JP2006102720A - Filter for bioseparation and kit for bioseparation using the filter - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a filter for bioseparation giving no damage to a test object with high B/F (Bound form/Free form) separation efficiency and low filter resistance, and capable of easily separating a target object, and a kit for bioseparation. <P>SOLUTION: The filter 1 for bioseparation has a filter 2 having pores 3 each having a straight axis and having a pore diameter distribution of a straight line and ribs 4 substantially integrated with the filter 2. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a bioseparation filter that separates biological components such as nucleic acids, proteins, and cells by B / F separation, and a bioseparation kit 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 sample on a carrier on which a probe or a ligand is immobilized, the carrier is separated from other substances not captured by the carrier. form / Free form) separation or affinity 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 (Non-Patent Document 1). 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.

  In addition, affinity chromatography that captures and separates a target substance using a column in which a probe-fixed gel is used as a carrier and is filled with agarose gel or the like 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 addition, a method of separating probe-carrying particles such as beads with a filter is widely used (Patent Document 1). As a filter for separation, glass fiber filter paper, membrane filter or the like is used, and the thickness of these filters is usually 100 μm to several mm. These filters have a structure in which the pores of the filter are three-dimensionally complicated and the surface area of the pores is very large, so the filtration resistance is very large, usually 100 to 1000 hPa (0.1 to 1 atmosphere). Filtration resistance is required. For this reason, it is necessary to increase the mechanical strength of the container for storing the filter so that the connecting portion between the container or the container and the filter can withstand this filtration resistance, and it is difficult to use a minute container. In addition, because the surface area inside the filter is very large, there are many non-specific adsorbents in the filter after the sample has been circulated, but filtration and washing must be carried out in one direction. There is. In other words, when washing is performed by feeding in the reverse direction, non-specifically adsorbed substances adhering to the inside of the filter adhere to the probe-supporting particles again, so it is necessary to carry out washing with liquid feeding from one direction. It is difficult to perform sufficient cleaning by feeding liquid from

  Filters with holes formed by electron beams on a film of polyester, polycarbonate, etc. (trade name: Cyclopore Whatman Co., Ltd.) Company-made) is commercially available. However, this filter is hydrophobic because it is made of polyester, polycarbonate, etc., and the aqueous solution as a biosolvent is difficult to permeate, and the pore spacing is very wide and random, and the aperture ratio is very low, so the filtration resistance is low. Get higher.

  A filter (trade name: manufactured by Anopore Whatman Co., Ltd.) having a pore diameter of 200 μm or less obtained by anodizing aluminum is also commercially available. However, in this filter, there is a high possibility that the filter hole will be clogged with impurities of the specimen. When this filter is used, since the mechanical strength of the filter is small, the filter is used in a state where the filter is placed on a mesh-like support provided on the bottom surface of the resin container. For this reason, a subject or the like may enter the gap between the support and the filter.

In the current situation as described above, in bioseparation, there is a need for a technique that does not damage an object, has high B / F separation efficiency, has low filtration resistance, and can easily separate target substances.
JP-A 63-270000 Nucleic Acid Research Volume 14 p5037-5048 (1986)

  An object of the present invention is to provide a bioseparation filter and a bioseparation kit that do not damage a subject, have high B / F separation efficiency, low filtration resistance, and can easily separate a target substance. It is said.

  A bioseparation filter of the present invention is characterized by comprising a filter in which pores having straight axes are formed so that the pore diameter has a distribution, and a rib substantially integrated with the filter. .

The thickness of the filter is preferably 1 to 100 μm.
The aperture ratio of the filter is preferably 15 to 60%.
The material of the filter is preferably silica, titania, alumina, nickel, gold, titanium, aluminum, or stainless steel.

In a preferred aspect of the invention, the first filter is provided on one end side of the rib, and the second filter is provided on the other end side with the rib interposed therebetween.
In a preferred aspect of the invention described above, a dispersion liquid in which probe-carrying particles (including particles carrying a ligand) are dispersed is accommodated in the well.

  When a dispersion liquid in which probe-carrying particles are dispersed is accommodated in the well, the particle diameter of the probe-carrying particles is substantially uniform, the particle diameter d and the pore diameter h of the pores formed in the filter And the hole interval p of the pores is preferably d> h + p.

In a preferred embodiment of the above invention, a probe is bound using the filter as an immobilization carrier.
The bioseparation kit of the present invention is characterized by comprising the above-described bioseparation filter and a container for storing the bioseparation filter.

In a preferred embodiment of the above invention, the container is provided with a plurality of independent spaces that are independent of each other corresponding to the plurality of wells in the bioseparation filter.
In a preferred aspect of the invention described above, the container is provided with a shared space that shares the flow of the liquid through the filter with respect to the plurality of wells in the bioseparation filter.

In the preferable aspect of said invention, the both sides through the said filter for bio separation in the said container are open | released outside.
It is preferable that the inner diameter or inner width of the internal space for storing the bioseparation filter in the container, or the height of the upper side internal space of the container formed above the rib or the filter is 5 mm or less. .

  The bioseparation filter and bioseparation kit of the present invention do not damage the analyte during B / F separation, have high B / F separation efficiency, low filtration resistance, and can easily separate target substances. is there.

Hereinafter, embodiments of the present invention will be described with reference to the drawings. 1 (a) and 1 (b)
It is sectional drawing of the filter for bio separation in one Embodiment of this invention. As shown in the figure, the bioseparation filter 1 of FIG. 1A is provided with a filter 2 on its upper surface, and a rib 4 that is substantially integrated with the filter 2 is formed on the lower side of the filter 2. . The filter 2 is formed with pores 3 having a straight hole axis.

  The bioseparation filter 1 shown in FIG. 1B is provided with a filter 2 at the bottom. In the filter 2, as in the bioseparation filter of FIG. 1A, pores 3 having straight pore axes are formed. A rib 4 substantially integrated with the filter 2 is provided on the upper surface side of the filter 2, and the well 5 is configured with the rib 4 as a side surface and the filter 2 as a bottom surface.

  The rib 4 functions as a side wall of the well 5 and functions as a reinforcing material for reinforcing the filter 2. 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 each well 5. It is desirable that That is, the 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 most preferable in terms of mechanical strength. An example in which such a rib 4 is actually formed is shown in FIG.

  The number of wells provided in the bioseparation filter is arbitrary, and may be one or many. In the embodiment of FIG. 1 (a), a structure having a filter on the top surface is shown, and in FIG. 1 (b), a structure having a filter on the bottom surface is shown. May be.

  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 the method of integrally forming the filter and the rib include a method of manufacturing the filter and the rib from the same material by a subtractive method such as etching, an additive method of electroforming, and a method of imprint (nanoimprint). It is done.

  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 is increased, and impurities such as an analyte do not enter between the filter and the rib, and the filter can have a high B / F separation capability. .

  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 can be filtered with a differential pressure of 10 to 50 gf / cm ^{2. In} the bioseparation kit described later, as a container for storing the bioseparation filter It is possible to make a thin container wall structure with a minimum mechanical pressure resistance, and extremely small containers can be used.

  In the present invention, the pores of the filter have a pore size distribution. In the present specification, “the pores of the filter have a pore size distribution” represent two cases of a random pore size pattern and a predetermined pore size pattern. In any of these cases, the CV (Coefficient of Variation) value of the pore diameter is larger than 20%, preferably 30 to 300%, more preferably 50 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 the distribution to the pore diameter of the filter in this manner, it is possible to effectively prevent an increase in filtration pressure due to the probe-carrying particles blocking the pores during filtration.
As one form of the hole diameter pattern having the hole diameter distribution, there is a pattern in which the hole diameters of 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 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.

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

As another form of the 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 sparse to reduce the opening area of this region, and the pores near the periphery of the well. There is a pattern in which the opening area of this region is increased by increasing the hole diameter of the holes or by increasing the hole interval. In this case, the filterability of the filter can be improved because the analyte uniformly passes through the filter pores.

  The pore diameter of the pores is preferably 1 μm or more, more preferably 1 μm 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. Also, if the pore diameter exceeds 50 μm, the surface area of the filter pores is reduced when fixing the probe to the filter surface, and large particles are used when fixing the probe to the particles. In either case, the probe amount is reduced, and the filterability of the target material 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 20 μm, still more preferably 2 μm to 10 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 filter aperture ratio is preferably 15% to 60%. When the aperture ratio is less than 15%, the filtration resistance may be excessively increased. When the aperture ratio exceeds 60%, the mechanical strength as a filter may be insufficient. Here, the aperture ratio is a percentage of the ratio of the total area of the filter surface in the region where these pores are formed to the sum of the average areas in the cross section of each pore formed in the filter. In general, the aperture ratio is for the entire surface of the filter portion constituting the bottom or top surface of one well, and is a number excluding the side wall area portion of the well.

The material of the filter is not particularly limited as long as it can form a filter. For example, a metal, a metal oxide, an organic material, or the like can be used, but a material having high mechanical strength is particularly preferable. Specifically, engineering plastics such as liquid crystal polymer, polycarbonate, polyamide resin, polyimide, polyethylene terephthalate, polyethylene naphthalate, polyarylate, polysulfone, 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 alloy, aluminum magnesium silicon alloy, aluminum zinc magnesium Metals such as alloys and iron-nickel alloys; metal oxides 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 and Diamond Like Examples thereof include carbon materials such as Carbon (DLC); glass such as soda glass, borosilicate glass, Pyrex (registered trademark), and quartz glass.

  Among these, silica, titania, alumina, nickel, gold, titanium, or stainless steel is preferable. These have high mechanical strength, can make the filter thin, and are relatively hydrophilic, so they have a high affinity with aqueous biosolutions, and can easily put the sample into the filter pores. Can do. In addition, you may use these as a base material, may oxidize the surface, or may surface-treat with a surface treating agent by another material. Corona treatment may be performed as the surface treatment. Examples of the surface treatment agent include nonspecific adsorption inhibitors for biomaterials such as alcohol, hydrophilizing agent, hydrophobizing agent, and protein.

  The bioseparation filter of the present invention can be produced by a method of stacking filters and the like by electroplating. In this method, a substrate that has been previously provided with conductivity is prepared, patterned thereon by a photolithography method or an imprint method, and the portion that is not electroplated is protected with a resist, and the substrate and the plating solution A metal material is formed only on a predetermined portion of the substrate by passing an electric current between them.

  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, the metal is electrically filled between these posts by applying 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. For example, in the case of a nickel sulfamate bath, an electroplated material having a thickness of 5 μm can be obtained by applying a direct current for about 10 to 20 minutes at a voltage of 6 V and a current density of 3 A / dm ² .

  After the filter is produced in this manner, the rib is further formed by electroplating in the same manner as the filter forming 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.

Further, the bioseparation filter of the present invention can be produced by producing at least one of a filter and a rib by an etching method. The object to be etched is not particularly limited as long as it can form a filter, such as a metal, a metal oxide, and an organic substance. Particularly preferable is a material having high mechanical strength. Specifically, for example, a liquid crystal polymer, Engineering plastics such as polycarbonate, polyamide resin, polyimide, polyethylene terephthalate, polyethylene naphthalate, polyarylate, polysulfone, 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 alloy, aluminum magnesium silicon alloy, aluminum zinc magnesium copper alloy, iron nickel alloy, etc. Metals: Metal oxides such as silica, alumina, titania, zirconia and tantalum oxide; Metal nitrides such as SiN, TiN and TaN; Metal carbides such as SiC and WC; Carbon such as diamond, graphite and Diamond Like Carbon (DLC) 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 exhibits a large crystal plane dependency with respect to an etching solution such as KOH aqueous solution, ethylenediamine / pyrocatechol (EDP), or 4-methylammonium 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, a particularly preferable method is pattern etching from both sides of plates made of a plurality of materials having different compositions, such as aluminum / alumina, metal silicon / silica, or metal titanium / titania. This is a method of forming a filter and a rib by performing the above. 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 A silicon wafer with a film or an SOI (Silicone On Insulator) wafer can be used.

  Moreover, the filter for bioseparation of the present invention can be produced by an 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.

  A method for producing a filter having a hole diameter pattern having a hole diameter distribution is obtained by designing the mask pattern or the convex mold so as to have a hole diameter distribution in the photoresist mask or imprint mold. be able to.

  FIG.1 (c) is sectional drawing which showed the filter for bio separation in other embodiment of this invention. As shown in the figure, the bioseparation filter 1 is provided with a first filter 2a at the bottom and a second filter 2b on the opposite side of the first filter 2a across the rib 4. ing.

  With the structure as shown in FIG. 1C, a plurality of completely independent wells are formed. For example, each particle combined with different probes can be divided and stored in each well. In addition, since the well height is uniform, the reaction space between the analyte and the probe carried on the particles is controlled in a narrow range, and the reaction rate can be improved.

This bio-separation filter can be produced by joining a pair of bio-separation filters, each having a filter on one side of the ribs, butting these ribs together. For example, after the probe-carrying particles are housed in the well of one bioseparation filter, the other bioseparation filter is attached. Examples of the attachment method include an adhesive, magnetic force, mechanical fitting, surface bonding between smooth portions, and mechanical pressurization. In addition, the bioseparation kit including the bioseparation filter of FIG. 1 (c) and a container for storing the bioseparation filter is, for example, one of a pair of bioseparation filters provided with a filter on one side. The other container is attached to the lower container, the upper container and the lower container are made to face each other, and these are attached by mechanical pressurization with an adhesive, a magnet, a clamp, or the like.
<Accommodation of probe-supported particle dispersion>
In the bioseparation filter of the present invention as shown in FIGS. 1A to 1C, a dispersion liquid in which probe-carrying particles are dispersed is accommodated in the well. Examples of the manner in which the probe-carrying particles are accommodated in each well include a mode in which particles carrying the same probe are contained in each well and a mode in which particles carrying different probes are contained in each well. it can.

  When the particles carrying different probes are accommodated in each well, as shown in FIG. 2, the particles 21 a to 21 d carrying different probes may be placed in each well 5 with the filter 2 at the bottom side. it can.

  Further, when the particles carrying the same probe are accommodated in each well, as shown in FIG. 3A, the particle 21 carrying the same probe for each well 5 with the filter 2 at the bottom side. Alternatively, as shown in FIG. 3 (b), the filter 2 is placed on the upper side, and the space formed by the side wall of the container 12 of the kit and the filter 2 is used as a well to contain particles. You may do it.

  Further, as shown in FIGS. 4A and 4B, particles in which the same probe is carried in each space (well 5) in which the pair of filters 2a and 2b are opposed to each other with the rib 4 interposed therebetween. 21 (FIG. 4 (a)) or particles 21a-21d (FIG. 4 (b)) carrying different probes can be inserted.

  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.

  Also, 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 cellulose, starch, agarose, galactose, etc. Natural cross-linked gels and synthetic cross-linked gels such as 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 1.5 μm to 120 μm, more preferably 1.5 μm to 30 μm, and even more preferably 3 μm to 15 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 diameter is large, the surface area of the particle per well unit volume becomes relatively small, and the amount of probe may decrease, or the reactivity may decrease due to steric hindrance.

  Preferably, the particles carrying the probe have a substantially uniform particle diameter. As shown in FIG. 5, the particle diameter, the diameter of the pore 3 formed in the filter 2, and two adjacent The relationship between the pore spacing between the pores 3 and 3 is d> h + p. Here, d is the particle diameter of the probe-carrying particles 21, h is the hole diameter of the pores 3 formed in the filter 2, and p is the interval between the adjacent pores 3 and 3. Here, the pore diameter of the pores 3 and the interval between the pores 3 and 3 are average values for 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.

  As a method for storing the probe-carrying particles in each well, there may be mentioned a method in which various probes corresponding to each well are fixed to the particles in advance and put into the corresponding well by a spotter or the like. Alternatively, particles having probe binding sites on the surface may be introduced into each well by a spotter or the like, and then different types of probes corresponding to a predetermined well may be introduced into the corresponding well by a spotter or the like. .

The absolute number of probe-carrying particles stored in each well is controlled by individually controlling the particles with a flow cytometer when the particle dispersion is charged, and by charging while actually counting each particle with a CCD camera or the like. Can do. Alternatively, the particle concentration of the particle dispersion is measured in advance, the number of particles is calculated from the particle concentration of the dispersion and the specific gravity of the particles, and the amount of the particle dispersion calculated backward from this result is charged with a spotter or the like. Thus, an approximate number of particles can be introduced into the well. Alternatively, a predetermined number of particles may be spotted in a plurality of times, the number of spotted particles may be counted each time to calculate an insufficient amount, and spotting may be performed until the insufficient amount is approximately zero. Here, the number of particles accommodated in each well may have a certain degree of approximate identity, but the error range is preferably within 20% in terms of CV value, and more preferably within 10%. .
<Immobilization of probe to filter>
6 and 7 are cross-sectional views illustrating a bioseparation filter according to another embodiment of the present invention. As shown in the figure, in this embodiment, the filter 2 in the bioseparation filter 1 is used as an immobilizing carrier, and probes 22 are bonded to both surfaces and the inner surface of the pore.

  In FIG. 6A, the same probe 22 for each well 5 is fixed to the filter 2 provided at the bottom of each well 5. On the other hand, in FIG. 6B, the same probe 22 for each well 5 is fixed to the filter 2 provided on the top of each well 5.

  In FIG. 7A, different types of probes 22 for each well 5 are immobilized on the filter 2 provided at the bottom of each well 5. On the other hand, in FIG. 7B, different types of probes 22 for each well 5 are immobilized on the filter 2 provided on the top of each well 5.

  In this embodiment, since the probe is immobilized on a filter in which straight pores are formed, the filtration resistance is low, so that the filtration time can be shortened, and damage to the target material is minimized. Can do. Moreover, compared with the case where a conventional membrane filter is used, the reaction between the probe and the target substance is very fast, and filtration and cleaning are easy.

  Thus, when immobilizing a probe to a filter, in order to increase the surface area of the filter, it is preferable to increase the thickness of the filter appropriately, and the thickness of the filter is preferably 5 μm to 100 μm. When the film thickness is less than 5 μm, the surface area as the immobilization carrier may be too small, and when it exceeds 100 μm, the filtration resistance may be excessively increased.

In this way, the bioseparation filter in which the probe is immobilized on the filter may have a form in which the pore diameter is uniform in addition to the form in which the pore diameter of the filter has a distribution.
Immobilization of the probe or ligand to the filter can be performed, for example, as follows. First, a probe or ligand linker is introduced into the filter with a spotter or the like, and the linker is immobilized on the filter. Next, after washing away the unfixed linker, the probe or ligand is added to bind to the linker, and finally the unbound probe or ligand is washed away.

If the probe is an oligo DNA or peptide, a phosphoramidite or amino acid NCA monomer is introduced into the well using a spotter or the like, and solid-phase polymerization is performed using the filter as a solid phase under a predetermined condition, so that the probe is formed by so-called in situ polymerization. It is also possible to do. Specifically, such probe formation can be performed by a method described in, for example, Japanese Patent Publication No. 7-53749 and Japanese Patent Application Laid-Open No. 61-205298.

By making the probe or probe precursor to be introduced the same for each well or different from each other, the same probe can be formed in each well as shown in FIG. 6, or each well as shown in FIG. Different probes can be formed.
<Bioseparation kit>
The above-described bioseparation filter of the present invention is used as a separation kit together with a container that houses the filter. 8 to 17 are cross-sectional views showing embodiments of the bioseparation kit.

The container for storing the bioseparation filter may have various shapes as long as it has a structure for storing the bioseparation filter therein. For example, in FIG. 8A, the entire upper surface is open. In FIG. 8 (b), the upper surface has a partially opened shape, that is, an open portion 13 to the outside having a diameter smaller than the filter diameter is formed on the upper surface of the container (note that The open portion 13 means a communication hole branched from the storage space of the container in which the bioseparation filter is stored and having one end opened to the outside of the container or a separate space provided in the container) . In FIG. 9, the upper surface is formed in a tapered shape, and an open portion 13 to the outside is formed at the top. In addition, the bottom surface may be closed, or the bottom surface may be entirely or partially open. Here, the expressions “upper surface” and “lower surface” are for convenience, and the container surface may be used upside down depending on the case. Moreover, the form which provided the open surface of the container on either side may be sufficient as a filter surface made into the vertical direction like FIG.

  Further, as shown in FIGS. 15 to 17, it is also possible to provide an opening in which the entire surface is opened or an opening part 13 that is partially opened to the outside on both sides of the bioseparation filter 1. Good. In FIG. 15 (a), a container 12 is used in which openings that are open to the entire surface are provided on both sides, and the inner space is straight from the upper surface to the lower surface of the container. . In FIG. 15 (b), a container provided with an opening that is open on the entire surface on one side and an opening 13 that is partly open to the outside and that has an inner diameter smaller than the inner diameter of the filter on the other side. 12 is used. In FIG. 16, a container 12 is used in which open portions 13 that are open to the outside are provided on the side surfaces on both sides of the bioseparation filter 1. In FIG. 17, a container 12 is used in which an open portion 13 having an inner diameter smaller than the filter inner diameter and partially opened to the outside is provided on both upper and lower portions through the bioseparation filter 1. Yes.

  In this bioseparation kit, an analyte is put into a bioseparation filter (or a bioseparation filter in which a probe is immobilized on a filter as shown in FIGS. 6 and 7) in which probe-carrying particles are housed in a well and allowed to react. Next, the target substance is covered by removing the non-captured substance by bringing the cleaning liquid into contact with the probe-carrying particles on which the target substance is captured (in the case of FIGS. 6 and 7, the probe-carrying filter on which the target substance is captured). Selectively separate from other substances in the specimen. It is also possible to put a stripping solution into the wells so that the target material in each well is stripped from the probe-carrying particles and taken out into a predetermined storage space formed in the container.

  The bioseparation kit of the present invention is used as follows, as described in detail below. For example, containers such as analytes, cleaning liquids, and stripping liquids are stored in the container, and the bioseparation filter and the container are moved relative to each other, thereby bringing the liquid interface into contact with the filter and introducing these liquids into the wells. Then, the probe-carrying particles and these liquids are agitated by relative movement (vertical movement) between the bioseparation filter and the container or by other methods. Alternatively, a differential pressure is applied from the open part to the outside provided in the container to move the interface of the liquid such as the specimen, the cleaning liquid, the stripping liquid, etc. in the container, thereby bringing the liquid interface and the filter into contact with each other. These liquids are introduced into the inside, and the liquid is moved in the forward and reverse directions by differential pressure, or the probe-carrying particles and these liquids are stirred by another stirring method.

  In this way, an open part to the outside provided in the container (branched from the storage space of the container in which the bioseparation filter is stored, and one end thereof was opened to the outside of the container or a separate space provided in the container The communication hole) plays a role such as a hole for introducing and discharging each liquid such as a specimen, a cleaning liquid, and a stripping liquid into the storage space of the container, and a differential pressure control.

  For example, in order to discharge the liquid to the outside of the container, an exhaust liquid receiving container filled with liquid is installed on one end side of the open portion, and the liquid in the exhaust liquid receiving container and the liquid discharge hole of the kit container are used. The liquid in a container can be discharged | emitted by making it contact with said opening part which functions.

For example, when the inner diameter of the container is a narrow inner diameter of about 1 mm, it is difficult to discharge the liquid due to capillarity, but for example, as shown in FIG. 19, below the storage space for storing the bioseparation filter 1 in the container 12 The contact protrusions 18 provided in the isolation spaces corresponding to the respective wells 5 in the lower container 12 that receives the discharged liquid are brought into contact with the filter 2 so that the liquid in the wells can be instantaneously supplied to the lower container. 12 can be moved.

  Further, differential pressure control can be performed by connecting a differential pressure pump to the open portion (liquid inlet / outlet hole) or an inlet / outlet container installed at one end of the open portion. By this differential pressure pump, the liquid can be poured into the kit container from the inlet / outlet container, the liquid can be moved in the container, and the liquid can be discharged from the 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, crosslinked polyvinyl alcohol, polyglycolic acid, polyamide, polyimide, cellulose acetate, triacetyl cellulose, cellulose nitrate, epoxy, and a copolymer of 2-methacryloyloxyethyl phosphorylcholine and methacrylate. 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 bioseparation filter is fixed to the container. In this case, for example, as shown in FIG. 11A, a step portion 14 is provided in the container 12, and the bioseparation filter 1 can be fitted and joined to the step portion 14. Alternatively, as shown in FIG. 11B, the bioseparation filter 1 may be sandwiched and sealed in the container 12 via a sealing material such as an O-ring 15.

  Examples of the method for joining and integrating the bioseparation filter with the container include a method of bonding the container and the bioseparation filter with an adhesive, and a method of insert molding together with the bioseparation filter when molding the container. It is done.

In one embodiment of the bioseparation kit of the present invention, a space for sharing the liquid flow through the filter (hereinafter also referred to as a shared space) is provided for a plurality of wells in the bioseparation filter. A bioseparation kit provided with such a shared space is shown in FIGS.

  The shared space 17 is configured inside the container 12 on the lower surface side of the filter 2. When the bioseparation filter 1 is fixed to the container 12, the shared space 17 is formed by the space between the bioseparation filter 1 and the bioseparation filter 1 and the container 12 are independent from each other. The common space 17 is configured by a space formed by housing the bioseparation filter 1 in the container 12.

In the shared space 17, as shown in FIGS. 14A and 14B, the aforementioned opening 13 may be provided on the bottom surface side or the side surface side of the container 12.
By providing the shared space 17 in this way, each liquid such as a subject is accommodated in the shared space 17, and the bioseparation filter 1 and the container 12 are moved relative to each other, or pressure increase / decrease is performed from the open portion 13. Thus, the liquid can be introduced in parallel into the plurality of wells 5 through the filter 2. Further, the liquid can be circulated through each well 5 by putting the liquid in and out of each well 5 through the shared space 17.

  As a result, for example, the reaction can be caused to flow through each well 5 containing different types of probe-carrying particles through the shared space 17 while the analyte is circulated. Since the common space 17 and each well 5 are adjacent to each other through only one layer of the filter 2, the pressure loss is very small, and the analyte reaches each well 5 and the probe-carrying particles in each well 5. Is performed in parallel, the time required for contact and reaction between the probe-carrying particles and the analyte is very short.

In addition, in the bioseparation kit in which each liquid such as a specimen and a cleaning liquid is introduced into the above-mentioned common space and the liquid level is moved by the differential pressure to perform reaction, cleaning, etc., the internal container for storing the bioseparation filter in the container The inner diameter d or inner width d of the space in a plane parallel to the filter surface direction or the surface direction of the upper and lower surfaces of the container, or the height h of the inner space on the upper side of the container formed on the upper side from the rib or filter is 5 mm. Or less, more preferably 3 mm or less, and still more preferably 2 mm or less. Here, the “inner diameter d” 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 the cross-sectional shape is an ellipse. “d” represents the width between opposite sides when the cross-sectional shape is a square, and the width between opposite long sides when the cross-sectional shape is a rectangle. Are also defined in the same way. Examples of the inner diameter d (inner width d) and the height h are shown in FIGS. 20 (a) and 20 (b). In FIG. 20A, the inner diameter d (inner width d) and the height h of the inner space on the upper side of the container 12 formed on the upper side of the rib 4 are formed, and in FIG. The height h of the upper side internal space of the container 12 is shown. When a predetermined amount of liquid is introduced into the shared space and the liquid level of the shared space is moved up and down, depending on the material of the container, even in the case of a hydrophilic material such as glass, for example. When the above-mentioned inner diameter d, inner width d, and height h exceed 5 mm, it is usually difficult to raise the liquid level with a differential pressure caused by a gas such as air.

  The reaction between the target substance in the subject and the probe using the bioseparation kit of the present invention is performed, for example, as follows. Predetermined probe-carrying particles are stored in advance in the well of the bioseparation filter. The specimen is put into the well of the bioseparation filter so that the specimen and the probe-carrying particles in all the wells can come into contact with each other. In this process, for example, the well is moved up and down in the specimen in the container, or the interface between the specimen in the container is moved by a differential pressure or the like, thereby dispersing the specimen in the container and the probe-carrying particles in the well. The liquid is brought into contact with and integrated to diffuse (and react) the target substance and the probe in the subject. At this time, when a bioseparation filter as shown in FIG. 1B in which a filter is provided only at the bottom is used, the interface height of the liquid in the well must not exceed the height of the rib. is there.

  When the specimen is put into the well until the predetermined interface height is reached, the specimen is put in the container in advance using a kit with an independent bioseparation filter and container, and the bioseparation filter is placed there. It is possible to introduce the analyte into each well through a filter. At this time, if there is a pressure loss of the filter, the part where the bioseparation filter and the container are in contact with each other (for example, the outer periphery of the bioseparation filter and the inner periphery of the container) is sealed and applied to the filter. By applying a pressure or a depressurizing force, the analyte can be introduced into each well through the filter.

  In addition, the distance between the bottom of the container and the bottom surface of the filter can be shared from the open part formed on the low or side wall of the container using a bioseparation kit that is fixed at a fixed distance between the bottom of the container and the filter. The subject can be introduced from the lower surface side of the filter through the space (liquid storage chamber). For example, the subject can be introduced into the well by introducing pressurized air from an open portion provided in the shared space and pushing up the interface of the subject in the container.

  In addition, the subject may be introduced from the opening on the upper surface side of the well, and in this case, it is preferable to introduce an equal amount for each well with a spotter or the like. In addition, when using a bioseparation filter in which a rib is sandwiched between the first filter and the second filter as shown in FIG. Pressurize from the open part provided in the sample and send the subject to the well, or depressurize the shared space and send the sample from the opposite side of the shared space through the bioseparation filter Good.

  Next, the well is moved up and down in the specimen stored in the container of the bioseparation kit, or the interface of the specimen stored in the container is moved up and down, or the specimen is stirred to move into each well. The reaction between the target substance in the subject and the probe is advanced by exchanging the subject.

  As a method for stirring the specimen, existing methods such as stirring by a stirrer equipped with a stirring blade, stirring by sonic vibration or ultrasonic vibration, stirring by movement of air or paramagnetic material can be used. Among these, vibration agitation using sound waves or ultrasonic waves is particularly preferable. For example, a container or a bioseparation filter is vibrated, or a vibrator is placed in a subject to vibrate. In this case, the applied frequency of vibration is appropriately adjusted depending on the type of the probe and the subject, and is preferably 100 Hz to 1 GHz, more preferably 1 to 10 kHz or 300 kHz to 1 GHz. At frequencies in this range, object damage can be minimized.

  In addition, when the container and the bioseparation filter are independent, after the bioseparation filter is pulled up from the subject in the container, the subject in the container is stirred by any of the methods described above, and then the bioseparation filter is Replace the specimen in each well by re-immersing the separation filter in the specimen in the container, rotating the bio-separation filter on a horizontal plane, or moving the container and the bio-separation filter relative to each other. Can do. As described above, the analyte can be exchanged between the wells by guiding the analyte to the shared space through the filter and then guiding the analyte into another well via the filter. .

After the target material and the probe are reacted in this way, B / F separation is performed as follows. First, the interface height of the liquid in the container is lowered until it is below the lower surface of the filter, and non-capturing substances (substances other than the target substance captured by the probes) that have not reacted with the probes supported on the probe-supporting particles are removed. The contained liquid is removed from each well.

  Next, the cleaning liquid is introduced into the well, the cleaning liquid is circulated through the well through the well to allow the cleaning liquid to be taken in and out of the well, and then the cleaning liquid is discharged out of the well to wash away the non-capture substance.

  Here, as a method of making the liquid interface height less than the lower surface of the filter, the liquid in the well is pumped through the filter or from the open part (discharge hole) to the outside of the container by pressure increase / decrease, etc. The method of moving to space is mentioned. Further, when the container and the bioseparation filter are independent, the height of the liquid interface can be made lower than the lower surface of the filter by moving the container and the bioseparation filter relative to each other and moving up and down. In this case, the bioseparation filter may be transferred to an adjacent container or another independent container.

  When using a bioseparation filter in which ribs are sandwiched between the first filter and the second filter as shown in FIG. 1C, the probe-carrying particles are stored in the well. Since there is no need to worry about particle leakage from the liquid, the liquid level can be arbitrarily moved beyond the height of the well.

  Further, before and after the above operation, various cleaning agents, various buffer solutions, or chemical solutions such as secondary antibodies may be added to the wells as necessary. These inputs can be performed in the same manner as the input of the subject.

  After separating the target material from the specimen in this way, a release agent can be introduced into the well to separate the target material from the probe-carrying particles, and the target material can be taken out into a container. A bioseparation kit using a container for this purpose is shown in FIGS. It should be noted that a container as shown in the figure may be used to peel the target substance from the probe-carrying particles, and a separate container may be used for the reaction or washing between the probe and the specimen.

  The illustrated container 12 is provided with a plurality of independent spaces 16 a to 16 d corresponding to the plurality of wells 5 in the bioseparation filter 1. These independent spaces 16 a to 16 d constitute a storage chamber (well) that receives the target material peeled off from the probe-carrying particles, and is disposed immediately below the predetermined well 5. For example, a convex fitting portion and a concave fitting portion are formed on the lower surface of the bioseparation filter 1 and the upper surface of the container 12, and these are fitted to each other so as to be below a predetermined well. A predetermined independent space can be located.

  When the target substance is peeled off from the probe-supported particles and taken out into the independent space of the container, for example, from the opening on the upper surface of the well of the bioseparation filter, using a spotter or the like, individually or into each well at once. Then, the solution in the well is moved to the independent space by making contact with the separate liquid stored in the independent space on the filter surface, or by applying reduced pressure from the external opening provided at the bottom of the independent space. Let

The bottom surface of each independent space provided in the container 12 may be closed as shown in FIG. 12 (a), or as shown in FIG. 12 (b), a hole-shaped opening 13 penetrating from the bottom surface to the outside. May be formed. When such an opening 13 is provided on the bottom surface, the inner diameter of the hole is preferably 3 mm or less, more preferably 1 mm, in order to prevent liquid leakage from the bottom surface. For example, by applying a decompression force with a differential pressure pump or the like through the opening 13,
The liquid containing the target material (or the liquid for detecting whether or not the target material is contained) contained in each independent space can be collectively flowed down and taken out to the independent space.

  The separation liquid thus obtained is analyzed by each detection device such as a mass spectrometer, a Raman analyzer, a surface plasmon analyzer, an X-ray analyzer, an electrochemical detection device, a quartz oscillator microbalance detection device, etc.・ Identified. Alternatively, the target substance in the obtained separation liquid is used as a starting material for the next reaction step, or a purified product for the assay.

  The containers provided with independent spaces shown in FIGS. 12 (a) and 12 (b) are used for putting a sample into the well of a bioseparation filter containing probe-carrying particles and bringing them into contact and reacting with each other. It can also be used. That is, for example, different specimens are accommodated in each of the independent spaces 16a to 16d, and a bioseparation filter 1 in which a dispersion liquid of particles in which the same or different types of probes are supported in each well 5 is accommodated in the container 12. Can be brought into contact with each other and brought into contact with each other through the filter surface.

  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. 1A is a cross-sectional view of a bioseparation filter in one embodiment of the present invention, and FIG. 1B is a cross-sectional view of a bioseparation filter in another embodiment of the present invention. FIG. 2 is a cross-sectional view of a bioseparation filter containing a dispersion of probe-carrying particles in a well. FIG. 3 is a cross-sectional view of a bioseparation filter in which a dispersion of probe-carrying particles is contained in a well. FIG. 4 is a cross-sectional view of a bioseparation filter containing a dispersion of probe-carrying particles in a well. FIG. 5 is a cross-sectional view illustrating the relationship between the particle diameter of the particles carrying the probe, the hole diameters of two adjacent pores on the filter, and the hole spacing. FIG. 6 is a cross-sectional view of a bioseparation filter in which the same probe is immobilized on the bottom or top filter of each well. FIG. 7 is a cross-sectional view of a bioseparation filter in which different probes are immobilized on the bottom or top filter of each well. FIG. 8 is a cross-sectional view of the bioseparation kit with the container upper surface opened. FIG. 9 is a cross-sectional view of a bioseparation kit in which the upper surface of the container is tapered and the top is opened. FIG. 10 is a cross-sectional view of the bioseparation kit in which the filter surface of the bioseparation filter is arranged in the vertical direction and the left and right sides of the container are opened. FIG. 11 is a cross-sectional view of a bioseparation kit in which a bioseparation filter is fixed to a container. FIG. 12 is a cross-sectional view of a bioseparation kit in which an independent space corresponding to each well of the bioseparation filter is provided in the container. FIG. 13 is a cross-sectional view of a bioseparation kit in which a plurality of wells in the bioseparation filter have spaces in the container that share the flow of liquid through the filter. FIG. 14 is a cross-sectional view of a bioseparation kit in which a plurality of wells in a bioseparation filter are provided with a space for sharing the liquid flow through the filter. FIG. 15 is a cross-sectional view of the bioseparation kit with both sides of the container open. FIG. 16 is a cross-sectional view of the bioseparation kit with both sides of the container open. FIG. 17 is a cross-sectional view of the bioseparation kit with both sides of the container open. FIG. 18 is an electron micrograph of a bioseparation filter having a structure in which ribs are continuously formed integrally and a well having a filter as a bottom surface exists therein. FIG. 19 is a cross-sectional view of a bioseparation kit in which both sides of the container are opened, and a discharged liquid receiving container provided with contact protrusions for discharging the liquid in the well by contacting the filter. FIG. 20 is a cross-sectional view for explaining the inner diameter d or inner width d of the internal space for accommodating the bioseparation filter in the container, and the height h of the upper internal space of the container formed above the filter or rib. .

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Bioseparation filter 2 Filter 2a 1st filter 2b 2nd filter 3 Pore 4 Rib 5 Well 11 Bioseparation kit 12 Container 13 Opening part 14 Step part 15 O-ring 16a-16b Independent space 17 Shared space 18 Contact Protrusion 21 Probe-carrying particles 21a to 21d Probe-carrying particles 22 Probes 22a to 22d Probe 23 Dispersion liquid

Claims (13)

  A bioseparation filter comprising: a filter having pores with straight axes and a pore diameter distribution, and a rib substantially integrated with the filter.   The bioseparation filter according to claim 1, wherein the filter has a thickness of 1 to 100 μm.   The bioseparation filter according to claim 1 or 2, wherein the filter has an aperture ratio of 15 to 60%.   The bioseparation filter according to any one of claims 1 to 3, wherein a material of the filter is silica, titania, alumina, nickel, gold, titanium, aluminum, or stainless steel.   The bio according to any one of claims 1 to 4, wherein a first filter is provided on one end side of the rib, and a second filter is provided on the other end side of the rib. Separation filter.   The bioseparation filter according to any one of claims 1 to 5, wherein a dispersion liquid in which probe-carrying particles are dispersed is accommodated in the well.   The particle diameter of the probe-carrying particles is substantially uniform, and the relationship between the particle diameter d, the pore diameter h of the pores formed in the filter, and the pore spacing p of the pores is d> h + p. The bioseparation filter according to claim 6, wherein the filter is a bioseparation filter.   The bioseparation filter according to any one of claims 1 to 5, wherein a probe is bound using the filter as an immobilization carrier.   A bioseparation kit comprising: the bioseparation filter according to any one of claims 1 to 5; and a container for housing the bioseparation filter.   The bioseparation kit according to claim 9, wherein the container is provided with a plurality of independent spaces independent of each other corresponding to a plurality of wells in the bioseparation filter.   The bioseparation kit according to claim 9, wherein the container is provided with a shared space for sharing the flow of the liquid through the filter with respect to the plurality of wells in the bioseparation filter. .   The bioseparation kit according to any one of claims 9 to 11, wherein both sides of the container through the bioseparation filter are open to the outside.   An inner diameter or an inner width of an internal space for storing the bioseparation filter in the container, or a height of an upper side internal space of the container formed on the upper side from the rib or the filter is 5 mm or less. The bioseparation kit according to any one of claims 9 to 12.