion.
[0040]
The voltage can be applied to the electrode by using a wiring of platinum or the like from a high voltage supply source (for example, a battery or a power supply), but from the viewpoint of simplicity of operation, a flat cover member ( When a reinforcing flat member is provided on the flat cover member side, an electric circuit (for example, a conductive wire, conductive ink, or conductive paste, etc.) is further formed on the surface of the reinforcing flat member. Is preferred. The electric circuit needs to be formed close to the electrodes.
In the electrophoresis chip of the present invention, the flat cover member and the flat base member are bonded to the narrow groove-shaped through-hole forming surface of the flat intermediate member. In some cases, completeness occurs, and the problem of liquid leakage is likely to occur. Therefore, it is preferable that the electric circuit is formed on the surface of the plate-shaped cover member on the side opposite to the surface on which the narrow groove-shaped through holes are formed.
[0041]
The method for forming the electrodes and the electric circuit is not particularly limited, and various conventionally known methods, for example, a plating method, a printing method, a vapor deposition method, and an adhesion method can be applied. When the flat cover member (the reinforcing flat plate member is provided on the flat cover member side) is a transparent resin, the plating method is difficult to handle in terms of protection against a chemical solution and the like. From the viewpoint of productivity, a printing method or a vapor deposition method is preferable. In particular, in the case of forming on the inner wall of the through hole for forming a liquid reservoir, a vapor deposition method, for example, vacuum vapor deposition, sputtering, or ion plating is preferable.
[0042]
The electrode and the electric circuit can also be formed as an integrated product in which both are integrated. The material of the electrode and the electric circuit is not particularly limited as long as it is a conductive material, and examples thereof include gold, silver, copper, platinum, aluminum, carbon, and stainless steel.
The material of the electrode is a material having good corrosion resistance, such as gold, silver, platinum, carbon or Stainless steel or the like is preferred. Considering that the device is a disposable device, carbon or stainless steel is preferable from the viewpoint of economy.
Further, the material of the electric circuit is preferably copper or aluminum from the viewpoint of cost and ease of use. When the flat cover member is made of resin, a silver paste or the like using a resin binder having good adhesion to the resin is preferable.
[0043]
The thicknesses of the electrodes and the electric circuit are not particularly limited as long as they do not hinder energization. For example, in the case of a conductive film formed by a printing method, the thickness is preferably 1 to 100 μm, more preferably 5 μm to 50 μm. In the case of a metal film for sputtering or ion plating, the thickness is preferably 0.005 μm to 20 μm, and more preferably 0.01 μm to 5 μm. In the case of bonding a stainless steel film, the thickness is preferably 200 μm or less.
The width of the electric circuit is preferably 0.1 mm to 20 mm, more preferably 0.5 mm to 10 mm, and still more preferably 1 to 5 mm. The width of the electrode is preferably 1 mm to 2 mm.
[0044]
In the electrophoresis chip of the present invention, electrodes can be provided on the chip itself, or no electrodes can be provided on the chip itself. When electrodes are formed on the flat cover member (or when the reinforcing flat plate member is provided on the flat cover member side), the electrodes can be easily electrophoresed without performing the conventional cumbersome electrode connection work. This is preferable because the electrode on the chip for use can be brought into contact with the wiring. Further, when an electric circuit is formed in the flat cover member (in the case where a reinforcing flat member is provided on the flat cover member side, an electric circuit is formed), the wiring is further simplified, This is more preferable because the wiring does not directly touch the sample solution, so that washing for each analysis is not required.
When electrodes are not provided on the chip itself, for example, electrophoresis can be performed by providing an electrode for electrophoresis in a device for electrophoresis that can be inserted into the liquid reservoir of the chip for electrophoresis of the present invention.
[0045]
In the electrophoresis chip of the present invention, for example, a separation medium containing, for example, an electrophoresis buffer and / or a polymer for molecular sieving is preliminarily provided in a channel formed from the narrow groove-shaped through-hole of the plate-shaped intermediate member. Can be filled.
As the support for the separation medium, for example, a polymer gel such as agar, starch, acrylamide, or agarose can be used.
[0046]
Further, the separation medium may contain, for example, a hydrophilic polymer compound capable of forming a sol, or a low molecular compound capable of imparting high viscosity.
Examples of the sol-formable hydrophilic polymer compound include, for example, linear polymers (for example, linear polyacrylamide, linear hydroxyethylcellulose, linear hydroxypropylmethylcellulose, linear hydroxypropylcellulose, Examples thereof include straight-chain methylcellulose, straight-chain polyethylene glycol, straight-chain polyethylene oxide, and polyvinyl alcohol), and natural polymers (for example, mannan, curdlan, cellulose, or agarose). When agarose is used as a hydrophilic polymer compound capable of forming a sol, the agarose is used at a concentration lower than the gelling concentration.
Examples of the low-molecular compound capable of imparting high viscosity include urea or derivatives thereof, polyhydric alcohols (eg, glycerin), and oligosaccharides (eg, cellobiose).
[0047]
In the method for producing an electrophoresis chip according to the present invention, a flat cover member having a through hole for forming a liquid reservoir, a flat intermediate member having a through hole for forming a narrow groove and a through hole for forming a liquid reservoir, and a flat plate The base member is joined in this arrangement order in a state of being superposed in three layers. As a method of joining these flat members, the opening of the narrow groove-shaped through hole of the flat intermediate member is closed by the flat cover member and the flat base member to form a flow path, and In particular, if the liquid reservoir portion forming through hole of the cover member is connected to the liquid reservoir portion forming through hole of the plate-shaped intermediate member, and further closed by the plate-shaped base member to form a liquid reservoir portion, Although there is no limitation, for example, a method of press-contacting the surfaces facing each other mechanically, a method of bonding using an adhesive, a method of heat-sealing a flat member, and the like can be given.
[0048]
In the case where the flat intermediate member is thin and has elasticity, for example, the flat intermediate member is pressed and fixed by the flat cover member and the flat base member, or the flat intermediate member is displaced (compressed). A tight seal around the narrow groove-shaped through-hole can be easily formed by a method such as fixing in a state.
When joining the plate-shaped intermediate member and the plate-shaped cover member by bonding, there is a concern that the adhesive may flow in depending on the size of the narrow groove-shaped through-hole, and the conduit may be lost or greatly changed. Special care must be taken for application.
Since the plate-shaped intermediate member has both the through hole for forming a liquid reservoir portion and the through hole having a narrow groove shape, a microchip in which a conventionally known member having a groove and a member having a liquid reservoir hole are bonded together (for example, Compared with the microchip described in JP-A-2001-157855, the trouble of adjusting the position of the groove and the hole can be omitted. In the manufacturing method of the present invention, the positioning at the time of joining the plate-shaped intermediate member and the plate-shaped cover member does not need to be so precise, so that the work is easy and the design and selection with high flexibility are possible. . In addition, there is an advantage that reproducibility is high and analysis accuracy is improved.
[0049]
When the flat intermediate member and the flat base member are joined by heat fusion, it is preferable to use a film-shaped sealing member as the flat base member. The film can be heat-sealed with a small amount of heat by making it thin, and the small amount of heat hardly causes deformation of the narrow groove-shaped through-hole. Further, from the viewpoint of noise reduction at the time of detection by fluorescence or UV absorption, the film is preferably used. Even when the narrow groove-shaped through-hole is a fine groove, a film-shaped sealing member is suitable.
[0050]
In the method for producing an electrophoresis chip according to the present invention, when the electrophoresis chip has an electrode (and further, an electric circuit if desired), the present invention is not limited thereto. Can be mentioned. For example, a method in which a flat cover member to which electrodes (and further an electric circuit is attached, if necessary) are attached and a flat intermediate member, and a flat base member is bonded thereto, a flat base member and a flat intermediate member, A flat cover member formed with electrodes (and, if desired, an electric circuit) in accordance with the through-hole for forming the liquid pool portion of the flat intermediate member, or a flat cover member, a flat plate, or the like. After joining the plate-shaped intermediate member and the plate-shaped base member, a method of forming an electrode (and further, an electric circuit if desired) on the surface opposite to the joint surface of the plate-shaped cover member can be used. Here, the formation of the electric circuit is preferably performed as the last step after joining the plate members and attaching the electrodes from the viewpoint of circuit protection.
[0051]
The device for electrophoresis of the present invention is not particularly limited as long as it includes the chip for electrophoresis of the present invention.In addition to the chip for electrophoresis of the present invention, for example, the separation flow of a sample containing a substance to be separated is performed. A means for applying a voltage to an electrode (and, if desired, an electric circuit) for applying a potential difference to both ends of the path to cause the separation target substance to electrophorese, a means for irradiating the separation target substance with light, and a separation target substance Means for analyzing the detection light from the detector or means for positioning the electrophoresis chip. In this specification, “analysis” includes “measurement” for quantitatively or semi-quantitatively determining the amount of an analyte (for example, detection light) and “measurement” for determining the presence of an analyte (for example, detection light). Both "detection" for determining the presence or absence are included.
[0052]
One embodiment of the electrophoresis device of the present invention is schematically shown in FIG. An electrophoresis apparatus 10 of the present invention shown in FIG. 3 includes an electrophoresis chip 11 of the present invention, a stage 12 for arranging the electrophoresis chip, a light source 13 provided below the stage, It includes a digital still camera 14 and a close-up lens 15 provided above the migration chip.
[0053]
The means for applying a voltage is, for example, a means including a power source (for example, a battery or a power supply) for generating a voltage and a wiring. From the viewpoint of miniaturizing the apparatus, the electrophoretic device of the present invention is used. It is preferred to be integrated into the device for use. Alternatively, the power supply and the electrophoresis apparatus of the present invention may be separately prepared without being integrated.
[0054]
The means for irradiating the analysis target substance with light is not particularly limited as long as it has at least a light source for generating light, and a light condensing means for efficiently irradiating light emitted from the light source. Preferably, it further comprises means. This part is usually not disposable. However, considering the purpose of the device of the present invention, a device not large-scale is desirable.
The performance of the light source is not particularly limited, and examples thereof include a mercury lamp, a QI lamp (quartz-iodine lamp), a photodiode, a light emitting diode (LED), and an EL (electroluminescence). Further, a laser light source can also be used.
[0055]
The light collecting means is not particularly limited, and examples thereof include a dichroic mirror, an optical filter, a lens (for example, an objective lens, a prism lens, or a microlens), and an optical fiber. They can be used in combination.
When a lens is used as the light condensing means, either a spherical lens or an aspherical lens can be used.However, from the viewpoint of easy focusing, when the light condensing area is small, an aspherical lens is preferable. One or two or more lenses can be used depending on the condensing area.
When irradiating with excitation light, it is preferable to use a dichroic mirror or an optical filter.
When a laser light source is used, it is preferable to provide a plurality of prism lenses and enlarge the dot-like laser light source so as to include all scanning lines.
[0056]
The means for analyzing the detection light from the substance to be analyzed is not particularly limited as long as it has at least a photodetector.From the viewpoint of measurement sensitivity, it may further include a light condensing means. preferable. When the excitation light is applied, the light to be detected becomes fluorescent light.
The photodetector is not particularly limited, and examples thereof include a fluorescence detector, a photomultiplier (photomultiplier), a CCD, and a photodiode. There is no particular limitation on the light condensing means, but examples thereof include a dichroic mirror, an optical filter, and a lens (for example, a spherical or aspherical lens, a microlens, or the like). The detection light analyzing means which can be used most simply is, for example, a combination of a commercially available digital camera and a close-up device, or a combination of a microscope and a digital camera.
[0057]
The means for positioning the electrophoresis chip is not particularly limited. For example, a positioning shape designed to match the shape of the electrophoresis chip (for example, a protrusion, a dent, a hole, or a pin), Examples thereof include a coil and a spring-like substance. One of these may be used alone, or a plurality of them may be provided, or a plurality of kinds of means may be provided in parallel, and they can be used as needed. However, they do not need to be precise, and a register line may be attached to a part of the electrophoresis tank.
[0058]
The electrophoresis apparatus of the present invention may include a means for moving the electrophoresis chip so that the analysis can be performed by moving the electrophoresis chip. Further, a moving unit may be provided in a unit for irradiating light and / or a unit for measuring detection light so that the positions of the light irradiation unit and the detection unit can be changed. An analyzer for analyzing the measured detection light can be integrated inside the device, or can be provided connected to the outside.
[0059]
It is preferable that the chip for electrophoresis of the present invention is simply set and positioned on the apparatus for electrophoresis. The electrophoresis chip is usually positioned by bringing the end face of the flat cover member into contact with the positioning means of the electrophoresis apparatus. In order to fix the electrophoresis chip so that it does not move during measurement, it is preferable to have a positioning means not only on the electrophoresis device side but also on the electrophoresis chip side. In this case, it is effective that the shape of the positioning means of the electrophoresis apparatus matches the shape of the positioning means of the electrophoresis chip. The means for positioning the electrophoresis chip is not particularly limited. For example, the plate-like cover member has at least one of a protrusion, a hole, and a hole for positioning. Is preferred. The size of the projection, dent, or hole can be arbitrarily designed, but in the case of a single shape, for example, an inner diameter of about 1 to 5 mm, and in the case of a linear shape, a width of 1 to 1 It is preferably about 3 mm.
[0060]
In the separation method of the present invention, the electrophoresis chip of the present invention or the electrophoresis apparatus of the present invention is used. The substance to which the separation method of the present invention can be applied is not particularly limited as long as it is a substance that can be electrophoresed, and is applicable to both charged substances and electrically neutral substances. can do.
[0061]
Examples of the chargeable substance include a chargeable compound and a chargeable particle. Examples of the charged compound include a charged low-molecular compound (for example, a low-molecular ion, an organic acid, or an amino acid) or a charged high-molecular compound (for example, a protein, a nucleic acid, or a sugar). it can. Examples of the charged particles include biological particles (eg, viruses or cells), natural or synthetic polymer compound particles (eg, latex, cross-linked dextran, or cellulose, etc.), or inorganic compound particles [eg, silica gel. (Or aerosil), colloidal gold, pigments and the like].
[0062]
Examples of the electrically neutral substance include, for example, alcohols and esters. As a method for separating an electrically neutral substance, for example, micellar electrokinetic chromatography can be mentioned. By adding an ionic surfactant micelle to the electrophoresis solution and performing electrophoresis, an electrically neutral substance can be obtained. Sexual substances can be separated.
[0063]
Further, according to the separation method of the present invention, it can be applied to separation of latex beads sensitized with a protein. Specifically, for example, a separation medium (for example, a separation buffer, a separation polymer gel, and / or a separation electroconvergence buffer, etc.) is provided in the flow path of the electrophoresis chip of the present invention. And a latex bead, which is a substance to be separated, can be separated by moving a potential difference in the separation medium by filling a mixed solution of the mixture and the sensitized latex and applying a voltage to both ends of the flow path.
[0064]
When a sample containing the substance to be separated is filled in the flow path of the electrophoresis chip, according to a known filling method, for example, it can be filled in the form of a mixture with a separation medium, or can be used for separation. After the medium is filled in the channel in advance, only the sample can be filled alone.
In order to fill the mixture of the separation medium and the sample into the flow path of the electrophoresis chip, for example, from the liquid storage portion of the liquid storage portion forming through hole of the flat cover member, via the separation medium, It can be introduced into the channel. Such an introduction method is not particularly limited, but includes, for example, a method utilizing a capillary phenomenon, a pressure injection method using a syringe, or the like, or a method in which a medium dropped in one liquid reservoir portion is filled in another liquid reservoir. And a pressure reducing injection method in which the pressure is reduced by using a water flow pump or a vacuum pump from the section. Excess solution existing outside the flow path can be removed with, for example, a trace pipette. At this time, it is important to prevent other contaminants such as bubbles and dust from entering the flow path.
[0065]
As described above, the substance to be separated is mixed with the separation medium in advance, and a predetermined amount (that is, the capacity of the flow path) can be introduced into the separation flow path by an appropriate introduction method. In this case, the introduction amount is very accurate as compared with a case where the sample is filled alone, which will be described later.
[0066]
Further, in the separation method of the present invention, even when only the sample is filled alone, the sample is filled in the liquid pool portion of the through hole for forming the liquid pool portion of the flat cover member, and a voltage is applied for a predetermined time. Thus, the sample can be introduced into the separation channel.
[0067]
The substance to be separated subjected to electrophoretic separation using the electrophoresis chip of the present invention can be analyzed (detected or measured) by utilizing, for example, its optical characteristics, electrochemical characteristics, and the like. For example, nucleic acid fragments such as DNA and RNA can be detected by measuring absorbance using the UV absorption properties of nucleic acid molecules, or by labeling nucleic acid molecules with a fluorescent dye and measuring fluorescence. Can be separated and the size of the fragment analyzed.
[0068]
【Example】
Hereinafter, the present invention will be described specifically with reference to Examples, but these do not limit the scope of the present invention.
Embodiment 1
<< Preparation of electrophoresis chip (1) >>
The electrophoresis chip of the present invention can be formed, for example, by bonding a film or thin plate coated with an adhesive to a plastic (polystyrene) flat plate in which various narrow grooves or narrow tanks are formed. In this example, an electrophoresis chip having the shape shown in FIGS. 1 and 2 was manufactured.
Specifically, one narrow groove-shaped through hole having a width of 200 μm, a depth of 50 μm, and a length of 18 mm is formed in the plate-like intermediate member as a separation channel, and the inner diameter is 2 mm at both ends. Was formed. In the flat cover member, a cylindrical through hole having an inner diameter of 2 mm was formed at a position corresponding to the cylindrical through hole of the flat intermediate member. Subsequently, the narrow groove-shaped through-hole formed on the plate-shaped intermediate member was joined and sealed with a plate-shaped cover member and a plate-shaped base member, thereby producing an electrophoresis chip of the present invention.
[0069]
Embodiment 2
<< Separation of protein solution by isoelectric focusing (1) >>
In this example, a commercially available isoelectric point standard protein solution was separated using the electrophoresis chip of the present invention prepared in Example 1.
Specifically, 4 volumes of an electrophoresis buffer containing 0.5% hydroxypropylmethylcellulose (manufactured by Sigma) and 5% ampholine (pH 3.5-10; manufactured by Amersham Biosciences) and a storage isoelectric point standard protein 0.5 μL of a sample solution mixed with 1 volume of a solution (IEF standard; manufactured by Bio-Rad) is dropped from one of the liquid pools of the electrophoresis chip, and the pressure is reduced from the other liquid pool. Inside. The above-mentioned isoelectric point standard protein solution for storage includes phycocyanin (pI = 4.45, 4.65, 4.75; blue), myoglobin (pI = 6.8, 7.0; brown), hemoglobin A (pI = 7.1; red), hemoglobin C (pI = 7.5; red), and cytochrome c (pI = 9.6; red) and β-lactoglobulin B (pI = 5. 1) Colorless proteins of bovine carbonic anhydrase (pI = 6.0), human carbonic anhydrase (pI = 6.5), and lentil lectin (pI = 7.8, 8.0, 8.2) And is contained.
[0070]
The sample liquid remaining in the liquid reservoir was removed, and a 10 mmol / L phosphoric acid aqueous solution was dispensed as an anolyte into one liquid reservoir, and a 40 mmol / L sodium hydroxide aqueous solution was dispensed as a catholyte into the other liquid reservoir. Isoelectric focusing was performed by applying a voltage of 444 V / cm to both ends of the capillary. The separated colored proteins were photographed and recorded at regular intervals by a digital still camera (RICHO RR1; manufactured by Ricoh Co., Ltd.). After the experiment, the electrophoresis image was analyzed using an image analysis program (Scion Image β4.02; manufactured by Scion Corporation).
[0071]
The results are shown in FIGS. FIG. 4 is a photograph instead of a drawing, showing electrophoretic images recorded over time at 10-second intervals by a digital still camera. FIG. 5 is a densitometric diagram obtained by computer processing of the electrophoretic image. It is.
4 and 5, the left end of each electrophoresis image or densitometry corresponds to a liquid reservoir filled with an anolyte (10 mmol / L phosphoric acid aqueous solution), and the right end is a catholyte (40 mmol). / L sodium hydroxide aqueous solution).
The numbers shown in FIGS. 4 and 5 indicate the elapsed time since the start of the electrophoresis, and the unit is “minute” and “second”. For example, the number “1:30” indicates that 1 minute and 30 seconds have elapsed since the start of electrophoresis.
[0072]
As is clear from FIGS. 4 and 5, the protein separation was completed in a short time of 2 minutes or less. In the case of a normal polyacrylamide-coated capillary (inner diameter = 75 μm, length = 500 mm), it takes 11 minutes to converge to the isoelectric point and 10 minutes or more to move the converged band to the detector. Was.
[0073]
Embodiment 3
<< Preparation of electrophoresis chip (2) >>
The chip for electrophoresis of the present invention is formed, for example, by bonding a film or thin plate coated with an adhesive to a flat plate made of PMMA (polymethyl methacrylate) in which various narrow grooves or narrow tanks are formed. Can be. In this example, an electrophoresis chip having the shape shown in FIGS. 1 and 2 and having a size different from that of Example 1 was produced.
Specifically, a single narrow groove-shaped through hole having a width of 100 μm, a depth of 50 μm, and a length of 6.5 mm is formed as a separation channel in the plate-shaped intermediate member. A cylindrical through-hole having an inner diameter of 3.5 mm was formed. A cylindrical through-hole with an inner diameter of 3.5 mm was formed in the plate-shaped cover member at a position corresponding to the cylindrical through-hole of the plate-shaped intermediate member. Subsequently, the narrow groove-shaped through-hole formed on the plate-shaped intermediate member was joined and sealed with a plate-shaped cover member and a plate-shaped base member, thereby producing an electrophoresis chip of the present invention.
[0074]
Embodiment 4
<< Separation of protein solution by isoelectric focusing (2) >>
Using the electrophoresis chip of the present invention prepared in Example 3, isoelectric focusing was performed on the same sample solution used in Example 2. A 5 mmol / L phosphoric acid aqueous solution was used as an anolyte and a 20 mmol / L sodium hydroxide aqueous solution was used as a catholyte, and a voltage of 450 V / cm was applied to both ends of the capillary.
[0075]
The results are shown in FIGS. The recording of the electrophoresis image and the computer processing were performed in the same manner as in Example 2. As is clear from FIGS. 9 and 10, the protein separation was completed in a short time of 1 minute or less.
[0076]
【The invention's effect】
The electrophoresis chip of the present invention is extremely inexpensive and is suitable for mass production. Therefore, it is suitable for use only for one-time analysis and disposal, that is, for disposable use.
[0077]
Further, according to the electrophoresis chip of the present invention, since a three-layer structure is employed, the separation accuracy can be improved at extremely low cost without employing a complicated manufacturing process.
For example, by forming the plate-like intermediate member from a material through which light does not transmit, the narrow groove-shaped through-hole can have a function as a slit, and therefore, the effect of stray light from the surroundings is reduced, and the target substance is reduced. Can be increased in detection sensitivity.
In addition, since the depth of the flow path can be easily adjusted by changing the thickness of the flat intermediate member, for example, a narrow groove deeper than the width of the flow path can be easily formed. For this reason, in optical detection, the optical path length can be made longer than the conventional wide flow path, and the detection sensitivity is expected to be improved.
[Brief description of the drawings]
FIG. 1 is a perspective view showing one embodiment of an electrophoresis chip according to the present invention in a state where it is separated into a flat cover member, a flat intermediate member, and a flat base member.
FIG. 2 is a cross-sectional view of one embodiment of the electrophoresis chip 11 according to the present invention shown in FIG.
FIG. 3 is a configuration diagram of an apparatus for continuously recording an electrophoretic image.
FIG. 4 is a photograph instead of a drawing, showing an electrophoretic image recorded over time with a digital still camera in Example 2.
FIG. 5 is a densitometry diagram obtained by computer processing of the electrophoresis image shown in FIG.
FIG. 6 is a schematic plan view schematically showing a combination of a through-hole for forming a liquid reservoir and a narrow groove-like through-hole provided in a flat intermediate member.
FIG. 7 is a schematic plan view schematically showing another combination of a through-hole for forming a liquid reservoir and a through-hole having a narrow groove provided in a flat intermediate member.
FIG. 8 is a schematic plan view schematically showing still another combination of a through hole for forming a liquid reservoir portion and a through hole having a narrow groove provided in a flat intermediate member.
FIG. 9 is a photograph instead of a drawing, showing an electrophoretic image recorded over time by a digital still camera in Example 4.
FIG. 10 is a densitometric diagram obtained by computer processing of the electrophoretic image shown in FIG. 9;
[Explanation of symbols]
1 ... flat cover member; 2 ... flat intermediate member;
3 ... flat base member; 4 ... through hole for forming a liquid reservoir;
5 ... through-hole for forming a liquid pool portion; 6 ... through-hole with a narrow groove; 7 ... electrode;
8: electric circuit; 9: liquid reservoir; 10: electrophoresis device;
11: electrophoresis chip; 12: stage; 13: light source;
14: Digital still camera; 15: Close-up lens.

Claims (18)

A flat base member, a flat intermediate member provided on one surface thereof, and a flat plate provided on the opposite surface of the flat intermediate member where the flat intermediate member comes into contact with the flat base member Including a cover member,
The plate-shaped intermediate member is provided at at least one narrow groove-shaped through hole penetrating in the thickness direction thereof, and a liquid provided at both ends of the narrow groove-shaped through hole, and penetrating in the thickness direction of the plate-shaped intermediate member. And a through hole for forming a reservoir,
A liquid reservoir penetrating in the thickness direction of the flat cover member, wherein the flat cover member forms a liquid pool portion together with the liquid reservoir forming through hole provided in the flat intermediate member. Having a through hole for forming a part,
The narrow groove-shaped through hole and the liquid reservoir forming through hole provided in the plate-shaped intermediate member are in contact with the plate-shaped base member, one opening of each of which is sealed,
The electrophoresis chip, wherein the other opening of the narrow groove-shaped through-hole provided in the flat intermediate member is sealed by contact with the flat cover member. On the surface of the plate-shaped cover member opposite to the surface in contact with the plate-shaped intermediate member, a liquid pool portion is formed together with the liquid reservoir portion forming through hole provided in the plate-shaped cover member, It has a reinforcing plate-shaped member provided with a through-hole for forming a liquid reservoir portion penetrating in the thickness direction, and / or on a surface of the plate-shaped base member opposite to a surface in contact with the plate-shaped intermediate member. 2. The electrophoresis chip according to claim 1, further comprising a reinforcing flat member. The electrophoresis chip according to claim 1, wherein the plate-like cover member has an electrode. The electrophoresis chip according to claim 2, wherein the reinforcing plate member provided on the surface of the plate cover member opposite to the surface in contact with the plate intermediate member has an electrode. The electrophoresis chip according to any one of claims 1 to 4, wherein the flat intermediate member is made of a member through which light does not pass. The electrophoresis chip according to any one of claims 1 to 5, wherein one of the flat base member and the flat cover member is formed of a member containing a substance that emits light when irradiated with ultraviolet light. The material of at least one of the flat base member, the flat intermediate member, and the flat cover member is an acrylic resin, a styrene resin, an epoxy resin, or a polyvinyl chloride resin. An electrophoresis chip according to any one of claims 1 to 6. (1) A flat plate having at least one narrow groove-shaped through hole penetrating in the thickness direction, and a liquid reservoir portion forming through hole provided at both ends of the narrow groove-shaped through hole and penetrating in the thickness direction. And (2) a through hole for forming a liquid reservoir portion, which can be formed together with the through hole for forming a liquid reservoir portion provided in the plate-like intermediate member and which penetrates in the thickness direction. And (3) a flat base member,
The flat plate-shaped cover member is in contact with one surface of the flat-shaped intermediate member, and the flat-plate base member is joined so as to be in contact with the other surface of the flat-shaped intermediate member. The method for producing an electrophoresis chip according to claim 1. The method for producing an electrophoresis chip according to claim 3, wherein the electrode is formed by printing, vacuum deposition, sputtering, ion plating, or adhesion. The joining of the flat members is performed by bonding with an adhesive and / or heat fusion of at least one of a flat base member, a flat intermediate member, and a flat cover member. Production method. An electrophoresis device, comprising the electrophoresis chip according to claim 1. A method for separating a substance, comprising using the chip for electrophoresis according to claim 1. A method for separating a substance, comprising using the electrophoresis apparatus according to claim 11. 14. The separation method according to claim 12, wherein the substance is a charged molecule or a charged particle. The separation method according to any one of claims 12 to 14, wherein a polymer gel is used as a support for the separation medium. The separation method according to any one of claims 12 to 14, wherein the separation medium contains a hydrophilic polymer compound capable of forming a sol or a low molecular compound capable of imparting high viscosity. Hydrophilic polymer compound, linear polyacrylamide, linear hydroxyethyl cellulose, linear hydroxypropyl methylcellulose, linear hydroxypropyl cellulose, linear methyl cellulose, linear polyethylene glycol, linear polyethylene oxide, 17. The separation method according to claim 16, wherein the separation method is a linear polymer selected from the group consisting of polyvinyl alcohol and polyvinyl alcohol, or a natural polymer selected from the group consisting of mannan, curdlan, cellulose, and agarose. 17. The separation method according to claim 16, wherein the low molecular compound imparting high viscosity is urea or a derivative thereof, a polyhydric alcohol, or an oligosaccharide.

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