JP7351065B2 - Biological particle concentration device, concentration system and concentration method, and biological particle detection method - Google Patents

Description

The present invention relates to a concentration device, a concentration system, and a concentration method used for concentrating biological particles, and a method for detecting biological particles.

Patent Document 1 discloses that a bundle of polyethylene porous hollow fiber membranes whose surfaces are coated with an ethylene-vinyl alcohol copolymer is used to capture microorganisms.

Patent Document 2 discloses the use of a filter membrane with a bubble point pore size not exceeding 1.0 μm to trap microorganisms.

Patent Document 3 discloses a method for producing a hydrophilized microporous polymer membrane, in which a hydrophilic monomer is subjected to a radiation graft reaction on the surface of a microporous polymer membrane made of a hydrophobic resin.

Patent Document 4 describes a hydrophilic microporous membrane obtained by copolymerizing a hydrophilic monomer having one vinyl group and a crosslinking agent having two or more vinyl groups in a microporous polymer membrane by a graft polymerization method. A membrane is disclosed.

Patent Document 5 discloses the use of a porous hollow fiber membrane in which a polyolefin porous hollow fiber substrate is coated with glycerin fatty acid ester as a membrane for concentrating and separating bacterial cells.

Patent Document 6 describes a polyethylene resin having a viscosity average molecular weight exceeding 1 million, containing at least one crystal component having a melting peak temperature of 145°C or higher, having a porosity of 20 to 95%, and an average pore diameter of 0. Microporous membranes are disclosed that are between .01 and 10 μm.

Patent Document 7 states that it is made of polyethylene resin, has a film thickness of more than 25 μm and 1 mm or less, has an average pore diameter of 0.01 to 10 μm, and has a structure factor F of 1.5×10 ⁷ sec ^-2・m ⁻¹・Pa ^-2 A medical separation filter including the highly permeable microporous membrane described above is disclosed.

US Pat. No. 5,001,302 discloses a method for removing aggregates present in a fluid containing biological products by passing it through a membrane filter that has been pretreated with a surfactant.

Patent Document 9 discloses a method for capturing and recovering oral microorganisms in a test liquid on a filter membrane.

Patent Document 10 discloses a hydrophilic composite porous membrane comprising a porous structural matrix made of polyolefin and an ethylene/vinyl alcohol copolymer coating layer covering the pore surfaces of the matrix.

Japanese Patent Application Publication No. 11-090184 Special Publication No. 2013-531236 Japanese Patent Application Publication No. 2009-183804 Japanese Patent Application Publication No. 2003-268152 Special Publication No. 06-057143 Japanese Patent Application Publication No. 2004-016930 Japanese Patent Application Publication No. 2002-265658 Special table 2016-534748 publication Japanese Patent Application Publication No. 2006-71478 Japanese Unexamined Patent Publication No. 61-271003

For example, viruses or bacteria may be isolated from specimens collected from living organisms for the purpose of diagnosing infectious diseases. One of the methods for separating viruses or bacteria is the centrifugation method, which involves repeating centrifugation by changing the centrifugal force, centrifuging the sample by placing it on a buffer solution with a density gradient, or performing ultracentrifugation. This is a method that requires equipment, effort, and time. Another method for separating viruses or bacteria is to use a porous membrane to separate them, but conventional methods only aim to completely remove viruses, etc. from the filtrate; The method is complicated, requiring adsorption and backwashing to remove viruses, etc., and lacks the perspective of efficiently concentrating and recovering viruses, etc. in the specimen.

The embodiments of the present disclosure were made under the above circumstances. That is, the embodiments of the present disclosure aim to provide a concentration device, a concentration system, and a concentration method that easily, quickly, and efficiently concentrate biological particles, and a method for detecting biological particles. The task is to solve this problem.

Specific means for solving the above problem include the following aspects.
[1] It has an inlet and an outlet, and a liquid to be treated containing biological particles and water is injected from the inlet and discharged from the outlet due to a pressure difference between the inlet and the outlet. a housing; a hydrophilic porous membrane provided within the housing to separate the inlet and the outlet to which the biological particles are not adsorbed; a concentrating membrane that transmits a discharged liquid that is a liquid with a reduced concentration of biological particles from the liquid to be treated; and a space upstream of the concentrating membrane in the housing, wherein a concentration space for accommodating a concentrate, which is a liquid with an increased concentration of the biological particles.

[2] The biological particle concentration device according to [1], wherein in the housing, the volume of the concentration space is 0.05 to 5 cm ³ .

[3] The device for concentrating biological particles according to [1] or [2], wherein in the housing, the filtration area of the concentrating membrane is 1 to 20 cm ² .

[4] The biological particles according to any one of [1] to [3], wherein in the housing, a guide groove continuous from the inlet is formed in the inner wall portion facing the concentration space. concentration device.

[5] The housing according to any one of [1] to [4], wherein the inner wall facing the concentration space has a shape that gradually increases in diameter from the inlet toward the concentration membrane. Biological particle concentration device.

[6] The concentration membrane includes a hydrophilic composite porous membrane comprising a porous base material and a hydrophilic resin that coats at least one main surface and the inner surface of the pores of the porous base material. The biological particle concentration device according to any one of [1] to [5].

[7] The biological particle concentration device according to [6], wherein the porous substrate is a microporous polyolefin membrane.

[8] The hydrophilic resin has a main chain of the polymer consisting only of carbon atoms, and has one or more functional groups selected from the group consisting of a hydroxy group, a carboxy group, and a sulfo group in the side chain, [6] or The biological particle concentration device according to [7].

[9] The hydrophilic resin may be polyvinyl alcohol, olefin/vinyl alcohol resin, acrylic/vinyl alcohol resin, methacrylic/vinyl alcohol resin, vinylpyrrolidone/vinyl alcohol resin, polyacrylic acid, polymethacrylic acid, or The biological particle concentration device according to any one of [6] to [8], which contains one or more selected from the group consisting of fluorosulfonic acid resin and polystyrene sulfonic acid.

[10] The biological particle concentration device according to any one of [6] to [9], wherein the hydrophilic resin is an olefin/vinyl alcohol resin.

[11] The concentration membrane is any one of [1] to [10], wherein the ratio t/x of the membrane thickness t (μm) to the average pore diameter x (μm) measured with a palm porometer is 50 to 630. A device for concentrating biological particles according to claim 1.

[12] The device for concentrating biological particles according to any one of [1] to [11], wherein the concentration membrane has a thickness t of 10 to 150 μm.

[13] The biological particle concentration device according to any one of [1] to [12], wherein the concentration membrane has an average pore diameter x of 0.1 to 0.5 μm as measured by a palm porometer.

[14] The biological particle concentration device according to any one of [1] to [13], wherein the concentration membrane has a bubble point pore diameter y of more than 0.8 μm and 3 μm or less as measured by a palm porometer.

[15] The concentration membrane has a ratio f/y of water flow rate f (mL/(min cm ² MPa)) to bubble point pore diameter y (μm) measured with a palm porometer of 100 to 480. , the biological particle concentration device according to any one of [1] to [14].

[16] The biological particle concentration device according to any one of [1] to [15], wherein the concentration membrane has a surface roughness Ra of 0.3 to 0.7 μm.

[17] Biological particles, comprising the biological particle concentration device according to any one of [1] to [16], and means for applying a differential pressure between the inlet and the outlet. concentration system.

[18] Supplying the liquid to be treated to the concentration device according to any one of [1] to [16], and applying a pressure difference between the inlet and the outlet of the concentration device. A method for concentrating biological particles, comprising the steps of: obtaining the concentrate in the concentration space; and recovering the concentrate from the concentration space.

[19] Supplying the liquid to be treated to the concentration device according to any one of [1] to [16], and applying a pressure difference between the inlet and the outlet of the concentration device. , comprising the steps of obtaining the concentrate in the concentration space, collecting the concentrate from the concentration space, and detecting the biological particles contained in the collected concentrate. , a method for detecting biological particles.

According to embodiments of the present disclosure, a concentration device, a concentration system, a concentration method, and a method for detecting biological particles that easily, quickly, and efficiently concentrate biological particles are provided.

FIG. 1 is a perspective view schematically showing an example of a biological particle concentration device according to the present disclosure. 2 schematically shows a cross section of the concentration device of FIG. 1; In the concentration device of FIG. 2, a state in which the liquid to be treated is supplied from the inlet is schematically shown. A state in which a differential pressure is applied between the inlet and the outlet from the state in FIG. 3 is schematically shown. A state in which a concentrated liquid is obtained from the state in FIG. 4 is schematically shown. A state in which the concentrated liquid is recovered from the state in FIG. 5 is schematically shown. FIG. 7 is a perspective view schematically showing another example of the overall shape of the housing. FIG. 7 is a perspective view schematically showing another example of the overall shape of the housing. FIG. 7 is a perspective view schematically showing another example of the overall shape of the housing. FIG. 7 is a perspective view schematically showing another example of the overall shape of the housing. FIG. 7 is a perspective view schematically showing another example of the shape of the entrance. FIG. 7 is a perspective view schematically showing another example of the shape of the entrance. FIG. 7 is a perspective view schematically showing another example of the shape of the entrance. FIG. 7 is a perspective view schematically showing another example of the shape of the entrance. FIG. 7 is a perspective view schematically showing another example of the shape of the outlet. FIG. 7 is a perspective view schematically showing another example of the shape of the outlet. FIG. 7 is a perspective view schematically showing another example of the shape of the outlet. FIG. 7 is a perspective view schematically showing another example of the shape of the outlet. FIG. 7 is a perspective view schematically showing another example of the positional relationship between an inlet and an outlet. FIG. 7 is a perspective view schematically showing another example of the positional relationship between an inlet and an outlet. FIG. 7 is a perspective view schematically showing another example of the positional relationship between an inlet and an outlet. FIG. 7 is a perspective view schematically showing another example of the shape of the internal space of the housing. 22 schematically shows the cross section of FIG. FIG. 7 is a perspective view schematically showing another example of the shape of the internal space of the housing. The cross section of FIG. 24 is schematically shown. FIG. 7 is a perspective view schematically showing another example of the shape of the internal space of the housing. FIG. 7 is a perspective view schematically showing another example of the shape of the internal space of the housing. The cross section of FIG. 27 is schematically shown. FIG. 7 is a perspective view schematically showing another example of the shape of the internal space of the housing. The cross section of FIG. 29 is schematically shown. FIG. 2 is a perspective view schematically showing an example of a method for collecting a concentrated liquid. FIG. 3 is a perspective view schematically showing another example of a method for collecting a concentrated liquid. FIG. 33 is a perspective view schematically showing a state in which the concentrated liquid has been collected from the state in FIG. 32. FIG. 3 is a perspective view schematically showing an example of the shape of the concentration membrane 30. FIG. 3 is a perspective view schematically showing another example of the shape of the concentration membrane 30. FIG. 3 is a perspective view schematically showing another example of the shape of the concentration membrane 30. FIG. 3 is a perspective view schematically showing another example of the shape of the concentration membrane 30. FIG. FIG. 1 is a perspective view schematically showing an example of a concentration system. FIG. 3 is a perspective view schematically showing another example of the concentration system. FIG. 3 is a perspective view schematically showing another example of the concentration system. FIG. 3 is a perspective view schematically showing another example of the concentration system. FIG. 3 is a perspective view schematically showing another example of the concentration system. FIG. 3 is a perspective view schematically showing another example of the concentration system.

Embodiments of the invention will be described below. These descriptions and examples are illustrative of embodiments and are not intended to limit the scope of the invention. The mechanism of action described in this disclosure includes speculation, and its validity does not limit the scope of the invention.

In the present disclosure, when embodiments are described with reference to drawings, the configuration of the embodiments is not limited to the configuration shown in the drawings. Furthermore, the sizes of the members in each figure are conceptual, and the relative size relationships between the members are not limited thereto.

In the present disclosure, a numerical range indicated using "~" indicates a range that includes the numerical values written before and after "~" as the lower limit and upper limit, respectively.

In the numerical ranges described step by step in this disclosure, the upper limit or lower limit described in one numerical range may be replaced with the upper limit or lower limit of another numerical range described step by step. . Furthermore, in the numerical ranges described in this disclosure, the upper limit or lower limit of the numerical range may be replaced with the values shown in the Examples.

In the present disclosure, the term "step" is included not only in an independent step but also in the case where the intended purpose of the step is achieved even if the step cannot be clearly distinguished from other steps.

In the present disclosure, each component may contain multiple types of corresponding substances. When referring to the amount of each component in the composition in this disclosure, if there are multiple types of substances corresponding to each component in the composition, unless otherwise specified, the amount of each component in the composition is means the total amount of substance.

In the present disclosure, "(meth)acrylic" means at least one of acrylic and methacrylic, and "(meth)acrylate" means at least one of acrylate and methacrylate.

In the present disclosure, the term "monomer unit" refers to a component of a polymer, which is formed by polymerizing monomers.

In the present disclosure, "machine direction" means the longitudinal direction of a membrane, film, or sheet manufactured in a long shape, and "width direction" means a direction perpendicular to the "machine direction." In the present disclosure, the "machine direction" is also referred to as the "MD direction", and the "width direction" is also referred to as the "TD direction".

In the present disclosure, the "principal surface" of a membrane, film, or sheet means a wide outer surface of the membrane, film, or sheet other than the outer surface extending in the thickness direction. The membrane, film or sheet has two major surfaces. In the present disclosure, the "side surface" of a membrane, film, or sheet refers to an outer surface of the membrane, film, or sheet that extends in the thickness direction.

In the present disclosure, the side of the concentration membrane into which the liquid to be treated flows in is referred to as "upstream," and the side from which the liquid to be treated flows out is referred to as "downstream."

<Concentration device>
The present disclosure provides a concentration device for use in concentrating biological particles. The concentration device of the present disclosure processes a "liquid to be treated" which is a water-containing liquid that may contain biological particles, and concentrates it into a "concentrated liquid" with an increased concentration of biological particles. do.

Here, regarding the liquid to be treated, "containing water" means that water is used as a solvent or a component, and the content thereof is not particularly limited. Regarding the liquid to be treated, "containing biological particles" refers to a state in which the biological particles are not dissolved in the liquid to be treated but are floating, suspended, or precipitated.

Biological particles as used in the present disclosure conceptually include particles possessed by living things, particles released by living things, particles parasitic on living things, microscopic living things, vesicles with lipid membranes, and fragments thereof. included. Specifically, viruses, parts of viruses (for example, particles obtained by removing the envelope from an enveloped virus), bacteriophages, bacteria, spores, fungi, molds, yeasts, cysts, protozoa, unicellular algae, Plant cells, animal cells, cultured cells, hybridomas, tumor cells, blood cells, platelets, organelles (e.g. cell nuclei, mitochondria, vesicles), exosomes, apoptotic bodies, particles of lipid bilayers, particles of lipid monolayers , liposomes, protein aggregates, and fragments thereof. Furthermore, the biological particles referred to in the present disclosure include not only natural products but also artificial products.

As shown in the schematic perspective view of FIG. 1, the device 10 for concentrating biological particles 50 according to the present disclosure has an appearance including an inlet 21 and an outlet 22 opening into a housing 20 having an internal space. In this figure, an example of the shape of the housing 20 is shown as a cylinder having a longer diameter than its height. More specifically, as shown in the schematic cross-sectional view of FIG. 2, in the internal space of the housing 20, a cylindrical inlet 21 that protrudes upward on the upstream side opens, and a cylindrical inlet 21 that protrudes downward on the downstream side opens. The outlet 22 is open. In the internal space of the housing 20, an inlet 21 and an outlet 22 are separated by a concentrating membrane 30. A space in the housing 20 on the upstream side of the concentration membrane 30 is a concentration space 24 .

In other words, the housing 20 has an inlet 21 and an outlet 22. Further, due to the pressure difference between the inlet 21 and the outlet 22, the liquid to be treated 40 containing biological particles 50 and water is injected from the inlet 21 and discharged from the outlet 22.

Further, the concentration membrane 30 is provided within the housing 20 so as to separate the inlet 21 and the outlet 22. The concentration membrane 30 is a hydrophilic porous membrane to which biological particles 50 are not adsorbed, and a liquid obtained by reducing the concentration of biological particles 50 from the liquid to be treated 40 is spread from the surface on the inlet 21 side to the surface on the outlet 22 side. The discharge liquid 42 is allowed to pass through.

The concentration space 24 is a space within the housing 20 on the upstream side of the concentration membrane 30 (in other words, a region defined by the inner wall 23 of the housing 20 and the main surface of the concentration membrane 30 on the upstream side). The concentration space 24 accommodates a concentrated liquid 41 that is a liquid obtained by increasing the concentration of biological particles 50 from the liquid to be treated 40 using the concentration membrane 30 .

The liquid to be processed 40 injected into the concentration device 10 of the present disclosure includes animal (especially human) body fluids (e.g., blood, serum, plasma, cerebrospinal fluid, lacrimal fluid, sweat, urine, pus, nasal discharge, sputum); dilutions of animal (especially human) body fluids; liquid compositions of animal (especially human) excrement (e.g. feces) suspended in water; animal (especially human) gargle fluids; animal (especially human) organs , buffer solutions containing extracts from tissues, mucous membranes, skin, squeezed specimens, swabs, etc.; tissue extracts of fish and shellfish; water collected from fish and shellfish culture ponds; surface swabbing liquids or tissue extracts of plants; Examples include soil extracts; plant extracts; food extracts; pharmaceutical raw material liquids.

[Concentration method and detection method of biological particles 50]
A method for concentrating biological particles 50 using the concentration device 10 of the present disclosure is as follows.

As shown in FIG. 3, this concentration device 10 undergoes a step in which a liquid to be treated 40 containing biological particles 50 and water is supplied from an inlet 21.

Next, by applying a pressure difference between the inlet 21 and the outlet 22, a step of obtaining a concentrate in the concentration space 24 is carried out.

That is, by applying a pressure difference between the inlet 21 and the outlet 22 to the injected liquid to be treated 40, the drained liquid 42 that has passed through the concentration membrane 30 is discharged, as shown in FIG. . The differential pressure at this time can be generated by increasing the pressure from the inlet 21, reducing the pressure from the outlet 22, or both. As described above, the concentration of biological particles 50 in the discharged liquid 42 is reduced compared to the liquid to be treated 40.

Then, as shown in FIG. 5, a concentrated liquid 41 is obtained within the concentration space 24. As described above, the concentrated liquid 41 has a higher concentration of biological particles 50 than the liquid to be treated 40 .

Next, a step of recovering the concentrated liquid 41 from the concentration space 24 is performed. That is, as shown in FIG. 6, the concentrated liquid 41 is collected from the concentrated space 24 using an appropriate tool or device such as a micropipette.

Finally, a step of detecting biological particles 50 contained in the collected concentrate 41 is performed. Biological particles 50 contained in the collected concentrated liquid 41 are detected by appropriate means depending on the type and properties thereof. For example, when the target to be detected by the biological particles 50 is a nucleic acid (DNA or RNA), polymerase chain reaction (PCR), Southern blotting, Northern blotting, etc. are performed. When the detection target of the biological particles 50 is a protein, mass spectrometry, Western blotting, immunochromatography, etc. are performed. When the detection target of biological particles 50 is sugar or lipid, mass spectrometry or the like is performed.

In the housing 20, the volume of the concentration space 24 can be determined as appropriate depending on the properties and amount of the liquid to be treated 40, but in consideration of convenience of use, it is preferably 0.05 to 5 cm ³ .

In the housing 20, the filtration area, which is the part where the concentration membrane 30 actually comes into contact with the liquid to be treated 40, can be determined as appropriate depending on the properties and amount of the liquid to be treated 40, but it should be determined in consideration of the convenience of use. For example, it is preferably 1 to 20 cm ² .

<Overall shape of housing 20>
The overall shape of the housing 20 is not limited to the cylindrical shape as shown in FIG. 1, but can have various shapes.

For example, the shape may be a triangular prism (FIG. 7), a quadrangular prism (FIG. 8), or another polygonal prism, such as a hexagonal prism (FIG. 9). In either case, an inlet 21 is provided on one of the prismatic bottoms (for example, the upper base), and an outlet 22 is provided on the other (for example, the lower base).
Further, the overall shape of the housing 20 can also be made into a spherical shape as shown in FIG. In this case as well, an inlet 21 is provided at one pole of the spherical shape (for example, the upper pole), and an outlet 22 is provided at the other pole (for example, the lower pole).

The material of the housing 20 is not particularly limited, but synthetic resins, particularly polypropylene resins, polyethylene resins, vinyl chloride resins, fluorine resins, ABS resins, MBS resins, polycarbonate resins, acrylic resins, and polystyrene resins are desirable. Although there is no particular limitation on the method of molding the housing 20, an upstream member including the inlet 21 and a downstream member including the outlet 22 are formed by injection molding, and the concentration membrane 30 is inserted between these members. The housing 20 can be formed by combining both members with the two members sandwiched therebetween by an appropriate method such as adhesion, welding, or screwing.

<Shape of entrance 21>
The shape of the inlet 21 is not limited to the upwardly projecting cylindrical shape shown in FIG. 1, but can be made into various shapes.

For example, as shown in FIG. 11, the inlet 21 can be formed as a simple hole without an upwardly protruding structure. In this case, a transport pipe for the liquid to be treated 40 can be inserted into the inlet 21 .

Furthermore, as shown in FIG. 12, a threaded groove (male thread) may be provided on the outer peripheral surface of the cylindrical inlet 21 that projects upward. In this case, by providing a female screw threadedly engaged with this thread groove at the end of the transport path for the liquid to be treated 40, the transport path and the inlet 21 can be prevented from coming off.

Furthermore, as shown in FIG. 13, the male side of the Luer lock can be provided on the outer peripheral surface of the tip of the cylindrical inlet 21 that projects upward. In this case, by providing a female side that fits with the male side of the Luer lock at the end of the transport path for the liquid to be treated 40, the transport path and the inlet 21 can be prevented from coming off.

Further, as shown in FIG. 14, the inlet 21 can be formed in a lid-like shape that closes the housing 20 whose top surface is open from above.

<Shape of outlet 22>
The shape of the outlet 22 is not limited to the downwardly protruding cylindrical shape as shown in FIG. 1, but can be made into various shapes.

For example, as shown in FIG. 15, the tube diameter can be different from that of the inlet 21.

Furthermore, as shown in FIG. 16, a threaded groove (male thread) may be provided on the outer peripheral surface of the cylindrical outlet 22 that projects downward. In this case, by providing a female screw threadedly engaged with this thread groove at the tip of the recovery path for the drained liquid 42, the recovery path and the outlet 22 can be prevented from coming off.

Furthermore, as shown in FIG. 17, the male side of the Luer lock can be provided on the outer peripheral surface of the tip end of the cylindrical outlet 22 that projects downward. In this case, by providing a female side that fits with the male side of this Luer lock at the tip of the recovery path for the drained liquid 42, the recovery path and the outlet 22 can be prevented from coming off.

Further, as shown in FIG. 18, the outlet 22 can be formed in a cylindrical shape that closes off the housing 20 whose bottom surface is open from the bottom surface. At this time, for example, a female thread is formed on the inner peripheral surface of the housing 20, a male thread is formed on the outer peripheral surface of the outlet 22, and these are screwed together to strengthen the connection between the housing 20 and the outlet 22. be able to.

<Positional relationship between inlet 21 and outlet 22>
Note that, as shown in FIG. 19, the inlet 21 and the outlet 22 can also be provided on the side surface of the cylindrical shape. In this case, since the inlet 21 and the outlet 22 need to be separated from each other by the concentration membrane 30, they need to be provided at different positions in the height direction of the columnar shape. As long as they are provided in such positions, the inlet 21 and the outlet 22 may be provided in opposite directions as shown in FIG. 19, or may be provided in the same direction as shown in FIG. Furthermore, as shown in FIG. 21, they may be provided so as to be separated from each other by an arbitrary plane angle.

<Internal shape of housing 20>
The inside of the housing 20 is also not limited to the shape shown in FIGS. 1 and 2, but can have various shapes.

For example, in the housing 20, a guide groove 25 continuous from the inlet 21 may be formed in the inner wall portion 23 (see FIG. 2) facing the concentration space 24. For example, as shown in the perspective view of FIG. 22 and the cross-sectional view of FIG. A guide groove 25 can be provided as a groove. Furthermore, as shown in the perspective view of FIG. 24 and the cross-sectional view of FIG. can. Furthermore, as shown in the perspective view of FIG. 26, a guide groove 25 is provided, which is a combination of a radial groove as shown in FIG. 22 and a concentric groove centered on the inlet 21 that intersects with the radial groove. You can also do it. By providing such a guide groove 25, the liquid to be treated 40 injected from the inlet 21 is easily guided to the concentration space 24 by the capillary force of the guide groove 25.

On the other hand, by forming the housing 20 into a tapered shape that tapers toward the inlet 21 as shown in the perspective view of FIG. The facing inner wall portion 23 may have a shape in which the diameter gradually increases from the inlet 21 toward the concentration membrane 30. Furthermore, by forming the housing 20 into a hemispherical shape that is convex toward the inlet 21 as shown in the perspective view of FIG. The facing inner wall portion 23 may have a shape in which the diameter gradually increases from the inlet 21 toward the concentration membrane 30. By forming the housing 20 in such a shape, the liquid to be treated 40 injected from the inlet can be guided to the concentrating membrane 30 through the slope of the inner wall portion 23 . At this time, if guide grooves 25 as shown in FIGS. 22 to 26 are provided in the inner wall portion 23, the liquid to be treated 40 can be guided more effectively.

<Recovery method of concentrated liquid 41>
The concentrated liquid 41 can be collected from the concentrated space 24 by inserting the tip of a suitable tool such as a micropipette through the inlet 21, as shown in FIG. 6 described above.

Further, as shown in FIG. 31, a breakable piece 14 can be formed on the upstream side of the housing 20. When this breaking piece 14 is broken off, a small hole appears on the upstream side of the housing 20. After the concentration of the liquid to be processed 40 by the concentration device 10 is completed, the tip of an appropriate tool such as a micropipette is inserted into this small hole to collect the concentrated liquid 41 from the concentration space 24. can do.

Furthermore, after the concentration of the liquid to be treated 40 by the concentration device 10 is completed, as shown in FIG. 32, by attaching the syringe 60 to the inlet 21 and sucking the plunger 61, as shown in FIG. Concentrate 41 can be collected in syringe 60 .

<Concentration membrane>
The concentration membrane 30 is made of an appropriate material and shape depending on the type and properties of the biological particles 50 contained in the liquid to be treated 40 . When the biological particles 50 are, for example, particles formed of a lipid bilayer (e.g., viruses, bacteria, or exosomes), the concentration membrane 30 comprises a porous base material and at least one of the porous base materials. It is desirable to include a hydrophilic composite porous membrane comprising a hydrophilic resin that covers the main surface and the inner surfaces of the pores. In addition, in the concentration membrane 30 of the present disclosure, "a hydrophilic porous membrane that does not adsorb biological particles" means a porous membrane that does not adsorb biological particles 50 and has hydrophilic properties. The property of "hydrophilicity that does not allow biological particles to be adsorbed" is not particularly limited as it also has a balance with the properties of the target biological particles 50, but the concentration rate when performing concentration processing is If it exceeds 100%, it means that concentration has been carried out, and it can be said that such a porous membrane has hydrophilicity to which the biological particles 50 are not adsorbed. For example, if the concentration membrane 30 contains a hydrophilic resin, which will be described later, or if the contact angle of water on the concentration membrane 30 is 90 degrees or less, it can be said to have "hydrophilicity," but the present disclosure The concentration membrane 30 in is not limited to this.

There is no limit to the size of the biological particles 50 to be concentrated by the concentration membrane 30 of the present disclosure. The diameter or major axis length of the biological particle 50 is, for example, 1 nm or more, 5 nm or more, 10 nm or more, or 20 nm or more, and, for example, 100 μm or less and 50 μm or less, It is 1000 nm or less, or 800 nm or less.

The concentration membrane 30 of the present disclosure may include members other than the hydrophilic composite porous membrane. Members other than the hydrophilic composite porous membrane include a sheet-like reinforcing member disposed in contact with a part or all of the main surface or side surface of the hydrophilic composite porous membrane; Examples include a guide member for mounting.

The hydrophilic composite porous membrane included in the concentration membrane 30 of the present disclosure only needs to be coated with a hydrophilic resin at least on the main surface on the upstream side during concentration treatment, and both main surfaces are coated with the hydrophilic resin. It is preferable that the Alternatively, the concentration membrane 30 may be a single-layer porous membrane containing a hydrophilic resin.

In the hydrophilic composite porous membrane, the main surface of the porous base material is coated with the hydrophilic resin, for example, a form in which the hydrophilic resin covers part or all of the main surface of the porous base material, a porous A form in which a part or all of the openings of a porous base material is filled with a hydrophilic resin, or a part of the main surface of a porous base material is covered with a hydrophilic resin and a part of the openings are filled with the hydrophilic resin. Examples include the form of When the hydrophilic resin fills the openings of the porous base material, it is preferable that the hydrophilic resin forms a porous structure. Here, porous structure means a structure that has many micropores inside and these micropores are connected so that gas or liquid can pass from one side to the other. .

In the hydrophilic composite porous membrane, the inner surface of the pores of the porous base material is coated with the hydrophilic resin, for example, by coating part or all of the wall surface of the pores of the porous base material with the hydrophilic resin. The hydrophilic resin fills some or all of the pores of the porous base material, or the hydrophilic resin covers part of the wall of the pores of the porous base material and fills some of the pores. An example is a form in which the portion is filled with a hydrophilic resin. When a hydrophilic resin fills the pores of a porous base material, it is preferable that the hydrophilic resin forms a porous structure. Here, porous structure means a structure that has many micropores inside and these micropores are connected so that gas or liquid can pass from one side to the other. .

Concentration of the biological particles 50 using the concentration membrane 30 of the present disclosure is performed when the liquid to be treated 40 is passed from one main surface to the other main surface of the hydrophilic composite porous membrane. A part or all of the biological particles 50 contained in the hydrophilic porous composite membrane do not pass through the hydrophilic composite porous membrane, and at least one of the upstream, upstream main surface, and pores of the hydrophilic composite porous membrane This is done by remaining in the liquid to be treated 40 at the site. Compare the liquid to be treated 40 before the concentration treatment and the liquid to be treated 40 recovered from at least one of the upstream, upstream main surface, and inside the pores of the hydrophilic composite porous membrane after the concentration treatment. Therefore, if the concentration of the biological particles 50 contained in the latter is high, it can be said that the biological particles 50 have been concentrated. The concentration ratio of biological particles 50 (see formula 1 below) achieved by the concentration membrane 30 of the present disclosure is more than 100%, preferably 200% or more, and more preferably 300% or more.

Concentration rate = (Biological particle concentration of the liquid to be treated recovered from at least one of the upstream, upstream main surface, and pores of the hydrophilic composite porous membrane after concentration treatment) ÷ (concentration treatment Biological particle concentration of previous treated liquid) x 100... (Formula 1)

Although the detailed mechanism is not necessarily clear, the hydrophilic composite porous membrane included in the concentration membrane 30 of the present disclosure has a hydrophilic composite porous membrane by having a hydrophilic resin on the upstream main surface and the inner surface of the pores. It is presumed that the biological particles 50 remaining on at least one of the upstream main surface of the porous membrane and inside the pores are easier to collect, and the concentration rate of the biological particles 50 is improved.

[Porous base material]
In the present disclosure, a porous base material means a base material having pores or voids inside. Examples of the porous base material include microporous membranes; porous sheets made of fibrous materials such as nonwoven fabric and paper; and the like. As the porous base material, a microporous membrane is preferable from the viewpoint of thinning and strength of the concentration membrane 30 of the present disclosure. A microporous membrane is a membrane that has many micropores inside and has a structure in which these micropores are connected, allowing gas or liquid to pass from one surface to the other. do.

The material of the porous base material may be either an organic material or an inorganic material.

The porous substrate may be either hydrophilic or hydrophobic. The concentration membrane 30 of the present disclosure exhibits hydrophilicity even if the porous base material is hydrophobic because the hydrophilic resin covers the porous base material.

One embodiment of the porous base material is a microporous membrane made of resin. The resins constituting the microporous membrane include polyesters such as polyethylene terephthalate; polyolefins such as polyethylene and polypropylene; heat-resistant resins such as fully aromatic polyamides, polyamideimides, polyimides, polyethersulfones, polysulfones, polyetherketones, and polyetherimides. ; etc.

One embodiment of the porous base material is a porous sheet made of a fibrous material, such as nonwoven fabric and paper. Fibrous materials constituting the porous sheet include polyester fibrous materials such as polyethylene terephthalate; polyolefin fibrous materials such as polyethylene and polypropylene; wholly aromatic polyamides, polyamideimides, polyimides, polyethersulfones, polysulfones, and polyesters. Examples include fibrous materials of heat-resistant resins such as etherketone and polyetherimide; cellulose; and the like.

The surface of the porous base material may be subjected to various surface treatments for the purpose of improving the wettability of a coating liquid used to coat the porous base material with a hydrophilic resin. Examples of surface treatments for the porous base material include corona treatment, plasma treatment, flame treatment, and ultraviolet irradiation treatment.

[Physical properties of porous base material]
The thickness of the porous base material is preferably 10 μm or more, more preferably 15 μm or more, and even more preferably 20 μm or more, from the perspective of increasing the strength of the porous base material and increasing the residual rate of biological particles 50. . The thickness of the porous base material is preferably 150 μm or less, more preferably 120 μm or less, and even more preferably 100 μm or less, from the viewpoint of shortening the processing time of the liquid to be treated 40. The method for measuring the thickness of the porous base material is the same as the method for measuring the thickness t of the hydrophilic composite porous membrane.

The average pore diameter measured with a palm porometer of the porous base material is determined from the viewpoint of shortening the treatment time of the liquid to be treated 40 and recovering the biological particles 50 remaining in the pores of the hydrophilic composite porous membrane. From the viewpoint of ease of use, the thickness is preferably 0.1 μm or more, more preferably 0.15 μm or more, and even more preferably 0.2 μm or more. The average pore diameter of the porous base material measured with a palm porometer is preferably 0.8 μm or less, more preferably 0.7 μm or less, and even more preferably 0.6 μm or less, from the viewpoint of increasing the residual rate of biological particles 50. . The average pore diameter of the porous base material measured with a Palm porometer is a value obtained by the half-dry method specified in ASTM E1294-89 using a Palm porometer. The measurement method is the same as that for the average pore diameter x.

The bubble point pore diameter measured with a palm porometer of the porous base material is used from the viewpoint of shortening the processing time of the liquid to be treated 40 and from the viewpoint of reducing the biological particles 50 remaining in the pores of the hydrophilic composite porous membrane. From the viewpoint of easy recovery, the particle size is preferably more than 0.8 μm, more preferably 0.9 μm or more, and even more preferably 1.0 μm or more. From the viewpoint of increasing the residual rate of biological particles 50, the bubble point pore diameter of the porous substrate measured with a palm porometer is preferably 3 μm or less, more preferably 2.8 μm or less, and even more preferably 2.5 μm or less. . The bubble point pore diameter measured with a palm porometer of a porous substrate is a value obtained by the bubble point method specified in ASTM F316-86 and JIS K3832 using a palm porometer. The measurement method is the same as that for the bubble point pore diameter y of the composite porous membrane.

The water flow rate (mL/(min cm ² MPa)) of the porous base material is preferably 20 or more, more preferably 50 or more, and even more preferably 100 or more, from the viewpoint of shortening the processing time of the liquid to be treated 40. . The water flow rate (mL/(min cm ² MPa)) of the porous base material is preferably 1000 or less, more preferably 800 or less, and even more preferably 700 or less, from the viewpoint of increasing the residual rate of biological particles 50. . The method for measuring the water flow rate of the porous substrate is the same as the method for measuring the water flow rate f of the hydrophilic composite porous membrane. However, if the porous base material is hydrophobic, use the porous base material that has been immersed in ethanol and dried at room temperature as a sample. Measurements are taken after wetting.

The porous base material preferably has a surface roughness Ra of 0.3 μm or more, more preferably 0.4 μm or more, on one or both surfaces. Further, the porous base material preferably has a surface roughness Ra of 0.7 μm or less, more preferably 0.6 μm or less, on one or both surfaces. The surface roughness Ra of the porous base material is the arithmetic mean height of the roughness curve, and the details of the measurement method are the same as the measurement method related to the surface roughness Ra of the hydrophilic composite porous membrane.

The Gurley value (sec/100 mL μm) per unit thickness of the porous base material is, for example, 0.001 to 5, preferably 0.01 to 3, and more preferably 0.05 to 1. be. The Gurley value of the porous base material is a value measured according to JIS P8117:2009.

The porosity of the porous base material is, for example, 70% to 90%, preferably 72% to 89%, and more preferably 74% to 87%. The porosity of the porous base material is determined according to the calculation method below. That is, regarding constituent material 1, constituent material 2, constituent material 3, ..., constituent material n of the porous base material, the mass of each constituent material is W ₁ , W _{2 ,} W ₃ , ..., W _n (g/cm ² ), the true density of each constituent material is d ₁ , d ₂ , d ₃ , ..., d _n (g/cm ³ ), and the film thickness is t (cm), then the porosity ε (% ) is determined by the following formula.

The BET specific surface area of the porous base material is, for example, 1 m ² /g to 40 m ² /g, preferably 2 m ² /g to 30 m ² /g, more preferably 3 m ² /g to 20 m ² /g. It is. The BET specific surface area of the porous substrate was measured using a specific surface area measurement device (Model: BELSORP-mini) manufactured by Microtrac Bell Co., Ltd., using a nitrogen gas adsorption method at liquid nitrogen temperature, with a set relative pressure of 1.0. This value was obtained by measuring an adsorption isotherm of ×10 ⁻³ to 0.35 and analyzing it using the BET method.

[Polyolefin microporous membrane]
As one embodiment of the porous base material, a microporous membrane containing polyolefin (referred to as a polyolefin microporous membrane in the present disclosure) is desirable. The polyolefin contained in the polyolefin microporous membrane is not particularly limited, and examples thereof include polyethylene, polypropylene, polybutylene, polymethylpentene, a copolymer of polypropylene and polyethylene, and the like. Among these, polyethylene is preferred, and high-density polyethylene, a mixture of high-density polyethylene and ultra-high molecular weight polyethylene, etc. are preferred. One embodiment of the microporous polyolefin membrane includes a microporous polyethylene membrane in which the polyolefin contained is only polyethylene.

The weight average molecular weight (Mw) of the polyolefin contained in the polyolefin microporous membrane is, for example, 100,000 to 5,000,000. When the Mw of the polyolefin is 100,000 or more, sufficient mechanical properties can be imparted to the microporous membrane. When the Mw of the polyolefin is 5 million or less, it is easy to form a microporous membrane.

As one embodiment of the polyolefin microporous membrane, a polyolefin composition (in the present disclosure, means a mixture of polyolefins containing two or more types of polyolefins, and when the polyolefin contained is only polyethylene, it is referred to as a polyethylene composition). Examples include microporous membranes containing The polyolefin composition forms a network structure as it fibrillates during stretching, and has the effect of increasing the porosity of the microporous polyolefin membrane.

The polyolefin composition is preferably a polyolefin composition containing 5% by mass to 40% by mass of ultra-high molecular weight polyethylene having a weight average molecular weight of 9×10 ⁵ or more based on the total amount of polyolefin, and preferably 10% by mass to 35% by mass. %, more preferably a polyolefin composition containing 15% by mass to 30% by mass.

The polyolefin composition consists of ultra-high molecular weight polyethylene having a weight average molecular weight of 9×10 ⁵ or more and high molecular weight polyethylene having a weight average molecular weight of 2×10 ⁵ to 8×10 ⁵ and a density of 920 kg/m ³ to 960 kg/m ³ . It is preferable to use a polyolefin composition in which density polyethylene is mixed at a mass ratio of 5:95 to 40:60 (more preferably 10:90 to 35:65, still more preferably 15:85 to 30:70).

The polyolefin composition preferably has a weight average molecular weight of 2×10 ⁵ to 2×10 ⁶ as a whole.

The weight average molecular weight of the polyolefin constituting the microporous polyolefin membrane can be determined by heating and dissolving the microporous polyolefin membrane in o-dichlorobenzene, and performing gel permeation chromatography (system: Alliance GPC 2000 model manufactured by Waters, column: GMH6-HT and GMH6-HTL) under conditions of a column temperature of 135°C and a flow rate of 1.0 mL/min. Molecular weight monodisperse polystyrene (manufactured by Tosoh Corporation) is used for molecular weight calibration.

One embodiment of the microporous polyolefin membrane includes a microporous membrane containing polypropylene from the viewpoint of having heat resistance that does not easily rupture when exposed to high temperatures.

One embodiment of the microporous polyolefin membrane includes a microporous polyolefin membrane containing a mixture of at least polyethylene and polypropylene.

One embodiment of the microporous polyolefin membrane includes a microporous polyolefin membrane having a laminated structure of two or more layers, at least one layer containing polyethylene and at least one layer containing polypropylene.

When the porous base material of the hydrophilic composite porous membrane included in the concentration membrane 30 of the present disclosure is a polyolefin microporous membrane (details will be described later), the biological particles 50 to be concentrated by the concentration membrane 30 are It is appropriate that the size is on the order of nanometers. In this case, the diameter or major axis length of the biological particle 50 is, for example, 10 nm or more, 20 nm or more, and, for example, 1000 nm or less, 800 nm or less, and 500 nm or less.

When the porous base material of the hydrophilic composite porous membrane included in the concentration membrane 30 of the present disclosure is a polyolefin microporous membrane, the concentration membrane 30 is suitable for concentrating viruses, bacteria, or exosomes.

[Method for manufacturing microporous polyolefin membrane]
A microporous polyolefin membrane can be produced, for example, by a production method including the following steps (I) to (IV).

Step (I): A step of preparing a solution containing a polyolefin composition and a volatile solvent having a boiling point of less than 210° C. at atmospheric pressure.
Step (II): A step of melt-kneading the solution, extruding the obtained melt-kneaded product from a die, cooling and solidifying it to obtain a first gel-like molded product.
Step (III): A step of stretching the first gel-like molded product in at least one direction (primary stretching) and drying the solvent to obtain a second gel-like molded product.
Step (IV): A step of stretching the second gel-like molded product in at least one direction (secondary stretching).

Step (I) is a step of preparing a solution containing a polyolefin composition and a volatile solvent having a boiling point of less than 210° C. at atmospheric pressure. The solution is preferably a thermoreversible sol-gel solution, and the polyolefin composition is heated and dissolved in a solvent to form a sol, thereby preparing a thermoreversible sol-gel solution. The volatile solvent having a boiling point of less than 210° C. at atmospheric pressure is not particularly limited as long as it can sufficiently dissolve the polyolefin. Examples of the volatile solvent include tetralin (206°C to 208°C), ethylene glycol (197.3°C), decalin (decahydronaphthalene, 187°C to 196°C), toluene (110.6°C), and xylene. (138°C to 144°C), diethyltriamine (107°C), ethylenediamine (116°C), dimethyl sulfoxide (189°C), hexane (69°C), etc., with decalin or xylene being preferred (temperatures in parentheses are: (It is the boiling point at atmospheric pressure.) The volatile solvents may be used alone or in combination of two or more.

The polyolefin composition (in this disclosure, it means a mixture of polyolefins containing two or more types of polyolefins, and when the polyolefin contained is only polyethylene, it is referred to as a polyethylene composition) used in step (I) contains polyethylene. A polyethylene composition is preferred, and a polyethylene composition is more preferred.

From the viewpoint of controlling the porous structure of the microporous polyolefin membrane, the solution prepared in step (I) preferably has a polyolefin composition concentration of 10% by mass to 40% by mass, and preferably 15% by mass to 35% by mass. It is more preferable that When the concentration of the polyolefin composition is 10% by mass or more, it is possible to suppress the occurrence of cutting in the film forming process of the microporous polyolefin membrane, and the mechanical strength of the microporous polyolefin membrane is increased and the handling properties are improved. When the concentration of the polyolefin composition is 40% by mass or less, pores in the polyolefin microporous membrane are likely to be formed.

Step (II) is a step of melt-kneading the solution prepared in step (I), extruding the obtained melt-kneaded product through a die, and solidifying it by cooling to obtain a first gel-like molded product. In step (II), for example, the polyolefin composition is extruded through a die at a temperature range of from the melting point to the melting point +65° C. to obtain an extrudate, and then the extrudate is cooled to obtain a first gel-like molded product. The first gel-like molded product is preferably shaped into a sheet. Cooling may be effected by immersion in water or an organic solvent, or by contact with a cooled metal roll, generally by immersion in the volatile solvent used in step (I). It will be done.

Step (III) is a step of stretching the first gel-like molded product in at least one direction (primary stretching) and drying the solvent to obtain a second gel-like molded product. The stretching step of step (III) is preferably biaxial stretching, and may be sequential biaxial stretching in which longitudinal stretching and lateral stretching are carried out separately, or simultaneous biaxial stretching in which longitudinal stretching and lateral stretching are carried out simultaneously. From the viewpoint of controlling the porous structure of the microporous polyolefin membrane, the stretching ratio of the primary stretching (the product of the longitudinal stretching ratio and the lateral stretching ratio) is preferably 1.1 times to 3 times, and 1.1 times to 2 times. More preferred. The temperature during the primary stretching is preferably 75°C or lower. The drying step of step (III) can be carried out without particular limitation as long as the temperature does not deform the second gel-like molded product, but it is preferably carried out at a temperature of 60° C. or lower.

The stretching step and drying step of step (III) may be performed simultaneously or in stages. For example, primary stretching may be performed while preliminary drying and then main drying, or primary stretching may be performed between preliminary drying and main drying. The primary stretching can also be carried out in a state where drying is controlled and the solvent remains in a suitable state.

Step (IV) is a step of stretching the second gel-like molded product in at least one direction (secondary stretching). The stretching step of step (IV) is preferably biaxial stretching. The stretching step of step (IV) includes sequential biaxial stretching in which longitudinal stretching and transverse stretching are carried out separately; simultaneous biaxial stretching in which longitudinal stretching and transverse stretching are carried out simultaneously; and transverse stretching after longitudinal stretching multiple times. A step of stretching in the longitudinal direction and a plurality of times in the transverse direction; A step of sequentially biaxially stretching and then further stretching one or more times in the longitudinal and/or transverse directions.

From the viewpoint of controlling the porous structure of the polyolefin microporous membrane, the stretching ratio (product of longitudinal stretching ratio and lateral stretching ratio) of the secondary stretching is preferably 5 times to 90 times, more preferably 10 times to 60 times. It's double. The stretching temperature of the secondary stretching is preferably 90°C to 135°C, more preferably 90°C to 130°C, from the viewpoint of controlling the porous structure of the microporous polyolefin membrane.

A heat setting treatment may be performed subsequent to step (IV). The heat setting temperature is preferably 110°C to 160°C, more preferably 120°C to 150°C, from the viewpoint of controlling the porous structure of the microporous polyolefin membrane.

After the heat-setting treatment, a treatment for extracting the solvent remaining in the polyolefin microporous membrane and an annealing treatment may be further performed. The residual solvent extraction process is performed, for example, by immersing the heat-set sheet in a methylene chloride bath and eluting the residual solvent into the methylene chloride. After the polyolefin microporous membrane immersed in a methylene chloride bath is withdrawn from the methylene chloride bath, the methylene chloride is preferably removed by drying. The annealing treatment is performed by conveying the polyolefin microporous membrane over a roller heated to, for example, 100° C. to 140° C. after the residual solvent extraction treatment.

By controlling each condition of steps (I) to (IV), a polyolefin microporous membrane having a ratio t/x of membrane thickness t (μm) to average pore diameter x (μm) of 50 to 630 is produced. becomes possible. For example, the ratio t/x can be controlled to 50 or more by reducing the longitudinal stretching ratio. For example, the ratio t/x can be controlled to 630 or less by increasing the longitudinal stretching ratio.

[Hydrophilic resin]
Although the hydrophilic resin is not particularly limited, examples thereof include resins having hydrophilic groups such as hydroxy group, carboxy group, and sulfo group.

From the viewpoint of not easily falling off from the porous base material and the concentration rate of the biological particles 50, the hydrophilic resin has a polymer main chain consisting only of carbon atoms, and side chains containing hydroxy groups, carboxy groups, and sulfonate groups. It is preferable that the resin has at least one functional group selected from the group consisting of groups.

Examples of hydrophilic resins include resins in which the main chain of the polymer contains not only carbon atoms but also oxygen atoms (e.g., polyethylene glycol, cellulose, etc.); is relatively easy to fall off from the porous substrate. From the viewpoint that it is difficult to fall off from the porous base material, a resin in which the main chain of the polymer consists only of carbon atoms is preferable, and the main chain of the polymer consists only of carbon atoms, and the side chain consists of hydroxy groups, carboxy groups, and sulfo groups. A resin having at least one functional group selected from the group is more preferred.

Hydrophilic resins include polyvinyl alcohol, olefin/vinyl alcohol resin, acrylic/vinyl alcohol resin, methacrylic/vinyl alcohol resin, vinylpyrrolidone/vinyl alcohol resin, polyacrylic acid, polymethacrylic acid, and perfluorosulfonic acid resin. It is preferable that at least one hydrophilic resin selected from the group consisting of resin and polystyrene sulfonic acid is included. Among these, it is more preferable to include an olefin/vinyl alcohol resin.

Examples of the hydrophilic resin include hydrophilic resins obtained by graft polymerizing a hydrophilic monomer onto the surface of a porous base material. In this case, the hydrophilic resin is in a form that is directly chemically bonded to the surface of the porous base material. Examples of the hydrophilic monomer graft-polymerized on the surface of the porous substrate include acrylic acid, methacrylic acid, vinyl alcohol, N-vinyl-2-pyrrolidone, and vinylsulfonic acid. From the viewpoint of manufacturability of hydrophilic composite porous membranes, it is preferable to form hydrophilic resins into porous forms by coating methods, etc., rather than in a form in which hydrophilic resins are chemically bonded directly to the surface of a porous substrate, such as in graft polymerization. A form in which the hydrophilic resin is attached to the surface of the base material (a form in which the hydrophilic resin is not chemically bonded to the surface of the porous base material) is preferable.

The number of hydrophilic resins may be one or two or more.

As the hydrophilic resin, from the viewpoint of less irritation to the biological particles 50 and from the viewpoint of easy recovery of the biological particles 50 remaining on the upstream main surface and pores of the hydrophilic composite porous membrane, Olefin/vinyl alcohol resins are preferred.

Examples of the olefin constituting the olefin/vinyl alcohol resin include ethylene, propylene, butene, pentene, hexene, heptene, octene, nonene, and decene. The olefin is preferably an olefin having 2 to 6 carbon atoms, more preferably an α-olefin having 2 to 6 carbon atoms, even more preferably an α-olefin having 2 to 4 carbon atoms, and particularly preferably ethylene. The number of olefin units contained in the olefin/vinyl alcohol resin may be one, or two or more.

The olefin/vinyl alcohol resin may contain monomers other than olefin and vinyl alcohol as constituent units. Examples of monomers other than olefins and vinyl alcohol include at least one acrylic monomer selected from the group consisting of (meth)acrylic acid, (meth)acrylate, and (meth)acrylic ester; styrene, methachlorostyrene; , parachlorostyrene, parafluorostyrene, paramethoxystyrene, meta-tert-butoxystyrene, para-tert-butoxystyrene, paravinylbenzoic acid, paramethyl-α-methylstyrene, and other styrenic monomers. The olefin/vinyl alcohol resin may contain one type or two or more types of these monomer units.

The olefin/vinyl alcohol resin may contain a monomer other than olefin and vinyl alcohol as a constituent unit, but from the viewpoint of causing less irritation to the biological particles 50, and from the viewpoint of causing less irritation to the biological particles 50, and from the viewpoint of being less irritating to the biological particles 50, From the viewpoint of easy recovery of the remaining biological particles 50, the combined ratio of olefin units and vinyl alcohol units is preferably 85 mol% or more, more preferably 90 mol% or more, and 95 mol%. % or more, and particularly preferably 100 mol%. As the olefin/vinyl alcohol resin, a binary copolymer of olefin and vinyl alcohol is preferable (here, the preferable embodiment of the olefin is as described above), and a binary copolymer of ethylene and vinyl alcohol is preferable. is more preferable.

The proportion of olefin units in the olefin/vinyl alcohol resin is preferably 20 mol% to 55 mol%. When the proportion of olefin units is 20 mol% or more, the olefin/vinyl alcohol resin is difficult to dissolve in water. From this viewpoint, the proportion of olefin units is more preferably 23 mol% or more, and even more preferably 25 mol% or more. On the other hand, when the proportion of olefin units is 55 mol% or less, the olefin/vinyl alcohol resin has higher hydrophilicity. From this viewpoint, the proportion of olefin units is more preferably 52 mol% or less, and even more preferably 50 mol% or less.

Examples of commercially available olefin/vinyl alcohol resins include "Soarnol" manufactured by Nippon Gosei Kagaku Kogyo Co., Ltd. and "Eval" manufactured by Kuraray Co., Ltd.

The amount of hydrophilic resin ^attached to the porous base material is, for example, 0.01 g/m ² to 5 g/m 2 , 0.02 g/m ² to 2 g/m ² , and 0.03 g/m ² to It is 1g/ ^m2 . The amount of the hydrophilic resin attached to the porous substrate is determined by subtracting the basis weight Wb (g/m ² ) of the porous substrate from the basis weight Wa (g/m ² ) of the hydrophilic composite porous membrane (Wa - Wb ).

[Production method of hydrophilic composite porous membrane]
The method for producing the hydrophilic composite porous membrane is not particularly limited. A common manufacturing method is to apply a coating solution containing a hydrophilic resin to a porous substrate, dry the coating solution, and then coat the porous substrate with the hydrophilic resin; Examples include a method in which a porous substrate is coated with a hydrophilic resin by graft polymerizing a hydrophilic monomer.

A coating solution containing a hydrophilic resin can be prepared by dissolving or dispersing the hydrophilic resin in the solvent by mixing the hydrophilic resin in a solvent heated to a temperature equal to or higher than the melting point of the hydrophilic resin and stirring the mixture. can. The solvent is not particularly limited as long as it is a good solvent for the hydrophilic resin, but specific examples include 1-propanol aqueous solution, 2-propanol aqueous solution, N,N-dimethylformamide aqueous solution, dimethyl sulfoxide aqueous solution. , ethanol aqueous solution, etc. The proportion of the organic solvent in these aqueous solutions is preferably 30% by mass to 70% by mass.

When applying a coating solution containing a hydrophilic resin to a porous substrate, the concentration of the hydrophilic resin in the coating solution is preferably 0.01% by mass to 5% by mass. When the concentration of the hydrophilic resin in the coating liquid is 0.01% by mass or more, hydrophilicity can be efficiently imparted to the porous substrate. From this viewpoint, the concentration of the hydrophilic resin in the coating liquid is more preferably 0.05% by mass or more, and even more preferably 0.1% by mass or more. When the concentration of the hydrophilic resin in the coating liquid is 5% by mass or less, the water flow rate in the produced hydrophilic composite porous membrane is large. From this viewpoint, the concentration of the hydrophilic resin in the coating liquid is more preferably 3% by mass or less, and even more preferably 2% by mass or less.

Applying the coating liquid to the porous substrate can be performed by a known coating method. Examples of the coating method include a dipping method, a knife coater method, a gravure coater method, a screen printing method, a Meyer bar method, a die coater method, a reverse roll coater method, an inkjet method, a spray method, and a roll coater method. A hydrophilic resin layer can be stably formed by adjusting the temperature of the coating liquid during coating. The temperature of the coating liquid is not particularly limited, but is preferably in the range of 5°C to 40°C.

The temperature at which the coating solution is dried is preferably 25°C to 100°C. When the drying temperature is 25° C. or higher, the time required for drying can be shortened. From this point of view, the dry concentration is more preferably 40°C or higher, and even more preferably 50°C or higher. When the drying temperature is 100° C. or lower, shrinkage of the porous base material is suppressed. From this point of view, the drying temperature is more preferably 90°C or lower, and even more preferably 80°C or lower.

The hydrophilic composite porous membrane may contain a surfactant, a wetting agent, an antifoaming agent, a pH adjuster, a coloring agent, and the like.

[Physical properties of concentration membrane 30]
It is preferable that the concentration membrane 30 has a water contact angle of 90 degrees or less measured under the following measurement conditions on one side or both sides, and the smaller the water contact angle, the more preferable it is. It is more preferable that the concentration membrane 30 is so hydrophilic that when attempting to measure the contact angle of water on one or both sides under the following measurement conditions, water droplets permeate into the inside of the membrane, making measurement impossible.

Here, the contact angle of water is a value measured by the following measurement method. After conditioning the concentration membrane 30 by leaving it in an environment with a temperature of 25°C and a relative humidity of 60% for 24 hours or more, inject 1 μL of ion-exchanged water onto the surface of the concentration membrane 30 with a syringe under the same temperature and humidity environment. A water drop is dropped, and the contact angle after 30 seconds is measured by the θ/2 method using a fully automatic contact angle meter (Kyowa Kaimen Kagakusha, model number Drop Master DM500).

The concentration membrane 30 used in the concentration device 10 of the present disclosure is a hydrophilic composite porous material that includes a porous base material and a hydrophilic resin that coats at least one main surface and the inner surface of the pores of the porous base material. It includes a membrane, and the ratio t/x of the membrane thickness t (μm) to the average pore diameter x (μm) measured with a palm porometer is 50 to 630.

If t/x of the concentration membrane 30 is less than 50, the membrane thickness t is too thin compared to the average pore diameter x, or the average pore diameter x is too large compared to the membrane thickness t. The biological particles 50 easily pass through the concentration membrane 30 and remain on at least one of the upstream, upstream main surface, and pores of the concentration membrane 30 (hereinafter simply referred to as "biological particles 50"). (referred to as "residual rate of biological particles 50") is inferior, and as a result, the concentration rate of biological particles 50 is inferior. From this point of view, t/x is 50 or more, preferably 80 or more, and more preferably 100 or more.

If t/x of the concentration membrane 30 is more than 630, the membrane thickness t is too thick compared to the average pore diameter x, or the average pore diameter x is too small compared to the membrane thickness t. It is difficult for the liquid to be treated 40 to pass through the concentration membrane 30, and it takes time for the liquid to be treated 40 to pass through the concentration membrane 30 (that is, it takes time to concentrate the liquid to be treated 40). From this viewpoint, t/x is 600 or less, preferably 500 or less, and more preferably 400 or less.

The thickness t of the concentration membrane 30 is preferably 10 μm or more, more preferably 15 μm or more, even more preferably 20 μm or more, from the viewpoint of increasing the strength of the concentration membrane 30 and increasing the residual rate of biological particles 50. More preferably, the thickness is 30 μm or more. The thickness t of the concentration membrane 30 is preferably 150 μm or less, and 100 μm or less, from the viewpoint of shortening the time required for the liquid to be treated 40 to pass through the concentration membrane 30 (hereinafter referred to as the processing time of the liquid to be treated 40). is more preferable, 80 μm or less is even more preferable, and even more preferably 70 μm or less.

The thickness t of the concentration membrane 30 is determined by measuring at 20 points using a contact type thickness meter and averaging the measurements.

The average pore diameter x of the concentration membrane 30 measured with a palm porometer is determined from the viewpoint of shortening the processing time of the liquid to be treated 40 and from the viewpoint of making it easier to collect the biological particles 50 remaining in the pores of the concentration membrane 30. , preferably 0.1 μm or more, more preferably 0.15 μm or more, and even more preferably 0.2 μm or more. The average pore diameter x of the concentration membrane 30 measured with a palm porometer is preferably 0.5 μm or less, more preferably 0.45 μm or less, and even more preferably 0.4 μm or less, from the viewpoint of increasing the residual rate of biological particles 50. .

The average pore diameter x of the concentration membrane 30 measured with a Perm porometer is determined by, for example, using a Perm porometer (PMI, Model: CFP-1200-AEXL) and using a PMI Gullwick (surface tension: 15. 9 dyn/cm) by the half-dry method specified in ASTM E1294-89. When only one main surface of the concentration membrane 30 is coated with a hydrophilic resin, the main surface coated with the hydrophilic resin is placed facing the pressurizing part of the palm porometer and the measurement is performed.

The bubble point pore diameter y measured with a palm porometer of the concentration membrane 30 is used from the viewpoint of shortening the processing time of the liquid to be treated 40 and making it easier to collect the biological particles 50 remaining in the pores of the concentration membrane 30. From the viewpoint, the thickness is preferably more than 0.8 μm, more preferably 0.9 μm or more, and even more preferably 1.0 μm or more. From the viewpoint of increasing the residual rate of biological particles 50, the bubble point pore diameter y of the concentration membrane 30 measured with a palm porometer is preferably 3 μm or less, more preferably 2.5 μm or less, and even more preferably 2.2 μm or less. .

The bubble point pore diameter y measured with a Palm porometer of the concentration membrane 30 is, for example, the bubble point pore diameter y measured by the bubble point method (ASTM F316-86, JIS K3832) using a Palm porometer (PMI, model: CFP-1200-AEXL). ). However, this value was obtained by changing the immersion liquid during the test to PMI's Gullwick (surface tension: 15.9 dyn/cm). When only one main surface of the concentration membrane 30 is coated with a hydrophilic resin, the main surface coated with the hydrophilic resin is placed facing the pressurizing part of the palm porometer and the measurement is performed.

The bubble point pressure of the concentration membrane 30 is, for example, 0.01 MPa or more and 0.20 MPa or less, and preferably 0.02 MPa to 0.15 MPa.

In the present disclosure, the bubble point pressure of the concentrated membrane 30 is determined by immersing a polyolefin microporous membrane in ethanol, applying it according to the bubble point test method of JIS K3832:1990, but changing the liquid temperature during the test to 24 ± 2 ° C. This value is obtained by performing a bubble point test while increasing the pressure at a pressure increase rate of 2 kPa/sec. When only one main surface of the concentration membrane 30 is coated with a hydrophilic resin, the main surface coated with the hydrophilic resin is placed facing the pressurizing part of the measuring device and the measurement is performed.

The water flow rate f (mL/(min cm ² MPa)) of the concentration membrane 30 is preferably 20 or more, more preferably 50 or more, and even more preferably 100 or more, from the viewpoint of shortening the processing time of the liquid to be treated 40. . From the viewpoint of increasing the residual rate of biological particles 50, the water flow rate f (mL/(min cm ² MPa)) of the concentration membrane 30 is preferably 1000 or less, more preferably 800 or less, and even more preferably 700 or less. .

The water flow rate f of the concentrating membrane 30 is, for example, when 100 mL of water permeates through a sample set in a liquid permeable cell having a constant liquid permeable area (cm ² ) at a constant differential pressure (20 kPa), and 100 mL of water permeates. The time (sec) required to do this is measured and converted into units. When only one main surface of the concentration membrane 30 is coated with a hydrophilic resin, measurement is performed by allowing water to permeate from the main surface coated with the hydrophilic resin to the main surface not covered with the hydrophilic resin. conduct.

The concentration membrane 30 has a ratio f/y between the water flow rate f (mL/(min cm ² MPa)) and the bubble point pore diameter y (μm) from the viewpoint of shortening the processing time of the liquid to be processed 40. It is preferably 100 or more, more preferably 150 or more, and even more preferably 200 or more. The ratio f/y of the concentration membrane 30 is preferably 480 or less, more preferably 400 or less, and still more preferably 350 or less, from the viewpoint of increasing the residual rate of biological particles 50. preferable.

From the viewpoint of increasing the recovery rate of the biological particles 50, the concentration membrane 30 preferably has a surface roughness Ra of 0.3 μm or more, at least on the main surface on the upstream side during concentration treatment, and preferably has a surface roughness Ra of 0.4 μm. It is more preferable that it is above. From the viewpoint of increasing the residual rate of biological particles 50, the concentration membrane 30 preferably has a surface roughness Ra of 0.7 μm or less, at least on the main surface on the upstream side during concentration treatment, and preferably has a surface roughness Ra of 0.6 μm. It is more preferable that it is below.

The surface roughness Ra of the concentrated film 30 was determined by measuring the surface of the sample at three random locations in a non-contact manner using a light wave interference surface roughness meter (Zygo, NewView 5032), and performing an analysis for roughness evaluation. Find it using software.

The Gurley value (sec/100 mL μm) per unit thickness of the concentration membrane 30 is, for example, 0.001 to 5, preferably 0.01 to 3, and more preferably 0.05 to 1. . The Gurley value of the concentration membrane 30 is a value measured according to JIS P8117:2009.

The porosity of the concentration membrane 30 is, for example, 70% to 90%, preferably 72% to 89%, and more preferably 74% to 87%. The porosity of the concentration membrane 30 is determined according to the following calculation method. That is, regarding constituent material 1, constituent material 2, constituent material 3, ..., constituent material n of the concentration membrane 30, the mass of each constituent material is _W1 , W2 _{, W3} ,..., _Wn (g/ ^cm2 ). , the true density of each constituent material is d ₁ , d ₂ , d ₃ , ..., d _n (g/cm ³ ), and the porosity is ε (%) when the film thickness is t (cm). is determined by the following formula.

From the viewpoint of handling properties, it is preferable that the concentration membrane 30 is hard to curl. From the viewpoint of suppressing curling of the concentration membrane 30, it is preferable that both main surfaces of the concentration membrane 30 are coated with a hydrophilic resin.

Since the concentration device 10 of the present disclosure uses the concentration membrane 30 as described above, the biological particles 50 can be concentrated easily and quickly compared to the centrifugation method. By using the concentration membrane 30 of the present disclosure, biological particles 50 can be concentrated more quickly and efficiently than conventional porous membranes.

[Shape of concentration membrane 30]
The shape of the concentration membrane 30 within the housing 20 can be flat, as shown in FIG. Moreover, as shown in FIG. 35, it may be folded in one direction, or as shown in FIG. 36, it may be folded in the circumferential direction.
Further, as shown in FIG. 37, an annular frame member 33 may be attached to the edge of the circular concentration membrane 30, and may be detachably attached to the housing 20 which is divided into two parts.

<Concentration system 70 for biological particles 50>
A biological particle 50 concentration system 70 is configured by combining any of the biological particle 50 concentration devices 10 described above with means for applying a differential pressure between the inlet 21 and the outlet 22.

For example, in the example shown in FIG. 38, a syringe 60 as a pressurizing means 71 is attached to the inlet 21 of the concentration device 10. It is desirable that the inlet 21 and the syringe 60 be reliably connected, for example, by a Luer lock (see FIG. 13). The liquid to be treated 40 is contained within the syringe 60 . On the other hand, a waste liquid tank 80 is installed below the outlet 22 of the concentration device 10. The concentration device 10 and the syringe are supported by a suitable device such as a stand (not shown). In this state, when the plunger 61 is pressed manually or by an appropriate device in the pressurizing means 71, the liquid to be treated 40 is pressurized, passes through the concentration membrane 30 in the housing 20, and is discharged from the outlet 22. 42, it falls into a waste liquid tank 80 installed below.

Further, in the example shown in FIG. 39, a syringe 60 as a pressurizing means 71 is attached to the inlet 21 of the concentration device 10. It is desirable that the inlet 21 and the syringe 60 be reliably connected, for example, by a Luer lock (see FIG. 13). The liquid to be treated 40 is contained within the syringe 60 . On the other hand, a waste liquid tank 80 is connected to the outlet 22 of the concentration device 10. It is also desirable that the outlet 22 and the waste liquid tank 80 be reliably connected by, for example, a Luer lock (see FIG. 17). In this example, the concentration device 10 is self-supporting along with the syringe 60 by a waste liquid tank 80. In this state, when the plunger 61 is pressed manually or by an appropriate device in the pressurizing means 71, the liquid to be treated 40 is pressurized, passes through the concentration membrane 30 in the housing 20, and is discharged from the outlet 22. 42, the liquid flows out to a waste liquid tank 80 installed below. Here, since the area from the outlet 22 to the waste liquid tank 80 is sealed from the outside world, the waste liquid 42 that has fallen into the waste liquid tank 80 is prevented from scattering around.

Furthermore, in the example shown in FIG. 40, a syringe 60 as a pressurizing means 71 is attached to the inlet 21 of the concentration device 10. It is desirable that the inlet 21 and the syringe 60 be reliably connected, for example, by a Luer lock (see FIG. 13). The liquid to be treated 40 is contained within the syringe 60 . On the other hand, a waste liquid tank 80 is connected to the outlet 22 of the concentration device 10. It is also desirable that the outlet 22 and the waste liquid tank 80 be reliably connected by, for example, a Luer lock (see FIG. 17). Further, the entire concentration device 10 is covered with a cylindrical guard unit 90 to prevent the liquid to be treated 40 from scattering. An exhaust port 81 is opened from the tip of the syringe 60 to the waste liquid tank 80 including the concentration device 10 for venting internal air. Note that an infection prevention filter (not shown) is installed in the middle of the exhaust port 81. In this state, when the plunger 61 is pressed manually or by an appropriate device in the pressurizing means 71, the liquid to be treated 40 is pressurized, passes through the concentration membrane 30 in the housing 20, and is discharged from the outlet 22. 42, the liquid flows out to a waste liquid tank 80 installed below. Here, since the area from the outlet 22 to the waste liquid tank 80 is sealed from the outside world, the waste liquid 42 that has fallen into the waste liquid tank 80 is prevented from scattering and contaminating the surrounding area. Further, since an infection prevention filter is attached to the exhaust port 81, the biological particles 50 that have passed through the concentration membrane 30 and fallen into the waste liquid tank 80 together with the discharged liquid 42 are prevented from scattering around. Note that this exhaust port 81 may be connected to a depressurizing means 72, which will be described later, so that pressing by the pressurizing means 71 and suction by the decompressing means 72 may be performed simultaneously.

Furthermore, in the example shown in FIG. 41, a syringe 60 that accommodates the liquid to be treated 40 is attached to the inlet 21 of the concentration device 10. It is desirable that the inlet 21 and the syringe 60 be reliably connected, for example, by a Luer lock (see FIG. 13). On the other hand, a waste liquid tank 80 is connected to the outlet 22 of the concentration device 10. It is also desirable that the outlet 22 and the waste liquid tank 80 be reliably connected by, for example, a Luer lock (see FIG. 17). The waste liquid tank 80 has an exhaust port 81 for venting air inside. Note that an infection prevention filter (not shown) is installed in the middle of the exhaust port 81. Further, a pressure reducing means 72 is connected to the exhaust port 81 . From this state, when the pressure reducing means 72 is operated to suck the air inside the waste liquid tank 80, the pressure inside the housing 20 is reduced. The liquid to be treated 40 in the syringe 60 is sucked into the housing 20, passes through the concentration membrane 30, and flows out from the outlet 22 as a discharged liquid 42 into a waste liquid tank 80 installed below. Here, since the area from the outlet 22 to the waste liquid tank 80 is sealed from the outside world, the waste liquid 42 that has fallen into the waste liquid tank 80 is prevented from scattering and contaminating the surrounding area. Furthermore, since an infection prevention filter is attached to the exhaust port 81, contamination of the depressurizing means 72 by biological particles 50 that have passed through the concentration membrane 30 and fallen into the waste liquid tank 80 together with the discharged liquid 42 is prevented. .

Note that, as in the example shown in FIG. 42, a plurality of sets of concentration devices 10 each having a syringe 60 as shown in FIG. It is also possible to process the liquid to be treated 40 of the specimen. Note that the exhaust port 81 and the pressure reducing means 72 are also similar to the example shown in FIG. 41.

Furthermore, in the example shown in FIG. 43, a syringe 60 containing the liquid to be treated 40 is attached to the inlet 21 of the concentration device 10. It is desirable that the inlet 21 and the syringe 60 be reliably connected, for example, by a Luer lock (see FIG. 13). On the other hand, a waste liquid tank 80 is connected to the outlet 22 of the concentration device 10. It is also desirable that the outlet 22 and the waste liquid tank 80 be reliably connected by, for example, a Luer lock (see FIG. 17). Further, in this example, a suction port 82 branches off from the outlet 22, and a pressure reducing means 72 using a water faucet is connected to the end thereof. Note that an infection prevention filter (not shown) is installed in the middle of the suction port 82. When the faucet serving as the pressure reducing means 72 is opened from this state, the air inside the housing 20 is sucked toward the suction port 82 by the water flow, thereby reducing the pressure inside the housing 20. The liquid to be treated 40 in the syringe 60 is sucked into the housing 20, passes through the concentration membrane 30, and flows out from the outlet 22 as a discharged liquid 42 into a waste liquid tank 80 installed below. Here, since the area from the outlet 22 to the waste liquid tank 80 is sealed from the outside world, the waste liquid 42 that has fallen into the waste liquid tank 80 is prevented from scattering and contaminating the surrounding area. Further, since the suction port 82 is equipped with an infection prevention filter, the water stream is prevented from being contaminated by the biological particles 50 that have passed through the concentration membrane 30 and fallen into the waste liquid tank 80 together with the discharge liquid 42.

Hereinafter, the concentration device of the present disclosure will be described in more detail using samples of concentration membranes that were actually produced. The materials, amounts used, proportions, processing procedures, etc. shown in the samples below can be changed as appropriate without departing from the spirit of the present disclosure. Therefore, the range of concentration membranes used in the concentration device of the present disclosure should not be construed as being limited by the specific examples shown below.

The composition and manufacturing process of samples 1 to 10 of the concentration membranes are listed in Table 1 below. Note that Samples 11 and 12 do not use a polyethylene microporous membrane, so they are not listed in Table 1 below.

Each sample will be described in detail below as necessary.

<Preparation of hydrophilic composite porous membrane>
[Sample 1]
-Preparation of microporous polyethylene membrane-
3.75 parts by mass of ultra-high molecular weight polyethylene (hereinafter referred to as "UHMWPE") with a weight average molecular weight of 4.6 million, and high density polyethylene ( ^hereinafter referred to as "HDPE") with a weight average molecular weight of 560,000 and a density of 950 kg/m 21. A polyethylene composition was prepared by mixing 25 parts by mass. A polyethylene solution was prepared by mixing a polyethylene composition and decalin so that the polymer concentration was 25% by mass.

The above polyethylene solution was extruded into a sheet from a die at a temperature of 147°C, and then the extrudate was cooled in a water bath at a water temperature of 20°C to obtain a first gel-like sheet.

The first gel-like sheet was pre-dried for 10 minutes in a temperature atmosphere of 70°C, then primary stretched at 1.8 times in the MD direction, and then main drying was carried out for 5 minutes in a temperature atmosphere of 57°C. A second gel-like sheet (base tape) was obtained (the residual amount of solvent in the second gel-like sheet was less than 1%). Next, as secondary stretching, the second gel-like sheet (base tape) was stretched in the MD direction at a temperature of 90°C and a magnification of 4 times, then in the TD direction at a temperature of 125°C and a magnification of 9 times, and then Immediately heat treatment (heat fixation) was performed at 144°C.

Decalin in the sheet was extracted while continuously immersing the heat-set sheet in two separate methylene chloride baths for 30 seconds each. After the sheet was taken out of the methylene chloride bath, methylene chloride was removed by drying in an atmosphere at a temperature of 40°C. In this way, a microporous polyethylene membrane was obtained.

-Hydrophilic treatment of polyethylene microporous membrane-
As a hydrophilic resin, an ethylene/vinyl alcohol binary copolymer (manufactured by Nippon Gosei Kagaku Kogyo, Soarnol DC3203R, ethylene unit: 32 mol %) (hereinafter referred to as EVOH) was prepared. EVOH was dissolved in a mixed solvent of 1-propanol and water (1-propanol:water = 3:2 [volume ratio]) so that the EVOH concentration was 0.2% by mass to obtain a coating liquid.

A microporous polyethylene membrane fixed to a metal frame was immersed in a coating solution to impregnate the pores of the microporous polyethylene membrane with the coating solution, and then pulled out. Next, excess coating liquid adhering to both main surfaces of the polyethylene microporous membrane was removed, and the membrane was dried at room temperature for 2 hours. Next, the metal frame was removed from the polyethylene microporous membrane. In this way, a hydrophilic composite porous membrane was obtained in which both main surfaces and the inner surfaces of the pores of the microporous polyethylene membrane were coated with a hydrophilic resin.

[Samples 2 to 7]
-Preparation of microporous polyethylene membrane-
A microporous polyethylene membrane was produced in the same manner as Sample 1 except that the composition of the polyethylene solution or the manufacturing process of the microporous polyethylene membrane was changed as shown in Table 1. In samples 3 to 6, after the sheets were taken out of the methylene chloride bath, the methylene chloride was removed by drying in an atmosphere at a temperature of 40°C, and annealing was performed while conveying the sheets on rollers heated to 120°C.

-Hydrophilic treatment of polyethylene microporous membrane-
EVOH was applied to a polyethylene microporous membrane in the same manner as Sample 1 to produce a hydrophilic composite porous membrane. However, in Samples 5 and 6, the EVOH concentration of the coating liquid was 1% by mass.

[Sample 8]
-Preparation of microporous polyethylene membrane-
A microporous polyethylene membrane was produced in the same manner as Sample 1, except that the composition of the polyethylene solution and the manufacturing process of the microporous polyethylene membrane were changed as shown in Table 1. In sample 8, after the sheet was taken out of the methylene chloride bath, the methylene chloride was removed by drying in an atmosphere at a temperature of 40°C, and annealing treatment was performed while conveying the sheet on a roller heated to 120°C.

-Hydrophilic treatment of polyethylene microporous membrane-
One side of the microporous polyethylene membrane was subjected to plasma treatment (AP-300 manufactured by Nordson MARCH: output 150 W, treatment pressure 400 mTorr, gas flow rate 160 sccm, treatment time 45 seconds) to obtain a hydrophilic composite porous membrane.

[Sample 9]
-Preparation of microporous polyethylene membrane-
A microporous polyethylene membrane was produced in the same manner as Sample 1 except that the manufacturing process of the microporous polyethylene membrane was changed as shown in Table 1.

-Hydrophilic treatment of polyethylene microporous membrane-
One side of the microporous polyethylene membrane was subjected to plasma treatment (AP-300 manufactured by Nordson MARCH: output 150 W, treatment pressure 400 mTorr, gas flow rate 160 sccm, treatment time 45 seconds) to obtain a hydrophilic composite porous membrane.

[Sample 10]
-Preparation of microporous polyethylene membrane-
A microporous polyethylene membrane was produced in the same manner as Sample 1, except that the composition of the polyethylene solution and the manufacturing process of the microporous polyethylene membrane were changed as shown in Table 1.

-Hydrophilic treatment of polyethylene microporous membrane-
EVOH was applied to a polyethylene microporous membrane in the same manner as Sample 1 to produce a hydrophilic composite porous membrane.

[Sample 11]
As Sample 11, a syringe filter SYNN0601MNXX104 manufactured by MDI was prepared. The porous membrane included in this syringe filter is made of nylon.

[Sample 12]
As sample 12, a syringe filter CA025022 manufactured by Membrane Solutions was prepared. The porous membrane included in this syringe filter is made of cellulose acetate.

<Measurement of physical properties of hydrophilic composite porous membrane>
The following physical properties were measured using each of the hydrophilic composite porous membranes of Samples 1 to 12 as samples. Note that for each of the hydrophilic composite porous membranes of Samples 8 and 9, the physical properties of the main surfaces subjected to plasma treatment were measured. In addition, for each porous membrane included in the syringe filters of Samples 11 and 12, the porous membrane was taken out from the syringe filter, and the physical properties of the main surface on the inlet side of the syringe filter were measured. Table 2 below shows its physical properties.

[Film thickness]
The thickness of the hydrophilic composite porous membrane or porous membrane was determined by measuring at 20 points using a contact type thickness meter (manufactured by Mitutoyo) and averaging the measurements. The contact terminal used was a cylindrical terminal whose bottom surface had a diameter of 0.5 cm. The measurement pressure was 0.1N.

[Average pore diameter x]
The average pore diameter x (μm) of the hydrophilic composite porous membrane or porous membrane was determined using a PMI palm porometer (model: CFP-1200-AEXL) and a PMI Galwick (surface tension 15 .9dyn/cm) by the half-dry method specified in ASTM E1294-89. The measurement temperature was 25° C., and the measurement pressure was varied in the range of 0 to 600 kPa.

[Bubble point pore diameter y]
The bubble point pore diameter y (μm) of the hydrophilic composite porous membrane or porous membrane is determined by the bubble point method (ASTM F316-86, JIS K3832) using a PMI palm porometer (model: CFP-1200-AEXL). ). However, this value was obtained by changing the immersion liquid during the test to PMI's Gullwick (surface tension: 15.9 dyn/cm). The measurement temperature was 25° C., and the measurement pressure was varied in the range of 0 to 600 kPa.

[Bubble point pressure]
The hydrophilic composite porous membrane or porous membrane was immersed in ethanol, and the test was conducted according to the bubble point test method of JIS K3832:1990, except that the liquid temperature during the test was changed to 24 ± 2°C, and the applied pressure was increased at a pressure increase rate of 2 kPa/ It was determined by performing a bubble point test while increasing the pressure in seconds.

[Water flow rate f]
The hydrophilic composite porous membrane was cut into a size of 10 cm in the MD direction x 10 cm in the TD direction, and set in a circular liquid permeable cell made of stainless steel with a liquid permeable area of 17.34 cm ² . 100 mL of water was permeated at a differential pressure of 20 kPa, and the time (sec) required for 100 mL of water to permeate was measured. The measurement was performed in a temperature atmosphere of room temperature 24°C. The measurement conditions and measured values were converted into units to determine the water flow rate f (mL/(min·cm ² ·MPa)).

[Surface roughness Ra]
The arithmetic mean height was measured under the following conditions using a light wave interference type surface roughness meter (Zygo, NewView 5032) to determine the surface roughness Ra.
・Objective lens: 20x Mirau ・Image zoom: 1.0x
・FDA Res: Normal or Low
-Analysis conditions: Zygo's standard application Stitch. After acquiring data at three locations on each sample in a non-contact manner using the Advanced Texture.app, which is an optional application for roughness evaluation. The surface roughness was analyzed using an app.

<Performance evaluation of concentration membrane>
A concentration test was conducted using each of the hydrophilic composite porous membranes or porous membranes of Samples 1 to 12 as concentration membranes. Note that when each of the hydrophilic composite porous membranes of Samples 8 and 9 was used as a concentration membrane, the main surface subjected to plasma treatment was placed on the upstream side. When each porous membrane included in the syringe filters of samples 11 and 12 was used as a concentration membrane, the porous membrane was taken out from the syringe filter, and the main surface on the inlet side of the syringe filter was placed on the upstream side. Table 2 shows the results. Details of the concentration test are as follows.

A virus suspension was prepared by suspending dengue virus in a buffer solution. The virus unit was 1×10 ⁴ FFU/mL. Dengue virus is an enveloped spherical virus with a diameter of approximately 40 nm to 60 nm.

A hydrophilic composite porous membrane or a porous membrane was punched into a circular shape with a diameter of 13 mm, and the punch was placed in the housing of a filter holder (Swinex 35, manufactured by Merck Millipore) to obtain a concentration device. This housing is provided with an inlet and an outlet, and a concentration space is provided within the housing upstream of the hydrophilic composite porous membrane or porous membrane used as the concentration membrane (see FIG. 2). 10 mL of the virus suspension was collected into a 10 mL syringe (manufactured by Terumo). Then, as in the example shown in FIG. 38, the tip of the syringe was connected to the concentration device, and the virus suspension was passed through the concentration device. The pressure applied to the plunger was approximately 30N. If the plunger did not move at this pressure, the applied pressure was gradually increased to apply the minimum pressure that would cause the plunger to move.

[processing time]
The time (seconds) from the time when the plunger was started to the time when the plunger was fully pressed was measured.

[Concentration rate]
After pushing the plunger all the way down, the plunger was moved back and forth several times with the concentration device facing up and the syringe facing down, and the virus suspension remaining upstream of the concentration membrane was collected. Using the collected virus suspension as a sample, total RNA was extracted using Viral RNA Mini Kit (QIAGEN). The extracted total RNA was reverse transcribed using ReverTra Ace (registered trademark) (Toyobo) to produce cDNA. qRT-PCR was performed using primers that specifically bind to dengue virus RNA and SYBR Green I (Takara Bio) (SYBR is a registered trademark) to quantify viral RNA in the sample. Concentration rate (%)=Cb÷Ca×100 was calculated from the virus RNA concentration Ca of the virus suspension before liquid passage and the virus RNA concentration Cb of the recovered virus suspension.

The results of the concentration test are shown in Table 2 below.

First, the virus concentration rates for samples 1 to 7 were as follows. In sample 1, it exceeded 900%. In sample 2, it exceeded 600%. In sample 3, it exceeded 300%. In sample 4, it exceeded 400%. In sample 5, it was 156%. In sample 6, it exceeded 700%. In sample 7, it exceeded 200%. From the above, virus concentration rates exceeding at least 150% were observed in the concentration devices using the concentration membranes of Samples 1 to 7, so the effect of concentrating biological particles was significant.

On the other hand, the virus concentration rates for samples 8 to 12 were as follows. In sample 8, it was 89%. In sample 9, it was 66%. In sample 10, it was 489%. In sample 11, it was 98%. In sample 12, it was 96%. From the above, the virus concentration rates in the concentration devices using the concentration membranes of Samples 8, 9, 11, and 12 were all less than 100%, and no biological particle concentration effect was observed at all.

The processing times for samples 1 to 7 were as follows. In sample 1, it was 40 seconds. In sample 2, it was 34 seconds. In sample 3, it was 72 seconds. In sample 4, it was 26 seconds. In sample 5, it was 60 seconds. In sample 6, it was 71 seconds. In sample 7, it was 96 seconds. From the above, the processing time for Samples 1 to 7 in the concentration device using the concentration membrane was 100 seconds or less, and the samples could be concentrated quickly.

The processing times for samples 8 to 12 were as follows. In sample 8, it was 47 seconds. In sample 9, it was 30 seconds. In sample 10, it was 162 seconds. In sample 11, it was 132 seconds. In sample 12, it was 126 seconds. From the above, the processing time of Samples 10 to 12 in the concentration device using the concentration membrane exceeded 100 seconds, and rapid concentration could not be achieved.

From the above results, all of the concentration devices using the concentration membranes of samples 1 to 7 have a virus concentration rate of over 150%, a processing time of less than 100 seconds, a high concentration rate of biological particles, and a rapid This method was considered to be useful for practical purposes because of its long processing time.

On the other hand, in the concentration devices using the concentration membranes of Samples 8 to 12, the virus concentration rate was less than 150%, the processing time exceeded 100 seconds, or both, and the biological particle concentration rate was high. However, it lacks either or both of the following: (1) rapid processing time, and was considered unsuitable for practical use.

10 Concentration device 14 Breaking piece 20 Housing 21 Inlet 22 Outlet 23 Inner wall 24 Concentration space 25 Guide groove 30 Concentration membrane 33 Frame member 40 Liquid to be treated 41 Concentrate 42 Effluent 50 Biological particles 60 Syringe 61 Plunger 70 Concentration system 71 Pressurizing means 72 Depressurizing means 80 Waste liquid tank 81 Exhaust port 82 Suction port 90 Guard unit

Claims (9)

It has an inlet and an outlet, and a liquid to be treated containing biological particles having a size of 10 nm to 1000 nm and water is injected from the inlet and discharged from the outlet due to the pressure difference between the inlet and the outlet. A housing that looks like this,
A hydrophilic flat porous membrane provided in the housing to separate the inlet and the outlet to which the biological particles are not adsorbed, from the inlet side surface to the outlet side surface; a concentration membrane that transmits a discharged liquid that is a liquid obtained by reducing the concentration of biological particles from the liquid to be treated;
a concentration space that is a space on the upstream side of the concentration membrane in the housing and stores a concentrated liquid that is a liquid obtained by increasing the concentration of the biological particles from the liquid to be treated by the concentration membrane;
Equipped with
The concentration membrane is made of ultra-high molecular weight polyethylene having a weight average molecular weight of 9×10 ⁵ or more and high density polyethylene having a weight average molecular weight of 2×10 ⁵ to 8×10 ⁵ and a density of 920 kg/m ³ to 960 kg/m ³ . A porous base material which is a polyethylene microporous membrane containing polyethylene at a mass ratio of 5:95 to 40:60, and a ratio of ethylene units covering at least one main surface and the inner surface of the pores of the porous base material is A hydrophilic composite porous membrane comprising 25 mol% to 50 mol% of an ethylene/ vinyl alcohol binary copolymer ,
The concentration membrane has a membrane thickness t of 10 μm to 70 μm, an average pore diameter x measured with a palm porometer of 0.1 μm to 0.5 μm, and the membrane thickness t (μm) and the average pore diameter x (μm) ^The ratio t/x of and bubble point pore diameter y (μm) measured with a palm porometer, the ratio f/y is 100 to 480. The device for concentrating biological particles according to claim 1 , wherein in the housing, the volume of the concentrating space is 0.05 to 5 cm ³ . The device for concentrating biological particles according to claim 1 or 2 ^, wherein in the housing, the filtration area of the concentrating membrane is 1 to 20 cm 2 . The biological particle concentration device according to any one of claims 1 to 3 , wherein in the housing, a guide groove continuous from the inlet is formed in an inner wall portion facing the concentration space. . The biological fluid according to any one of claims 1 to 4 , wherein, in the housing, an inner wall portion facing the concentration space has a shape that gradually increases in diameter from the inlet toward the concentration membrane. Particle concentration device. The biological particle concentration device according to any one of claims 1 to 5 , wherein the concentration membrane has a surface roughness Ra of 0.3 to 0.7 μm. A biological particle concentration device according to any one of claims 1 to 6 ,
means for applying a pressure differential between the inlet and the outlet;
Biological particle concentration system with. supplying the liquid to be treated to the concentration device according to any one of claims 1 to 6 ;
obtaining the concentrate within the concentration space by applying a pressure differential between the inlet and the outlet of the concentration device;
Recovering the concentrated liquid from the concentration space;
A method for concentrating biological particles, including: supplying the liquid to be treated to the concentration device according to any one of claims 1 to 6 ;
obtaining the concentrate within the concentration space by applying a pressure differential between the inlet and the outlet of the concentration device;
Recovering the concentrated liquid from the concentration space;
Detecting the biological particles contained in the collected concentrate;
A method for detecting biological particles, including: