JP4762521B2 - Bio separation kit and method of use thereof - Google Patents

Description

  The present invention relates to a bioseparation kit for separating biological components such as nucleic acids, proteins, and cells by B / F separation and a method for using the same.

  Bioseparation that separates biological components such as nucleic acids, proteins, and cells is an indispensable technique in the fields of medicine, diagnosis, and biotechnology. There are various methods for bioseparation, but after capturing a target substance in a specimen on a carrier on which a probe is immobilized, so-called B / F (Bound form / Free form) separation is still used as an effective method, and various methods are known.

  For example, the centrifugation method uses a specific gravity difference between a probe-immobilized carrier such as beads and a non-captured substance not captured by the immobilized carrier to precipitate the immobilized carrier by centrifugation, and then removes the supernatant. This method is widely used. In this method, by adjusting the centrifugal force and the time of centrifugation, separation is possible even when the gravity difference between the immobilization carrier and the non-captured substance is quite small. It may cause aggregation of the carrier and destruction of the trapping substance.

  A method of separating probe-carrying particles such as beads with a filter is also widely used. Filters for separation include glass fiber filter paper, membrane filters, polyester film or polycarbonate film with holes formed by electron beams (trade name: manufactured by Cyclopore Whatman Co., Ltd.), aluminum anodized holes with a pore size of 200 μm or less (Product name: Anopore Whatman Co., Ltd.) is used. In this case, in order to avoid the analyte adsorbing to the filter during the reaction, the probe-carrying particles and the analyte are reacted in another container, and the reaction solution is transferred from the container to the B / F separation container. It is said that it is preferable to transfer and perform B / F separation.

  Also, in the step of peeling the target material with the stripping solution after B / F separation, peeling in a container different from the B / F separation container may remove contamination caused by the B / F separation due to the analyte. It is preferable because it is not brought in.

However, since two containers are used in this way, when the object is very small, the object is worn out due to adhesion to the container.
Moreover, when transferring a liquid from a container to a container, the method which passes through a syringe is common, However, In this case, these are further worn out by adhesion of the particle liquid or a test substance to a syringe wall.

  In addition, since the movement of the liquid from the container to the container is performed manually, the operation is complicated. Even if a handling robot is used, the number of reaction solutions that can be moved in a batch is limited, and the number of reactions increases and the work time increases.

An affinity chromatograph that captures and separates a target substance using a gel-fixed probe as a carrier and a column packed with the gel is also widely used. In this method, the gel needs to be packed into the column, the packing operation is complicated and costly, and the flow rate of the liquid flowing through the column and the column back pressure are inversely related, so that the separation performance is increased. When the flow rate is increased, the filtration pressure of the analyte increases, and as a result, gel deformation, column breakage, and the like are likely to occur, so that the flow rate is limited.

In the current situation as described above, bioseparation does not damage the specimen, has high B / F separation efficiency, low filtration resistance, little specimen and particle liquid depletion, and easily and quickly separates target substances. There is a need for technology that can
JP-A 63-270000 Nucleic Acid Research Vol. 14, p5037-5048 (1986)

  The present invention provides a bioseparation kit capable of easily and quickly separating a target substance without damaging the specimen, having a high B / F separation efficiency, low filtration resistance, less subject and particle liquid depletion. And its usage.

The method for producing the bioseparation kit of the present invention comprises:
A metal filter part in which straight pores having a straight hole center line are formed, and a container for storing the filter part,
A method for producing a bioseparation kit having a reaction part that reacts a subject and probe-carrying particles in a region separated from a position where the filter part is arranged inside the container,
The filter unit prepares a substrate that has been previously imparted with conductivity, and resist patterning is performed on the substrate, and a portion that is not electroplated is protected with a resist, and a current flows between the substrate and the plating solution. Thus, a metal material is formed only on a predetermined portion of the substrate, and the filter is manufactured by a method of stacking by an electroplating method.
Further, the bioseparation kit of the present invention comprises:
A metal filter part in which straight pores having a straight hole center line are formed, and a container for storing the filter part,
In a region separated from the position where the filter unit is arranged inside the container, a reaction unit that reacts the analyte and the probe-carrying particles ,
The filter unit prepares a substrate that has been previously imparted with conductivity, and resist patterning is performed on the substrate, and a portion that is not electroplated is protected with a resist, and a current flows between the substrate and the plating solution. forming a metal material only a predetermined portion of the substrate by, wherein the this are those prepared by the method of stacking by electroplating the filter.

In the above invention, it is preferable that a rib formed of the same material as that of the filter and integrated is provided on the surface of the filter.
In the above invention, it is preferable that a stirring plate is provided at least in any position in the vicinity of the reaction unit and the filter unit in the container.
In the above invention, the filter includes two filter portions, a B / F separation filter portion for performing B / F separation and a separation filter portion for separating the target material captured by the probe-carrying particles. Is preferably housed.

In the above invention, both end faces of the filter part storage space for storing the B / F separation filter part and the peeling filter part in the container are opened to the outside,
An inner space sandwiched between the B / F separation filter portion and the separation filter portion in the filter portion storage space is open to the outside through at least one branch space that branches from the inner space. preferable.

  In the above invention, a branch space that branches off from the filter portion storage space for storing the B / F separation filter portion and the peeling filter portion in the container and has an opening opened to the outside on one end side thereof. At least one is provided, and at least one of the dispersion liquid of the probe-carrying particles, the specimen, the cleaning liquid, and the stripping liquid is introduced from the opening into the filter unit storage space through the branch space. It is preferable.

In the above invention, it is preferable that the branching space has the reaction part for reacting the analyte and the probe-carrying particles.
In the above invention, at least two of the branch spaces are provided, a cleaning solution is supplied from one of the branch spaces to the filter unit storage space, and a release solution is supplied from the other one of the branch spaces to the filter unit storage space. It is preferable to configure so as to.

In the above invention, a branch space that branches off from the filter portion storage space for storing the B / F separation filter portion and the peeling filter portion in the container and has an opening opened to the outside on one end side thereof. Have at least one,
For the three flow path spaces l, m, and n extending from the branch point between the branch space and the filter unit storage space, the cross-sectional area M of the flow path space m is made smaller than the cross-sectional area N of the flow path space n. It is preferable that the liquid is selectively moved from the channel space l to the channel space m of the channel space m and the channel space n.

In the above invention, it is preferable that a plurality of the bioseparation kits are integrated and mounted on the same substrate.
In the bioseparation method of the present invention, the probe-carrying particles and the analyte are reacted in the reaction unit using the bioseparation kit described above, and then the probe-carrying particles are moved to the filter unit, and then the washing liquid is used. By contacting the probe-carrying particles, the target material captured by the probe-carrying particles is separated from other substances.

  In the bioseparation method of the present invention, the probe-carrying particles and the analyte are reacted in the reaction unit using the bioseparation kit described above, and then the probe-carrying particles are moved to the B / F separation filter unit. Then, by bringing the cleaning liquid into contact with the probe-carrying particles, the target substance captured by the probe-carrying particles and the other substance are separated, and then the probe-carrying particles are moved to the separation filter unit, The target substance is peeled from the probe-carrying particles by bringing the peeling liquid into contact with the probe-carrying particles.

  In the above invention, the probe-carrying particles are positioned in the vicinity of the filter unit, and at least one of the analyte, the cleaning solution, and the stripping solution is reciprocated a plurality of times in the filter unit storage space. The liquid and the probe-carrying particles are preferably brought into contact with each other while the carried particles are brought into contact with and separated from the filter portion.

  In the above invention, after contacting at least one of the analyte, the cleaning liquid, and the peeling liquid with the probe-carrying particles, the liquid is discharged through the filter unit and the probe-carrying particles are placed on the filter unit. Then, it is preferable that the next liquid is introduced into the filter unit and brought into contact with the probe-carrying particles.

In the above invention, it is preferable that after the pressure in the filter unit storage space is reduced, at least one of a specimen, a cleaning solution, and a stripping solution is put into the space.
In the above invention, when moving at least one of the specimen, the cleaning liquid, and the stripping liquid inside the container, it is preferable to move the liquid in the horizontal direction or the gravity direction.

  In the above invention, it is preferable that the liquid is moved by a differential pressure with the inner diameter or inner width of the target space in the container to which the liquid is moved being 5 mm or less.

  The bioseparation kit of the present invention does not damage the specimen, has high B / F separation efficiency, low filtration resistance, little wear of the specimen and particle liquid, and allows easy and rapid separation of the target substance. It is.

  According to the bioseparation method of the present invention, the target substance is not easily and quickly separated without damaging the specimen, with high B / F separation efficiency, low filtration resistance, little wear of the specimen and particle liquid. Is possible.

Hereinafter, embodiments of the present invention will be described with reference to the drawings. FIG. 1 is a cross-sectional view of a bioseparation kit according to an embodiment of the present invention. As shown in the figure, the bioseparation kit 11 includes a container 12 in which a filter unit 1 is accommodated.
<Filter section>
The filter unit 1 allows each liquid such as a specimen, a cleaning solution, and a stripping solution to pass through the pores 3 and blocks passage of the probe-carrying particles. In this filter unit 1, B / F separation, from the probe-carrying particles The target material is peeled off.

A filter 2 is provided at the bottom of the filter unit 1. The filter 2 is formed with pores 3 having a straight hole axis.
A rib 4 substantially integrated with the filter 2 is provided on the upper surface side of the filter 2 as necessary. In this case, the well 5 may be configured with the rib 4 as a side surface and the filter 2 as a bottom surface.

  The rib 4 serves as a reinforcing material for reinforcing the filter 2 and also functions as a side wall of the well 5. The rib 4 can have any shape, but in order to maintain the strength of the filter 2, the rib 4 is a continuous wall formed with a closed hole that forms the side surface of the well 5. It is desirable to be. That is, a structure in which the ribs 4 are continuously formed integrally and the well 5 having the filter 2 as a bottom surface exists therein is preferable in terms of mechanical strength. An example in which such a rib 4 is actually formed is shown in the electron micrograph of FIG.

  The number of wells provided in the filter unit 1 is arbitrary, and can be an appropriate number as required. Note that the filter 4 may be the upper surface instead of the bottom surface, and the rib 4 may be below the upper surface.

  In this specification, “the filter and the rib are substantially integrated” means that the filter and the rib are integrally formed from the same material. Examples of a method for integrally forming the filter and the rib include a method in which the filter and the rib are manufactured from the same material by a subtractive method such as etching, an additive method by electroforming, a nanoprint method, or the like.

  In addition, an oxide film can be formed on the surface of a metal such as a silicon wafer, and a filter and a rib can be produced by utilizing the difference in etching rate between the metal and the oxide film. Since it is formed, it is considered that the filter and the rib are substantially integrated.

  By integrating the filter and the rib, the mechanical strength of the filter increases, and impurities such as analyte do not enter between the filter and the rib, and the filter section has a high B / F separation capability. Can do.

In the present specification, “straight” pores formed in the filter mean that the pores are formed without branching in the middle, and the axis (center line) of the pores is linear. For example, even if the perpendicular line from the center of the opening formed on one filter surface to the other filter surface and the perpendicular line from the center of the opening formed on the other filter surface to the one filter surface are shifted, Such pores may be used because they are not inferior in terms of pressure loss. The shape of the cross section of the pore perpendicular to the penetration direction is not particularly limited, and the shape of the pore may be any shape such as a cylinder, a quadrangular pyramid, a polygonal pyramid, etc. An obtuse or circular shape is desirable. By making the pores straight in this way, the length of the pores is minimized, so the contact area between the circulating fluid and the pore walls is reduced, and the pressure resistance accompanying filtration can be minimized. . In addition, the nonspecifically adsorbed substance can be minimized from adhering to the pores in the specimen, and the nonspecifically adsorbed substance can be easily washed and removed. For example, in the case of a silica layer filter having a thickness of 2 μm and a pore diameter of 4 μm, it is possible to filter with a differential pressure of 10 to 50 gf / cm ² , and the container containing the filter part is minimized in its mechanical pressure strength. Therefore, a very small container can be used.

  In the present invention, the pores of the filter may have a uniform pore size or a distribution. When the pore diameter is uniform, the CV (Coefficient of Variation) value of the pore diameter is preferably 10% or less. When the pore diameter has a distribution, the CV value of the pore diameter is larger than 20%, preferably 50 to 300%, more preferably 100 to 200%. Note that, when calculating this CV value, pores that are formed to have a particularly large diameter compared to many other pores are excluded.

By giving a distribution to the pore diameter of the filter, it is possible to effectively prevent an increase in filtration pressure due to the probe-carrying particles blocking the pores during filtration.
When the filter pores have a pore size distribution, the pore size pattern may be random, but may be a predetermined pore size pattern. As one form of the predetermined hole diameter pattern having the hole diameter distribution, there is a pattern in which the hole diameters of the adjacent pores are different and the hole diameter is the same every other hole. In this case, when the probe-carrying particles having a uniform particle diameter are used, the probability that the particles block the pores is reduced, so that the filterability of the filter is improved. In this case, the particles carrying the probe have a substantially uniform particle diameter, the particle diameter d, the pore diameter h of the pore formed in the filter, and the two adjacent pores 3 and 3. The relationship with the hole interval p is preferably d> h + p. Here, the hole diameter h and the hole interval p of the pores are average values in the entire filter portion constituting the upper surface or the lower surface of the well. When the above relationship is satisfied, the probability that the particles block the filter pores is significantly reduced.

  As another form of a predetermined hole diameter pattern having a hole diameter distribution, the hole area of the pores in the vicinity of the well center is increased or the hole interval is increased to increase the opening area of this region, and in the vicinity of the well periphery. The pattern which made the opening area of this area | region small by making the hole diameter of these pores small or making a hole space | interval sparse is mentioned. In this case, the strength of the filter can be increased.

  As another form of a predetermined hole diameter pattern having a hole diameter distribution, the hole area of the pores near the center of the well is reduced or the hole interval is reduced to reduce the opening area of this region and the vicinity of the periphery of the well The pattern which enlarged the opening area of this area | region by enlarging the hole diameter of these pores or making the space | interval of a hole dense is mentioned. In this case, the filterability of the filter can be improved because the subject passes through the filter holes uniformly.

  The pore diameter of the filter pores is preferably 1 μm or more, more preferably 1 to 50 μm, still more preferably 3 μm to 20 μm. When the pore diameter is less than 1 μm, the subject is easily clogged. On the other hand, if the pore diameter is larger than 50 μm, it is necessary to use large particles as the particles for supporting the probe, and the amount of the probe decreases, so that the filterability of the target substance from the subject is lowered.

  The pore spacing of the filter (the shortest distance between the adjacent pores where the pores are not open) is not particularly limited, but is preferably 1 μm to 10 μm. This is preferable. When the pore interval of the filter is less than 1 μm, the mechanical strength may be insufficient, and when the pore interval exceeds 10 μm, the aperture ratio of the filter may be excessively lowered.

  The thickness of the filter is preferably 1 μm to 100 μm, more preferably 1 μm to 25 μm, and still more preferably 2 μm to 15 μm. When the thickness is less than 1 μm, the strength of the filter may be insufficient, and when the thickness exceeds 100 μm, the area of the side surfaces of the pores may increase and the filtration resistance may increase excessively.

  The aperture ratio of the filter is preferably 3 to 50%, more preferably 5 to 40%, and still more preferably 7 to 35%. When the opening ratio is less than 3%, the filterability tends to be lowered, and when the opening ratio exceeds 40%, the strength of the filter tends to be lowered.

  The material of the filter is not particularly limited as long as it can form a filter, and examples thereof include metals, metal oxides, and organic substances. Particularly preferred are materials with high mechanical strength. Specifically, for example, engineering plastics such as liquid crystal polymer, polycarbonate, polyamide resin, polyimide, polyethylene terephthalate, polyethylene naphthalate, polyarylate, polysulfone, and polyethersulfone. Iron, nickel, copper, zinc, aluminum, silicon, titanium, tantalum, magnesium, molybdenum, tungsten, rhodium, palladium, silver, gold, platinum, stainless steel, brass, brass, bronze, phosphor bronze, aluminum copper alloy, aluminum magnesium Metals such as alloys, aluminum magnesium silicon alloys, aluminum zinc magnesium copper alloys, iron nickel alloys; metal oxides such as silica, alumina, titania, zirconia, tantalum oxide; SiN, TiN, TaN, etc. Metal nitride; SiC, metal carbides such as WC; diamond, graphite, carbon materials such as Diamond Like Carbon (DLC); soda glass, borosilicate glass, Pyrex (registered trademark), and a glass such as quartz glass.

  Among these, silica, titania, alumina, nickel, gold, titanium, or stainless steel is preferable. These have high mechanical strength and can make the filter thin. And since it is comparatively hydrophilic, it has high affinity with an aqueous biosolution, and the specimen can be easily put into the filter pores. Note that these materials may be used as a base material and the surface thereof may be oxidized, or may be surface-treated with a surface treatment agent using another material. Examples of the surface treatment method include corona treatment. Examples of the surface treatment agent include nonspecific adsorption inhibitors for biomaterials such as alcohol, hydrophilizing agent, hydrophobizing agent, and protein.

  The filter part can be manufactured by a method of stacking filters and the like by electroplating. In this method, a substrate that has been previously imparted with conductivity is prepared, and resist patterning is performed on the substrate by photolithography, imprinting, etc., and portions that are not electroplated are protected with resist, and then the substrate and the plating are plated. A metal material is formed only on a predetermined portion of the substrate by passing an electric current between the substrate and the solution.

  First, a substrate subjected to a treatment for imparting conductivity is prepared, and a resist is applied thereon to form a film. Next, a photomask is prepared and exposed and developed with UV light, and a resist post as a filter reversal pattern is formed on the substrate. Although the pattern limit depends on the resolution of the resist, a resist post having a pattern pitch of 5 μm and an aspect ratio of 2 can be produced by using, for example, THB-110N (trade name: manufactured by JSR Corporation).

Next, between these posts, the metal is electrically filled by flowing an alternating current or direct current by electroplating. Examples of the metal material filled by the electroplating method include gold, nickel, copper, iron, iron-nickel alloy, and the like. As a plating solution for nickel electroforming, a nickel sulfamate bath (a sulfamic acid 60% solution 700 g / l, nickel bromide 5 g / l, boric acid 35 g / l mixed solution, bath temperature 50 ° C.) and the like are used. As a plating solution for copper electroforming, a copper sulfate bath (copper sulfate 200 g / l, sulfuric acid 60 g / l, chlorine ion 30 mg / l mixed solution, bath temperature 30 ° C.) is used. As the plating solution, an iron sulfamate bath (iron sulfamate 400 g / l, ammonium sulfamate 30 g / l, formalin 100 mg / l mixed solution, bath temperature 46 ° C.) and the like are used. For example, in the case of a nickel sulfamate bath, an electroplated product having a thickness of 5 μm can be obtained by applying a direct current for 10 to 20 minutes at a voltage of 6 V and a current density of 3 A / dm ² .

  After producing the filter in this way, ribs are formed by an electroplating method, if necessary, in the same manner as the filter formation method. That is, a resist film is formed on a filter in the same manner as described above, a pattern is formed by photoetching, and then an electroplating material is filled. In this case, generally, the height of the rib is equal to or greater than the thickness of the filter, so that the resist film is also thickened. For this reason, the resist is preferably used by laminating a dry film resist. In general, it is difficult to form a plated product with an aspect ratio of line width and thickness exceeding 2 once. In such a case, a predetermined number of times is obtained by repeating the formation of the plated product by photoetching and electroplating several times. The height of the rib.

  The filter part can be manufactured by producing at least one of a filter and a rib by etching. The object to be etched is not particularly limited as long as it can form a filter or the like, but is not particularly limited, such as a metal, a metal oxide, and an organic substance. Particularly preferable is a material having high mechanical strength. , Polycarbonate, polyamide resin, polyimide, polyethylene terephthalate, polyethylene naphthalate, polyarylate, polysulfone, polyethersulfone and other engineering plastics; iron, nickel, copper, zinc, aluminum, silicon, titanium, tantalum, magnesium, molybdenum, tungsten Rhodium, palladium, silver, gold, platinum, stainless steel, brass, brass, bronze, phosphor bronze, aluminum copper alloy, aluminum magnesium alloy, aluminum magnesium silicon alloy, aluminum zinc magnesium copper alloy, iron nickel alloy Metals such as silica, alumina, titania, zirconia, and tantalum oxide; metal nitrides such as SiN, TiN, and TaN; metal carbides such as SiC and WC; diamond, graphite, diamond like carbon (DLC), etc. Carbon materials: Glasses such as soda glass, borosilicate glass, Pyrex (registered trademark), and quartz glass can be used.

  Etching can be performed by an ordinary method. However, etching with an excessively large aspect ratio may cause the pore diameter and shape to be nonuniform, and the aspect ratio obtained by ordinary chemical etching is substantially 5 or less, preferably 3 or less. However, the filterability can also be improved by using a filter in which an inversely tapered shape is positively formed within this aspect ratio range.

  In the case where the well, the rib, or both of them are formed by etching, the aspect ratio is desirably 5 or more, but etching having an aspect ratio of 5 or more can be performed by anisotropic etching. For example, it is utilized that a single crystal material such as silicon shows a large crystal plane dependency with respect to an etching solution such as a KOH aqueous solution, ethylenediamine pyrocatechol (EDP), or 4 methyl ammonium hydroxide (TMAH). In the case of silicon, the etching rate of the (111) plane is extremely slow compared to other crystal planes, and a silicon wafer with the (110) plane as the surface is used, and the side of the mask material opening is defined as the (111) plane direction. Etching with an aspect ratio of about 100 can be performed by performing chemical etching together. These methods are described in the Japan Society for Precision Engineering, edited by: “Nanoscale Processing Technology”, Nikkan Kogyo Shimbun (1993).

Further, if necessary, reactive ion etching (RIE) using plasma can be performed. For example, anisotropic etching perpendicular to the substrate can be performed with a gas containing Freon-based chlorine gas in SF ₆ . These methods are described in “'02 latest semiconductor process technology”, Press Journal (2001).

Among the methods for producing filters and ribs by etching, particularly preferable methods are plates made of a plurality of materials having different compositions, such as aluminum / alumina, metal silicon / silica,
Or it is the method of forming a filter and a rib by carrying out pattern etching from both sides about metal titanium / titania etc., respectively. Specifically, one side composition layer, for example, a metal oxide layer such as alumina, silica, titania, etc. is pattern etched to the boundary between the oxide layer and the metal layer to form a filter, and then aluminum, metal silicon, metal titanium The rib and the filter can be integrally formed by pattern-etching a metal layer such as the above to the boundary between the metal layer and the oxide layer.

As the material in which two layers having different compositions are integrated, for example, an aluminum / alumina plate obtained by oxidizing the surface of an aluminum plate to a predetermined thickness to obtain alumina, metal titanium / titania obtained by similarly oxidizing metal titanium, oxidation Silicon wafer with film or SOI (Silicone
An On Insulator wafer can be used.

  Moreover, a filter part can be produced by the imprint method. That is, the filter can be produced by press-molding a resin plate using a two-stage convex mold produced in advance by a MEMS process or the like.

The filter in which the hole diameter pattern having the hole diameter distribution is formed can be obtained by designing the mask pattern or the convex mold so as to have the hole diameter distribution in the photoresist mask or the imprint mold. .
<Reaction part>
In the bioseparation kit of the present invention, as shown also in FIG. 1, in a region separated from the position where the filter unit 1 is arranged inside the container 12 (reaction unit 41: a region surrounded by a dotted line in the figure), The specimen and the probe-carrying particles are allowed to react with each other. The reaction is performed by mixing the probe-carrying particle dispersion 31 and the specimen 32 by, for example, a method of moving the liquid in the forward and reverse directions. The reaction unit 41 may be located on either the upper surface side or the lower surface side of the filter unit 1.

  In this way, by adopting a configuration in which the reaction part is provided in a region separated from the position where the filter part is disposed, the filtration resistance of the filter, in particular, the filtration by the probe-carrying particles blocking a part of the pore 3 of the filter. Resistance load can be prevented or reduced, and non-specific adsorption of the analyte to the filter surface can be prevented or reduced.

It should be noted that two divided bodies having a configuration in which the reaction section and the filter section are separated and divided are prepared, and these divided bodies are combined to form a liquid flow path when the liquid is transferred from the reaction section to the filter section. You may comprise. Similarly, when using two filter parts as described later, prepare two divided bodies in the form of separating and dividing the two filter parts, and combine these divided bodies when moving the liquid between the two filter parts. A liquid flow path may be formed. As a method of combining the divided bodies, for example, a method of providing a concave portion and a convex portion at a coupling position of both the divided bodies obtained by dividing the space in which the liquid is moved and fitting the concave portions and the convex portions together. Is mentioned.
<Container>
An inorganic material, an organic material, or an organic-inorganic hybrid material can be used as the container forming material in the bioseparation kit of the present invention. Examples of the inorganic material include metals such as nickel, aluminum, silicon, titanium, gold, and stainless steel; metal oxides such as silica, alumina, and titania, soda glass, borosilicate glass, Pyrex (registered trademark), and quartz glass. Glass is mentioned.

Examples of organic materials include polyethylene, polypropylene, polystyrene, polymethyl methacrylate, polyacrylonitrile, liquid crystal polymer, polycarbonate, polyamide, polyimide, polyethylene terephthalate, polyethylene naphthalate, cycloolefin, polymethylpentene, polyarylate, polysulfone, and polyether. Copolymers of various acrylates such as sulfone, polyethylene vinyl alcohol, crosslinked polyvinyl alcohol, polyglycolic acid, polyamide, polyimide, cellulose acetate, triacetyl cellulose, cellulose nitrate, epoxy, and a copolymer of 2-methacryloyloxyethyl phosphorylcholine and methacrylate. A polymer is mentioned.

  Further, the surface of the container forming material may be subjected to plasma treatment, corona treatment, ion treatment or the like to form hydroxyl groups, carboxyl groups, or the like. Further, a hydrophilic metal or metal oxide may be formed on the surface by plating or the like, for example, a hydrophilic material such as a copolymer of 2-methacryloyloxyethyl phosphorylcholine and methacrylate, or a polyethylene glycol derivative may be coated. It is also possible to open the epoxy group after applying glycidyl methacrylate.

  The container can be produced by a general molding method such as press molding, injection molding, blow molding, etc., and drilling, laser, or etching may be applied.

  In one embodiment of the bioseparation kit of the present invention, the filter part is fixed to the container and integrated. For example, a step portion is provided inside the container, and the filter portion is fitted to the step portion to join the filter portion to the container. Alternatively, the filter part can be fixed to the container by sealing the container with the filter part sandwiched through an O-ring or other sealing material. The container and the filter part can be fixed to each other by a method of bonding with an adhesive, a method of insert molding together with the filter part when the container is formed, or the like.

Moreover, you may make it accommodate a filter part in a container so that attachment or detachment is possible. In this case, for example, a step portion is provided inside the container, and the filter portion is fitted to the step portion and the filter portion is attached to the container, or the filter portion is sandwiched between the containers via a sealing material such as an O-ring. The filter part can be attached to the container by sealing with.
<Probe-supported particles>
As the probe-carrying particles, each particle carrying the same probe may be used for reaction with the analyte, or each particle carrying different probes may be used for reaction with the analyte. Good. In the present specification, “probe-carrying particles” include particles carrying ligands.

  Examples of probes or ligands carried on the particles include nucleic acids, proteins having a molecular weight of 500 to 1,000,000, lipids, sugar chains, cells, protein-expressing cells, aptamers, viruses, enzymes, and leads having a molecular weight of 500 to 1,000,000. Compounds, or chemical substances that have or may have a specific physiological activity.

  Examples of the particles carrying the probe include organic particles, inorganic particles, and organic-inorganic composite particles. Examples of the organic particles include monomers such as butadiene, styrene, divinylbenzene, acrylonitrile, acrylate, methacrylate, acrylamide, benzoguanamine, nylon, polyvinyl alcohol, or fluorine alone, or Examples thereof include particles obtained by emulsion polymerization or suspension polymerization from monomer raw materials used in combination of two or more. Or the particle | grains obtained by the method by the membrane emulsification which extrude resin from the porous plate with a uniform hole and produce particle | grains can also be used. These particles may have the same particle size with a classifier as required.

Further, particles obtained by adding a magnetic substance such as ferrite during or after polymerization, or those obtained by subjecting particles to ferrite plating by a method such as JP-A-10-83902, or natural substances such as cellulose, starch, agarose, galactose, etc. Synthetic cross-linked gels such as product cross-linked gels and acrylamide can also be used.

  Examples of the inorganic particles include metal oxide particles, metal sulfide particles, and metal particles. The most preferable metal oxide particles are silica particles, and various commercially available silica particles can be used.

  The surface of the above particle is coated with amino group, carboxyl group, carbodiimide group, epoxy group, tosyl group, N-succinimide group, maleimide group, thiol group, sulfide group, hydroxyl group, trimethoxysilyl group, nitrile triacetic acid group, benzosulfo group. Surface modification with various functional groups such as amide group and polyethyleneimine group, or γ-glycidoxypropyltrimethoxysilane can be used as a probe binding site.

  When organic particles are used as the particles carrying the probe, the particle diameter is preferably 0.1 μm to 120 μm, more preferably 0.5 μm to 60 μm. When the particle diameter is small, the handling properties are difficult, making it difficult to produce a filter that captures these particles, and the analyte is easily clogged because the filter hole is small. In addition, when the particle size is large, the reactivity may decrease due to steric hindrance.

  Preferably, the particles carrying the probe have a substantially uniform particle diameter, the particle diameter d, the pore diameter h of the pore 3 formed in the filter 2, and the distance between two adjacent pores 3 and 3. D> h + p in relation to the hole interval p. Here, the hole diameter h of the pores 3 and the hole interval p between the pores 3 and 3 are average values of the entire filter portion constituting the upper surface or the lower surface of the well.

  By satisfying the above relationship and increasing the number of particles to be larger than the number of pores, the particles do not block many pores when washing and filtering by laminating particles on a filter in multiple stages. Therefore, a decrease in filterability can be effectively prevented.

  FIG. 2 is a cross-sectional view showing another embodiment of the bioseparation kit of the present invention. As shown in the figure, the two filter parts 1 may be accommodated in the internal space of the container 12, and in this case, for example, as will be described later, the subject and the probe-carrying particles in the space between these filter parts 1. (Reaction unit 41 in the figure), the probe-carrying particles are moved to one filter unit 1 and the cleaning liquid is brought into contact with the probe-carrying particles, so that the target substance other than the target substance captured by the probe-carrying particles can be obtained. The target substance can be peeled off from the probe-carrying particles by washing away the non-capturing substance and then moving the probe-carrying particles to the other filter unit 1 and bringing the stripping solution into contact with the probe-carrying particles.

The subject and the probe-carrying particles can be introduced into the reaction section 41 between the filter sections 1 from the branch space 15 branched from the space, which communicates with the filter section storage space of the container.
FIG. 23 (a) is a sectional view showing another embodiment of the bioseparation kit of the present invention. In the embodiment of FIG. 2, the reaction unit 41 and the filter unit 1 are located in the normal direction of the filter surface of the filter unit 1, and the opening to the outside of the reaction unit 41 and the container 12 is the surface direction of the filter unit. In this embodiment, the reaction part 41 and the opening to the outside of the container 12 are provided in parallel with the surface direction of the filter part. That is, from the space in which the filter unit 1 is stored, branch spaces 15A and 15B whose flow direction is perpendicular to this space are branched, and one ends of these branch spaces 15A and 15B are opened to the outside. The interior of the space 15A is a reaction part 41 between the subject and the probe-carrying particles.

In FIG. 23 (a), the cross-sectional diameter d1 (or cross-sectional area) of the space that houses the filter portion 1 and the cross-sectional diameter d2 (or cross-sectional area) of the branch space 15A may be the same or different, but d1 If <d2, the flow of the liquid from the branch space 15A to the storage space of the filter unit 1 is facilitated by the capillary action or the surface tension difference, and the flow rate of the liquid in the storage space of the filter unit 1 is changed to the branch space. Since it can be made larger than the flow rate in 15A, a liquid can be uniformly permeated into a filter part.

  In the embodiment of FIG. 2, when the filter unit 1 and the reaction unit 41 are connected in a straight container inner space having a uniform hole diameter in the flow path direction, the reaction unit increases when the cross-sectional area of the filter unit 1 increases. In this embodiment, even if the cross-sectional area of the filter unit 1 is large, the cross-sectional area of the filter unit 1 and the cross-sectional area of the reaction unit 41 can be set independently. .

  Further, as shown in FIG. 23B (the view of the kit as seen from the upper surface side of the filter), the branch space 15 is connected along the tangent portion of the filter unit 1, and the liquid charging speed of the reaction unit 41 is set. By controlling, this liquid flows into the center part of the filter part 1 while circling the part without the pores in the peripheral part of the filter part 1. As a result, the liquid can be poured uniformly over the entire surface of the filter.

  24, the basic configuration is the same as that in FIG. 23, but spaces (branching spaces 15 </ b> A to 15 </ b> D) branched from the storage space of the filter unit 1 and having one end opened to the outside of the container are used as the storage space of the filter unit 1. Provided on both sides. These branch spaces 15A to 15D may be provided such that two spaces above and below the filter are provided on the same plane, or at least one of the branch spaces 15A to 15D is circular. You may make it provide in a separate tangent part so that a tangent with the filter part 1 may not be shared.

  FIG. 3 is a sectional view showing another embodiment of the bioseparation kit of the present invention. As shown in the figure, the filter unit 1 may be arranged in the vertical direction. In this case, since the liquid moves in the inner space of the container 12 in the horizontal direction, the liquid is fed in either the left or right direction. Can be fed without being affected by gravity.

  4 and 5 are cross-sectional views showing other embodiments of the bioseparation kit of the present invention. In the present embodiment, a stirring plate is provided at at least one of the positions near the reaction section and the filter section in the internal space of the container. By providing this stirring plate, when contacting the probe-carrying particles with each liquid such as the specimen, the cleaning liquid, and the stripping liquid, a turbulent flow is generated to efficiently react, clean, and peel off the target material. It can be carried out.

  In FIG. 4, the stirring plate 17 is provided in the reaction part 41 between the subject and the probe-carrying particles, and the subject 31 and the probe-carrying particle dispersion 32 are passed from one side of the stirring plate 17 to the other side. The contact between the specimen and the probe-carrying particles is made efficient. By moving the liquid a plurality of times in the forward and reverse directions, the specimen and the probe-carrying particles are passed a plurality of times from one side of the stirring plate 17 to the other side and the other side to the one side, or the inside of the stirring plate 17 It may be moved in the forward and reverse directions.

In FIG. 5, the stirring plate 17 is provided in the B / F separation part 42 in the vicinity of the B / F separation filter part 1a, which is a region where the probe-carrying particles are washed to remove non-captured substances, and the cleaning liquid 33 and the probe-carrying particles are provided. 21 is passed from one side of the stirring plate 17 to the other side, so that the contact between the cleaning liquid 33 and the probe-carrying particles 21 is made efficient. In addition, the stirring plate 17 is also provided in the peeling portion 43 in the vicinity of the peeling filter portion 1b, which is a region where the target material is peeled from the probe-carrying particles, and after the probe-carrying particles are washed by the B / F separation unit 42, the probe-carrying particles Is moved to the peeling section 43, and the peeling liquid and the probe-carrying particles are passed from one side of the stirring plate 17 to the other side, so that the contact between the peeling liquid and the probe-carrying particles is made efficient.

  The structure of the stirring plate may be any structure as long as it allows the probe-carrying particles to pass therethrough and generate a turbulent flow, and a known structure known as a baffle plate structure can be applied. Moreover, the incorporation of the stirring plate into the container can be performed by the same method as the incorporation of the filter unit. The material of the stirring plate may be the same material as the container or may be different.

  6 and 7 are cross-sectional views showing another embodiment of the bioseparation kit of the present invention. In the present embodiment, a B / F separation filter section 1a for performing B / F separation in the filter section storage space 14 in the container 12 and a separation filter for separating the target substance captured by the probe-carrying particles. Portion 1b is housed, and both end faces 14a, 14b of the filter portion housing space 14 are open to the outside. A branch space 15 having one end opened to the outside is provided in the inner space sandwiched between the B / F separation filter portion 1a and the separation filter portion 1b in the filter portion storage space.

  The end faces 14a and 14b of the container 12 opened to the outside are differential pressure control for controlling the movement of the cleaning liquid and the peeling liquid, the discharge of the specimen and the probe-carrying particle dispersion liquid, or the movement of these liquids with the differential pressure. Functions as a hole.

  The branch space 15 communicating with the filter unit storage space 14 functions as a differential pressure control hole for controlling the movement of the cleaning liquid, the stripping liquid, the specimen, the probe-supported particle dispersion liquid, and the movement of these liquids with the differential pressure. To do. The branch space 15 is not provided at a single position as shown in FIG. 6, but is used for the input of each liquid, for example, the input of the specimen and the probe-supported particle dispersion, the supply of the cleaning liquid, the input of the stripping liquid, and the like. You may provide in two places or more.

  In this way, by separating the B / F separation filter part and the separation filter part, the filter part contaminated by the analyte during the B / F separation is not used for the separation, so that the separation is performed. Contamination between the target material and the analyte can be minimized.

  In order to minimize the contamination of the peeling filter part by the analyte, the reacted probe-carrying particles are introduced between the B / F separation filter part and the peeling filter part, and the B / F separation filter It is preferable to move the particle liquid containing the probe-carrying particles to the part to perform B / F separation, and then move the particle liquid to the separation filter part to perform separation. In order to perform such an operation, the peeled filter part may be attached to the container after the B / F separation is performed by moving the reacted probe-carrying particles to the B / F separation filter part.

  In addition, the movement of the particle liquid to each filter part is a method in which the container is turned upside down and the liquid is moved downward by gravity with the filter part to which particles are to be moved downward, or a valve is provided in the space inside the container. A differential pressure pump is installed in the open part to the outside of the container, a predetermined open part from the space inside the container to the outside is closed by a valve, and the pressure inside the container is reduced by the differential pressure pump. It can be performed by a method of moving the liquid.

  8 to 10 are cross-sectional views showing other embodiments of the bioseparation kit of the present invention. In the present embodiment, the reaction space 41 between the subject and the probe-carrying particles is provided in the branch space 15 that communicates with the filter unit storage space 14, and the subject and the probe-carrying particles are provided in the branch space 15 from the opening 16 to the outside. The dispersion is charged, and the reaction is carried out by mixing these liquids by repeatedly moving them in the forward and reverse directions by, for example, differential pressure control. After the reaction in the branch space 15, the probe-carrying particles are moved to the filter unit 1 and subsequent processes such as B / F separation are performed.

  As shown in FIGS. 8 and 9, when the cross-sectional area of the branch space 15 is smaller than the cross-sectional area of the filter unit storage space 14, these conditions are obtained under the condition that the branch space 15 is on the horizontal plane and the influence of gravity is uniform. The liquid remains in the branch space due to the difference in surface tension due to the difference in the cross-sectional area of the channel space. When the inner diameter or inner width of the filter unit storage space is 5 mm or more, after reacting the analyte and the probe-carrying particles in the branch space 15, the reaction solution containing the particles is transferred to the filter unit storage space 14 by the differential pressure. If pushed out, it can be dropped to the lower filter section 1 (the filter section 1a for B / F separation in FIG. 9) by gravity.

  On the other hand, when the cross-sectional area of the branch space 15 is larger than the cross-sectional area of the filter unit storage space 14 as shown in FIG. 10, the liquid is easily moved to the filter unit storage space 14 by capillary action or surface tension difference.

  FIG. 11 is a cross-sectional view showing another embodiment of the bioseparation kit of the present invention. In the present embodiment, three branch spaces 15 a, 15 b, 15 c communicating with the filter unit storage space 14 are provided. The branched space 15a constitutes a reaction unit 41 that reacts by introducing the subject and the probe-carrying particle dispersion from the opening 16. The branch space 15b constitutes a cleaning liquid input unit that supplies the cleaning liquid from the opening 16 to the B / F separation unit 42 of the filter unit storage space 14, and the branch space 15c releases the stripping solution from the opening 16 to the filter unit storage space 14. A stripping solution feeding unit to be fed into the unit 43 is configured.

  In the bioseparation kit 11, the target material is separated as follows. First, the specimen and the probe-carrying particle dispersion are introduced into the branch space 15a from the opening 16 to react the specimen and the probe-carrying particles. Next, the reaction solution is moved to the B / F separation filter 1a by a differential pressure or the like, the specimen is discharged to the outside through the B / F separation filter 1a, and the probe-carrying particles are left on the filter.

  Next, a cleaning liquid is introduced from the branch space 15b into the filter housing space 14, and is allowed to pass through the B / F separation filter 1a to come into contact with the probe-carrying particles. By contacting the cleaning liquid interface while changing the distance from the filter surface, the non-capturing substance can be efficiently removed from the probe-carrying particles.

  Next, the probe-carrying particles are moved to the separation filter part 1b, and a peeling solution is introduced from the branch space 15c into the filter part storage space 14 to contact the probe-carrying particles. By bringing the peeling liquid interface into contact while changing the distance from the filter surface, the target substance can be efficiently peeled from the probe-carrying particles.

  Hereinafter, each operation for separating the target substance from the subject using the bioseparation kit of each embodiment described above will be described with reference to FIGS. 17 to 19. First, as shown in FIG. 17, the specimen and the probe-carrying particles are reacted at a position separated from the filter portion in the container. As shown in the figure, the reaction is performed with the branch space 15 branched from the filter housing space 14 as a reaction portion 41, and the reaction is performed by reciprocating the liquid in the forward and reverse directions by the differential pressure, or the B / After introducing these into the space between the F separation filter portion 1a and the separation filter portion 1b, the reaction is performed by reciprocating the liquid in the forward and reverse directions by means of differential pressure or the like.

After the reaction, the probe-carrying particles 21 are moved to the B / F separation filter unit 1a as shown in FIG. 18, and the specimen passes through the filter and is discharged to the outside. Next, the cleaning liquid is supplied from the branch space 15b to the B / F separation filter unit 1a to perform B / F separation. By performing the reaction and the B / F separation at different positions in this way, the analyte can be mixed and reacted in a high concentration state, and the filter can be damaged due to repeated movement of the liquid through the filter. Nonspecific adsorption of the analyte to the filter can be minimized.

  When cleaning and removing non-capturing substances from the probe-carrying particles with the cleaning liquid, the cleaning liquid is reciprocated multiple times (including the filter) with the filter in between, so that a plurality of cleaning liquid and particles can be moved in the forward and reverse directions. It is preferable that the contact is made twice. That is, as shown in FIG. 22, after the liquid interface is moved in the A direction from the position 55a to the position 55b, the liquid interface is moved in the B direction, and a plurality of movements in the A direction and in the B direction are alternately performed. By repeating the steps, the cleaning liquid and the probe-carrying particles can be efficiently contacted.

  That is, since the probe-carrying particles come into contact with the cleaning liquid in a state of being separated from the filter, in addition to being in contact with the cleaning liquid while sticking to the filter, the contact efficiency between the particles and the cleaning liquid is increased and the cleaning property is improved.

  It should be noted that the pressure difference during the movement of the cleaning liquid in the A direction and the movement in the B direction or the movement distance of the particles need not be the same. For example, the cleaning liquid is moved by 10 in the A direction and then moved by 1 in the B direction. This may be repeated a plurality of times.

  The above-described operation is also preferably applied to the reaction between the analyte and the probe-carrying particles and the contact between the stripping solution and the probe-carrying particles, thereby improving the reactivity and the peelability. Further, by applying the above operation to the reaction between the analyte and the probe-carrying particles, it is possible to substantially achieve both filtration and high reactivity.

  At the time of washing, after bringing the washing liquid into contact with the probe-carrying particles, the washing liquid is moved in the direction A in FIG. 22 and discharged downward through the filter unit 1a, leaving only the probe-carrying particles on the filter, After that, it is preferable to perform one or more operations of supplying the next cleaning liquid to the filter unit 1a and performing the next cleaning.

By doing so, the cleaning operation of the probe-carrying particles and the cleaning liquid becomes a batch operation, and the cleaning can be performed efficiently with a small amount of liquid.
The series of operations of contacting the probe-carrying particles with the liquid, discharging the liquid, introducing the next liquid, and contacting the probe-carrying particles are performed by the reaction between the probe-carrying particles and the analyte, and the target substance from the probe-carrying particles. It is preferably applied to peeling. When this operation is applied to the reaction between the probe-carrying particles and the analyte, the reaction is a so-called batch reaction, and the reaction can be performed efficiently with a small amount of liquid.

  As described above, when the cleaning liquid is discharged and then the next cleaning liquid is input, it is preferable to discharge the cleaning liquid and then supply the next cleaning liquid after decompressing the space inside the container. By doing in this way, it can prevent that a bubble entraps into a filter pore and a washing | cleaning liquid does not enter into a filter pore, but filterability falls. This operation is also preferably applied to the reaction between the probe-carrying particles and the analyte and the separation of the target material from the probe-carrying particles.

  The degree of reduced pressure is usually 50 Pa to 90000 Pa, preferably 100 Pa to 50000 Pa, more preferably 500 Pa to 10000 pa, although it depends on the type of the analyte in the reaction between the probe-carrying particles and the analyte, for example. In addition, when this operation is applied to the reaction between the probe-carrying particles and the specimen, it is desirable that the time for decompression is the shortest in order to minimize damage to the specimen, and usually 0.1 seconds to 60 seconds, preferably 1 to 10 seconds.

After performing the B / F separation in the B / F separation unit 42, for example, the filter unit storage space 14 is depressurized, the cleaning liquid is introduced from the branch space 15b, and the probe-carrying particles are moved to the peeling unit 43 of FIG. 19 together with the cleaning liquid. Let When moving the particle liquid to the peeling part 43, as will be described later, it is possible to select the moving flow path of the particles by the difference in the cross-sectional area of each flow path extending from the flow path branch point or the valve provided at the flow path branch point. .

  After moving the probe-carrying particles to the peeling part 43, the cleaning liquid is discharged to the outside through the peeling filter part 1b, and then the filter part storage space 14 is decompressed as necessary, and the peeling liquid is introduced from the branch space 15c. Guide to the peeling portion 43. Also in this case, the stripping solution can be selectively moved to the stripping portion 43 side due to a difference in cross-sectional area of each channel extending from the channel branch point or by providing a valve at the channel branch point.

  After supplying the stripping solution, the target material is stripped in the vicinity of the stripping filter portion 1b. As described above, the target substance can be efficiently peeled from the particles by bringing the peeling liquid into contact with the probe-carrying particles present in the peeling filter portion 1b and reciprocating the peeling liquid a plurality of times with the filter interposed therebetween. . The B / F separation filter unit 1a may be contaminated by non-specifically adsorbed substances in the subject, but since the separation process is performed by another filter unit 1b, the non-specifically adsorbed substance in the subject Contamination with the target substance can be minimized.

  In the series of operations described above, when any one of the specimen, the cleaning liquid, and the stripping liquid is moved in the filter unit storage space 14, the moving direction of the liquid is set to the horizontal direction or from above to below. It is preferable to select appropriately depending on the case whether the direction of gravity is to be applied.

As a case where it is preferable that the moving direction of the liquid is a horizontal direction, the filter unit storage space 14 is open to the outside,
(I) When the moving liquid is not discharged to the outside of the container (ii) When the liquid is moved by the differential pressure (iii) As described later, the cross-sectional area of each flow path extending from the flow path branching point is different, and the liquid is obtained by capillary action. Is selectively moved to one channel. That is, the gravity difference between the point before the movement and the point after the movement can be eliminated, and the liquid does not move against the gravity, which is appropriate in the cases (i) to (iii).

In addition, as a case where it is preferable that the moving direction of the liquid is the gravity direction, the filter unit storage space 14 is open to the outside,
(I) When the moving liquid is discharged to the outside of the container (ii) When the liquid is moved by the differential pressure (iii) As will be described later, the cross-sectional area of each flow path extending from the flow path branch point is made different so that the liquid is generated by capillary action. Is selectively moved to one channel. That is, by moving the liquid using gravity, the liquids (i) to (iii) can be appropriately moved.

As shown in FIG. 20, by rotating the container, the moving direction of the liquid in the filter unit storage space can be changed according to the situation. As illustrated, the movement of the liquid can be reversed as shown in FIG. 20B by rotating the container 12 180 degrees around the X axis from the state shown in FIG. In addition, depending on the situation, the container 12 may be rotated 180 degrees around the Y axis from the state of FIG. 20A to the state of FIG. 20C, or the container 12 may be centered on a line perpendicular to the paper surface. If 12 is rotated 90 degrees, the moving direction of the liquid changes from the gravitational direction to the horizontal direction.

In addition, when the upper and lower end surfaces of the filter unit storage space 14 are open, in order to move the liquid by the differential pressure, the inner diameter or inner width of the target space to move the liquid by the differential pressure is preferably 5 mm or less, More preferably, it is 3 mm or less, More preferably, it is 2 mm or less. Here, the “inner diameter” means the diameter when the cross-sectional shape of the internal space of the container by the parallel plane is a circle, and the short axis diameter when it is an ellipse, and the “inner width” Means the width between the opposing sides when the cross-sectional shape is square, and the width between the opposing short sides when the cross-sectional shape is rectangular, even if the cross-sectional shape is otherwise As well as By doing in this way, a liquid can be easily moved by differential pressure control by gas.

  If the inner diameter or inner width of the space is large (depending on the hydrophilicity / hydrophobicity of the inner wall of the container, the guideline is 5 mm ± 2 mm or more), the open portions on both end faces of the container are positioned horizontally with respect to the liquid in the filter unit storage space. The liquid can be easily moved by making the moving direction of the horizontal direction horizontal.

  FIG. 12 is a cross-sectional view showing another embodiment of the bioseparation kit of the present invention. In this embodiment, due to the difference in the cross-sectional areas of the three flow passage spaces extending from the branch point between the filter unit storage space and the branch space, the other two pieces branched from the flow passage space from one flow passage space. The liquid is selectively moved to one of the channel spaces.

  In FIG. 12, the cross-sectional area L of the branch space 15a, the cross-sectional area M of the filter unit storage space 14 on the B / F separation filter unit 1a side, and the cross-sectional area N of the filter unit storage space 14 on the separation filter unit 1b side. And L> N> M. In this case, if the amount of the liquid charged into the branch space 15a exceeds the capacity of the branch space 15a or the liquid charged into the branch space 15a is moved to the branch point with the filter unit storage space 14 by pressurization. (For example, a specimen and a probe-supported particle dispersion or a reaction liquid in which these are reacted in the branch space 15a). This liquid has a cross-sectional area M smaller than a cross-sectional area N on the peeling filter portion 1b side by capillary action. It moves selectively to the B / F separation filter section 1a side (from the flow path 1 to the flow path m).

When the liquid containing the probe-carrying particles washed by the B / F separation filter unit 1a is moved from the filter unit storage space 14 on the B / F separation filter unit 1a side to the branch point with the branch space 15a, The liquid selectively moves to the filter portion storage space 14 on the peeling filter portion 1b side (from the flow channel m to the flow channel n), whose cross-sectional area N is smaller than the cross-sectional area L of the branch space 15a by capillary action. .

  In addition, when controlling the movement direction of the liquid at the flow path branch point using the capillary action in this way, if the movement of the liquid is the gravitational direction or the antigravity direction, this is taken into consideration and the capillary action is It is necessary to reduce the cross-sectional area of the flow path so that the inner diameter or inner width of the space is 5 mm or less, more preferably 3 mm or less (when each flow path is formed of the same material and has the same surface tension). is there.

  FIG. 13 is a cross-sectional view showing another embodiment of the bioseparation kit of the present invention. In the present embodiment, valves 18a to 18c are provided at the branch points of the filter unit storage space 14 and the branch spaces 15a to 15c, and from this one flow path to one of the other two flow paths at this branch point. The liquid is selectively moved.

For example, when moving the analyte and the probe-carrying particle dispersion or the reaction liquid obtained by reacting these in the branch space 15a from the branch space 15a to the B / F separation filter unit 1a, the passage B2 of the valve 18b is closed, The passage B1 and the passage B3 are opened. When the analyte after the reaction is separated from the probe-carrying particles through the B / F separation filter unit 1a, the passage C3 of the valve 18c is closed and the passage C1 and the passage C2 are opened. When the cleaning liquid is introduced from the branch space 15b to the B / F separation filter 1a side of the filter housing space 14, the valve 18
The passage C2 of c is closed, and the passage C1 and the passage C3 are opened. When the cleaned probe-carrying particles are moved to the separation filter portion 1b, the passage B1 of the valve 18b is closed, the passage B2 and the passage B3 are opened, the passage A3 of the valve 18a is closed, and the passage A1 Open the passage A2. When supplying the stripping solution from the branch space 15c to the stripping filter portion 1b side of the filter portion storage space 14, the passage A1 of the valve 18a is closed and the passage A2 and the passage A3 are opened.

  Thus, the liquid can be moved in a desired direction by switching the valve and moving the liquid with the differential pressure. Further, the number of valves installed in the container can be reduced by combining the control by the valve and the method of controlling the moving direction of the liquid by the capillary action described above. The valve may be provided with a part of the valve and function as a valve in combination with an external drive system.

  Specific examples of the valve provided at the branch point of the flow path include an electromagnetic valve, a piezoelectric element valve, a diaphragm seat valve, a pinch valve, and a valve using volume change caused by heating and cooling of the gel.

  14 and 16 are cross-sectional views showing other embodiments of the bioseparation kit of the present invention. In the present embodiment, containers 51a to 51e for introducing and discharging liquid are connected to the open end surfaces 14a and 14b of the container 12 and the openings 16 of the branch spaces 15a to 15c. FIG. 15 shows an example in which this liquid inlet / outlet container is arranged.

  When the liquid inside the container 12 (mixed liquid of the particle dispersion after the reaction and the specimen, cleaning liquid, stripping liquid, etc.) is discharged to the outside of the container 12, the container 51 previously accommodated in the container 51 of FIG. The liquid interface is positioned at the discharge port (open end surfaces 14a, 14b or opening 16), and the liquid from the inside of the container 12 is brought into contact with the receiving liquid to discharge the liquid. For example, the liquid in the container 12 can be instantaneously moved to the container 51 by bringing the container 51 filled with the receiving liquid into contact with the liquid from the inside of the container 12.

  The liquid inlet / outlet container 51 may be in a form opened to the atmospheric pressure through the container 51 or may be in a form in which the container 51 is hermetically sealed and blocked from the outside. A form in which the container 51 is opened to atmospheric pressure is shown in FIG. 15A, and a form in which the container 51 is hermetically sealed is shown in FIG. 15B.

  The above-mentioned liquid connected to or directly into the open end surfaces 14a and 14b of the container 12 or the openings 16 of the branch spaces 15a to 15c is used to supply and discharge the liquid into the container of the bioseparation kit. This can be done by connecting a differential pressure pump to the containers 51a to 51e for entering and discharging. The movement of the liquid inside the container of the kit can also be performed by applying a differential pressure by this differential pressure pump.

  Various known pumps can be used as the differential pressure pump. For example, a rotary pump, a gear pump, a syringe pump, a screw pump, a tube pump, a vacuum pump, a diaphragm pump, a piezoelectric element pump, and the like can be used.

  By using the differential pressure pump in this way, for example, each open portion such as the branch spaces 15b and 15c opened to the outside of the container is sealed, and the inside of the container is depressurized by the differential pressure pump to thereby disperse the probe-carrying particle dispersion liquid. And the subject can be introduced into the branch space 15a. Similarly, other liquids such as a cleaning liquid and a stripping liquid can be charged by sealing a predetermined opening and then reducing the pressure inside the container with a differential pressure pump.

  Thus, by decompressing the inside of the container and introducing these liquids, the liquid is introduced into the container without chewing bubbles. In addition, since the bubbles present in the filter pores can be removed and the filter pores can be filled with liquid, the filtration portion of the filter is not limited by the bubbles, and the entire pores formed in the filter are used. Thus, filtration can be performed.

In FIG. 16, the target material separation operation is performed as follows, for example. First, the probe-carrying particle dispersion and the specimen are charged into the branch space 15a, and the probe-carrying particles and the specimen are reacted in the branch space 15a. Next, the passage B2 of the valve 18b is closed, the passages B1 and B3 are opened, the passage C3 of the valve 18c is closed, and the passages C1 and C2 are opened. Moving to the B / F separation side, the probe-carrying particles are captured by the B / F separation filter unit 1a and discharged to the container 51d.

  Next, with the passage C2 of the valve 18c closed and the passages C1 and C3 opened, the cleaning liquid is introduced from the branch space 15b and contacts the probe-carrying particles captured by the B / F separation filter 1a. And wash.

Next, the passage C2 of the valve 18c is closed, the passages C1 and C3 are opened, the passage B1 of the valve 18b is closed, the passages B2 and B3 are opened, the passage A3 of the valve 18a is closed, and the passage In a state where A1 and A2 are opened, the cleaning liquid is introduced from the branch space 15b, the cleaning liquid containing particles is moved to the peeling filter unit 1b side, the probe-carrying particles are captured by the peeling filter unit 1b , and the cleaning liquid is stored in the container. Discharge to 51a.

  Next, with the passage A1 of the valve 18a closed and the passages A2 and A3 opened, a stripping solution is introduced from the branch space 15c and brought into contact with the probe-carrying particles captured by the stripping filter portion 1b. Then, the target material is peeled off from the probe-carrying particles. The stripping solution containing the target material is stored in a container attached to the container end surface 14a.

  As described above, the movement of the liquid in each operation is performed after the predetermined opening portion is closed with the valves 18a to 18c or the like and the inside of the container is decompressed using any of the differential pressure pumps 54a to 54e. Can do. Further, a method of selectively introducing the liquid to one of the channels may be combined depending on a difference in cross-sectional area of each channel extending from the channel branch point.

  The bioseparation kit described above may have a configuration in which a plurality of kits are integrated on the same substrate, an example of which is shown in FIG. In the bioseparation kit 11 in the figure, two kits 11a and 11b are connected and integrated. The number of connected kits can be an appropriate number depending on the case. By connecting a plurality of kits in this way, a plurality of analytes can be processed simultaneously, and the separation and purification throughput can be improved. In addition, you may provide the injection | throwing-in part of each liquid in the position of the upper surface of the same figure, a front surface, a back surface, and a side surface.

  The bioseparation kit in which a plurality of kits are integrated on the same substrate is a desired space such as a reaction unit, a cleaning unit, a filtration unit, a B / F separation filter storage space, and a separation filter storage space on the first substrate. It can be obtained by forming a portion and a groove to be a liquid passage channel, and then attaching a separate second substrate to the first substrate. Various methods such as adhesion with an adhesive, thermal lamination, and electromagnetic induction heating can be applied to the bonding of these substrates.

  As mentioned above, although embodiment of this invention was described, this invention is not limited to these embodiment, A various deformation | transformation and change are possible within the range which does not deviate from the summary.

FIG. 1 is a cross-sectional view showing an embodiment of the bioseparation kit of the present invention. FIG. 2 is a cross-sectional view showing another embodiment of the bioseparation kit of the present invention. FIG. 3 is a cross-sectional view showing another embodiment of the bioseparation kit of the present invention. FIG. 4 is a cross-sectional view of a bioseparation kit in which a reaction plate is provided with a stirring plate. FIG. 5 is a cross-sectional view of a bioseparation kit in which a stirring plate is provided in the vicinity of the filter portion. FIG. 6 is a cross-sectional view of a bioseparation kit in which both end surfaces of the container are opened and an opening is formed between the pair of filter portions that is open to the outside from the filter portion storage space. FIG. 7 is a cross-sectional view of a bioseparation kit in which both end surfaces of the container are opened and an opening that opens to the outside from the filter unit storage space is provided between a pair of filter units. FIG. 8 is a cross-sectional view of a bioseparation kit in which a reaction portion between a subject and probe-carrying particles is provided in a branch space that is branched from the filter portion storage space and one end is opened to the outside. FIG. 9 is a cross-sectional view of a bioseparation kit in which a reaction portion between a specimen and probe-carrying particles is provided in a branch space that is branched from a filter portion storage space and one end is opened to the outside. FIG. 10 is a cross-sectional view of a bioseparation kit in which a reaction part between a subject and probe-carrying particles is provided in a branched space that branches off from the filter unit storage space and has one end opened to the outside. FIG. 11 is a cross-sectional view of a bioseparation kit provided with a branch space for supplying a cleaning liquid and a stripping liquid. FIG. 12 is a cross-sectional view of a bioseparation kit configured to send a liquid in a predetermined direction by making the cross-sectional area of each flow path extending from the branch point different. FIG. 13 is a cross-sectional view showing an example in which a valve is provided in the flow path of the bioseparation kit. FIG. 14 is a cross-sectional view showing an example in which a liquid inlet / outlet container is provided at the opening of the bioseparation kit to the outside of the container. FIG. 15 is a view showing an example in which a container for liquid entry / exit is provided in the opening to the outside of the container. FIG. 16 is a cross-sectional view showing a separation system using the bioseparation kit of the present invention. FIG. 17 is a cross-sectional view showing a state in which probe-carrying particles are introduced into a reaction part in the bioseparation kit of the present invention and reacted with a subject. FIG. 18 is a cross-sectional view showing a state where the probe-supported particles after reaction are moved to the B / F separation unit from the state of FIG. FIG. 19 is a cross-sectional view showing a state in which the cleaned probe-carrying particles are moved to the peeling portion from the state of FIG. FIG. 20 is a cross-sectional view showing a state before and after the bioseparation kit is rotated about the vertical direction and the horizontal direction as axes. FIG. 21 shows a bioseparation kit in which a plurality of kits are integrated. FIG. 22 is a cross-sectional view illustrating an agitation operation in which the liquid is moved in the forward and reverse directions in the vicinity of the filter portion to bring the liquid and the probe-carrying particles into contact with each other a plurality of times. FIG. 23 is a cross-sectional view showing an embodiment of the bioseparation kit of the present invention. FIG. 24 is a cross-sectional view showing an embodiment of the bioseparation kit of the present invention. FIG. 25 is an electron micrograph of a filter part having a structure in which ribs are continuously formed integrally and a well having a filter as a bottom surface exists therein.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Filter part 1a B / F separation filter part 1b Separation filter part 2 Filter 3 Pore 4 Rib 5 Well 11 Bio separation kit 12 Container 14 Filter part storage space 14a, 14b End face 15 Branching space 15a Branching space (analyte) And particle input)
15b Branch space (for cleaning liquid input)
15c Branch space (for stripping solution input)
15A to 15D Branch space 16 Opening 17 Stirring plates 18a to 18c Valve 21 Probe-carrying particles 31 Probe-carrying particle dispersion 32 Subject 33 Washing liquid 41 Reaction unit 42 B / F separation unit 43 Separation unit 51, 51a to 51e Container 53 Liquid inlet / outlet passage 54a-54e Syringe pump 55a, 55b interface

Claims (18)

A metal filter part in which straight pores having a straight hole center line are formed, and a container for storing the filter part,
A method for producing a bioseparation kit having a reaction part that reacts a subject and probe-carrying particles in a region separated from a position where the filter part is arranged inside the container,
The filter unit prepares a substrate that has been previously imparted with conductivity, and resist patterning is performed on the substrate, and a portion that is not electroplated is protected with a resist, and a current flows between the substrate and the plating solution. A method for producing a bioseparation kit, characterized in that a metal material is formed only on a predetermined portion of a substrate and the filters are stacked by electroplating. The method for producing a bioseparation kit according to claim 1, wherein a rib formed of the same material as the filter and integrated with the filter is provided on the surface of the filter. A metal filter part in which straight pores having a straight hole center line are formed, and a container for storing the filter part,
In a region separated from the position where the filter unit is arranged inside the container, a reaction unit that reacts the analyte and the probe-carrying particles ,
The filter unit prepares a substrate that has been previously imparted with conductivity, and resist patterning is performed on the substrate, and a portion that is not electroplated is protected with a resist, and a current flows between the substrate and the plating solution. forming a metal material only a predetermined portion of the substrate by, kit Bioseparation, wherein the this are those prepared by the method of stacking by electroplating the filter. The bioseparation kit according to claim 3, wherein a rib formed of the same material as that of the filter and integrated is provided on a surface of the filter. The bioseparation according to any one of claims 3 to 4 , wherein a stirring plate is provided in at least one of positions in the vicinity of the reaction section and the filter section in the container. kit. The two filter parts are accommodated in the container, one of which is a B / F separation filter part for performing B / F separation, and the other part peels off the target material captured by the probe-carrying particles. The bioseparation kit according to any one of claims 3 to 5, wherein the bioseparation kit is an exfoliation filter part. Both end surfaces of the filter part storage space for storing the B / F separation filter part and the peeling filter part in the container are opened to the outside,
The inner space sandwiched between the B / F separation filter portion and the separation filter portion in the filter portion storage space is opened to the outside via at least one branch space that branches from the inner space. The bioseparation kit according to claim 6 , wherein the kit is a bioseparation kit. The container has at least one branch space branched from a filter part storage space for storing the B / F separation filter part and the peeling filter part in the container and provided with an opening opened to the outside on one end side thereof. , from the opening through the branch space, dispersion of probe-supported particles, wherein the analyte, the washing liquid, and at least one liquid of the stripping solution was so put into the filter unit accommodating space according Item 7. The bioseparation kit according to item 6 . The bioseparation kit according to claim 8 , wherein the branching space includes the reaction unit that causes a subject to react with a probe-carrying particle. Having at least two branch spaces, a cleaning solution is supplied from one branch space to the filter unit storage space, and a stripping solution is supplied from the other branch space to the filter unit storage space. kit Bioseparation according to any of claims 8 9, it said. The container has at least one branch space branched from a filter part storage space for storing the B / F separation filter part and the peeling filter part in the container and provided with an opening opened to the outside on one end side thereof. ,
For the three flow path spaces l, m, and n extending from the branch point between the branch space and the filter unit storage space, the cross-sectional area M of the flow path space m is made smaller than the cross-sectional area N of the flow path space n. The bioseparation kit according to claim 6 , wherein the liquid is selectively moved from the channel space l to the channel space m of the channel space m and the channel space n. The bioseparation kit according to any one of claims 3 to 11 , wherein a plurality of the bioseparation kits are integrated and mounted on the same substrate. The probe-carrying particles and the analyte are reacted in the reaction unit using the bioseparation kit according to any one of claims 3 to 12 , and then the probe-carrying particles are moved to the filter unit, and then a cleaning solution A bioseparation method characterized in that a target substance captured by a probe-carrying particle is separated from another substance by bringing the substance into contact with the probe-carrying particle. The probe-carrying particles are moved to the B / F separation filter unit after the probe-carrying particles and the analyte are reacted in the reaction unit using the bioseparation kit according to any one of claims 6 to 11. Then, by separating the target substance captured by the probe-carrying particles and other substances by bringing the cleaning liquid into contact with the probe-carrying particles, the probe-carrying particles are moved to the separation filter unit, A bioseparation method comprising peeling a target substance from probe-carrying particles by bringing a peeling solution into contact with the probe-carrying particles. The probe-carrying particles are positioned in the vicinity of the filter unit, and the probe-carrying particles are reciprocated a plurality of times in the filter unit storage space by moving at least one of the specimen, the cleaning liquid, and the stripping liquid in the filter unit storage space. The bioseparation method according to claim 13, wherein the liquid and the probe-carrying particles are brought into contact with each other while being brought into contact with or separated from the part. After contacting at least one of the analyte, the cleaning liquid, and the peeling liquid with the probe-carrying particles, the liquid is discharged through the filter unit and the probe-carrying particles are left on the filter unit, and then The bioseparation method according to any one of claims 13 to 15 , wherein the next liquid is introduced into the filter unit and brought into contact with the probe-carrying particles. The bioseparation method according to any one of claims 13 to 16 , wherein after the pressure in the filter unit storage space is reduced, at least one of a specimen, a cleaning solution, and a stripping solution is introduced into the space. Wherein the subject, the washing liquid, and at least one liquid of the stripping solution, when moved in the interior of the container, in any one of claims 13, characterized in that for moving the liquid in the horizontal direction or the direction of gravity 17 Bioseparation method.