Pipette tip and automated device using the same

Abstract

To provide a pipette tip which can produce concentrated solution with a high concentration of a target substance from sample solution including the target substance or a target substance-containing substance, and to provide a method for producing concentrated solution using the pipette tip.SOLUTION: A pipette tip includes: a tip main body housing liquid; and a filter placed on a lower opening part at a bottom end of the tip main body. An inclined part is provided in at least a part of the lower opening part where the filter is placed at the bottom end. The pipette tip can perform such an operation repeatedly that a carrier in the tip can be dispersed by suction and discharge, and consequently small dead volume is materialized.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a pipette tip and an automated device using the same. [Background technology]

[0002] Chromatography, which separates and purifies a target substance from a sample solution containing multiple substances, can be classified into two types: continuous chromatography using a packed column (hereafter referred to as column chromatography) and batch chromatography.

[0003] Batch chromatography is a method in which a carrier and a sample solution are mixed in a container, and then the carrier that has captured the target substance is separated from the sample solution. Methods for separating the carrier from the sample solution include centrifuging the mixture of the sample solution and the carrier to separate the carrier as a precipitate, placing the mixture of the sample solution and the carrier on a filter cartridge and centrifuging it to separate the carrier as a residue on the filter, and using magnetic beads as the carrier and a magnet to separate the carrier.

[0004] Batch chromatography generally has the advantage of being rapid, since it can separate even large amounts of sample liquid at once. Furthermore, because the carrier disperses easily in the sample liquid, it is easy to capture the target substance on the carrier. However, because the entire sample liquid comes into contact with the carrier at once, there is an excess of sample liquid relative to the carrier, and if the binding between the target substance and the carrier is not strong, the target substance may not be sufficiently captured. Furthermore, while centrifugation or the use of magnetic beads is essential, centrifugation carries the risk of sample liquid leaking or scattering due to tube breakage or loosening of the lid during centrifugation, leading to the risk of spreading contamination. Magnetic beads also have the problem of being difficult to recover when the concentration of the target substance in the sample liquid is low.

[0005] Column chromatography is a method in which a sample liquid is brought into contact with a carrier packed in a column online, and a target substance is captured on the carrier and then released. Column chromatography has the advantage that the sample liquid passes from top to bottom through a column packed with a carrier, making it easy for the target substance to be captured on the carrier during this process. Examples of using column chromatography to separate and purify a target substance from a large number of sample solutions containing proteins, nucleic acids, lipids, sugars, viruses, exosomes, etc. include methods using spin columns or column tips that use carriers with properties corresponding to the target substance. Column tips are specially shaped pipette tips that contain carriers with properties suited to the target substance and are attached to the end of a pipette. Because target substances can be separated and purified simply by aspirating and dispensing with the pipette, they are simple to operate and easily adaptable to automated robotic liquid handlers, making them widely used in situations where multiple samples must be processed simultaneously. An example of a commercially available column tip is Biotage's PhyTip (registered trademark).

[0006] Column tips typically have filters arranged above and below the carrier, sandwiching the carrier between the filters. This makes it difficult for the carrier to disperse in the liquid aspirated into the column tip. Therefore, the target substance in the liquid is unlikely to encounter the carrier, and often only a portion of the target substance is adsorbed onto the carrier. Furthermore, if air bubbles get into the column tip during aspirating and discharging, the adsorption efficiency is likely to decrease. Furthermore, column tips are narrow and prone to clogging, making repeated aspirating and discharging impossible unless the aspirating and discharging speed is significantly slowed. Furthermore, pressure may be applied to the filter, causing damage. For these reasons, the target substance may not be sufficiently purified.

[0007] A diffusible carrier-encapsulated flow tube (chip) with a turbulence-generating member capable of generating turbulence that diffuses the carrier has been reported as a column chip that can increase the uniformity of encounter between a target substance in a liquid and the carrier, thereby enabling efficient reaction or binding (Patent Document 1). However, this chip has a complex structure and a thin tube at the tip (e.g., Figure 3 of Patent Document 1), so a certain amount of liquid remains in the chip after discharge, resulting in a significant dead volume. If the dead volume is large, a certain amount of concentrated liquid remains in the chip, resulting in a reduced recovery rate of the concentrated liquid, especially when the amount of liquid during elution is significantly reduced compared to the sample liquid to concentrate the target substance. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Patent No. 5732407 Summary of the Invention [Problem to be solved by the invention]

[0009] Provided are a pipette tip that can repeatedly disperse a carrier in the tip by suction and discharge, has a small dead volume, and is capable of producing a concentrated solution with an increased concentration of a target substance from a sample solution containing a target substance or a substance containing a target substance, and a method for producing a concentrated solution using the pipette tip. [Means for solving the problem]

[0010] The present inventors have conducted extensive research to solve the above problems, and as a result have found that the above problems can be solved by providing the following configuration, which has led to the completion of the present invention. The present invention relates to, for example, the following [1] to [7]. [1] A tip body that contains a liquid; a filter disposed in a lower opening at the lower end of the chip body; A pipette tip having a sloped portion is provided in at least a part of a lower opening in which a filter is disposed at the lower end of the tip body; A pipette tip that holds a carrier within the tip body. [2] The pipette tip according to [1], wherein, when viewed from the front of the pipette tip, the inclination angle of the inclined portion is greater than 20° and less than 70° with respect to a line perpendicular to the axis of the pipette tip. [3] The pipette tip according to [1] or [2], wherein the carrier is hydroxyapatite or a magnetic carrier. [4] The pipette tip according to any one of [1] to [3], wherein the material constituting the filter is polytetrafluoroethylene (PTFE) or polyvinylidene fluoride (PVDF).

[0011] [5] The pipette tip according to any one of [1] to [4], wherein the melting point of the material constituting the filter is higher than the melting point of the material constituting the tip body, and the filter is fused to the lower opening. [6] A pipette tip according to any one of [1] to [5], for producing a concentrated solution with an increased concentration of substance α from a sample solution containing a target substance (substance α) or a substance α-containing substance (substance β). [7] A method for producing a concentrated solution having an increased concentration of substance α from a sample solution containing a target substance (substance α) or a substance α-containing substance (substance β), using the pipette tip according to any one of [1] to [6], a step A of aspirating and discharging the sample liquid through a lower opening at the lower end of the pipette tip to adsorb the substance α or the substance β onto the carrier; and A step B of aspirating and discharging the released liquid to release the substance α from the carrier, thereby obtaining a concentrated liquid having an increased concentration of the substance α. A method comprising: [Effects of the Invention]

[0012] A pipette tip according to one aspect of the present invention allows repeated dispersion of the carrier in the tip by suction and discharge, has a small dead volume, and can produce a concentrated solution having an increased concentration of the target substance from a sample solution containing the target substance or a substance containing the target substance. Furthermore, a method for producing a concentrated solution according to one aspect of the present invention allows a concentrated solution having an increased concentration of the target substance to be produced from a sample solution containing the target substance or a substance containing the target substance. [Brief explanation of the drawings]

[0013] [Figure 1] Figure 1 shows an example of the shape of the lower end of a pipette tip of the present invention. The dotted line indicates the filter. [Figure 2] FIG. 2 is a six-view diagram of the pipette tip according to the first embodiment. [Figure 3] FIG. 3 is a perspective view of the pipette tip according to the first embodiment shown in FIG. [Figure 4] FIG. 4 is a six-view diagram of the pipette tip according to the second embodiment. [Figure 5] FIG. 5 is a perspective view of the pipette tip according to the second embodiment shown in FIG. [Figure 6] FIG. 6 is a six-view diagram of a pipette tip according to the third embodiment. [Figure 7] FIG. 7 is a perspective view of the pipette tip according to the third embodiment shown in FIG. [Figure 8] FIG. 8 is a six-view diagram of a pipette tip according to the fourth embodiment. [Figure 9] FIG. 9 is a perspective view of the pipette tip according to the fourth embodiment shown in FIG. [Figure 10] FIG. 10 is a photograph showing the state in which the pipette tips of Examples 2, 3, 4, and 5 are used to aspirate and discharge solutions. [Figure 11] FIG. 11 is a photograph showing, at a larger scale than FIG. 10, how the pipette tips of Examples 2, 3, 4, and 5 are used to aspirate and discharge solutions. [Figure 12] FIG. 12 is a conceptual diagram of the dispersed state of the carrier in the pipette tip. [Figure 13] FIG. 13 is a photograph showing the state in which the pipette tips of Examples 1, 4, and 6 are used to aspirate and discharge solutions. [Figure 14] FIG. 14 is a photograph showing the state in which a solution is aspirated and discharged using the pipette tip of Example 7. [Figure 15] FIG. 15 shows the results of detecting CD9 by Western blotting in Experimental Example 7. [Figure 16] FIG. 16 shows the results of detecting CD9 by Western blotting in Experimental Example 8. [Figure 17] FIG. 17 shows the results of detecting CD9 by Western blotting in Experimental Example 9. [Figure 18] FIG. 18 shows the amplification curves in quantitative PCR in Experimental Example 10. [Figure 19] FIG. 19 shows the results of silver staining of SDS-PAGE gel in Experimental Example 11. [Figure 20] FIG. 20 shows the results of CBB staining of SDS-PAGE gel in Experimental Example 12. [Figure 21] FIG. 21 shows the results of CBB staining of SDS-PAGE gel in Experimental Example 13. DETAILED DESCRIPTION OF THE INVENTION

[0014] Next, the present invention will be described in detail. Unless otherwise specified, the expression "A to B" regarding a numerical range means that it is equal to or greater than A and equal to or less than B. Furthermore, % means % by mass.

[0015] [Pipette Tip] A pipette tip according to one aspect of the present invention is a pipette tip having a tip body for containing a liquid and a filter disposed in a lower opening at the lower end of the tip body, and at least a portion of the lower opening in which the filter is disposed is provided with an inclined portion for holding a carrier within the tip body.

[0016] The pipette tip can be used to produce a purified solution in which the purity of substance α has been increased from a sample solution containing a target substance (hereinafter also referred to as "substance α") or a target substance-containing substance (hereinafter also referred to as "substance β"), or to produce a concentrated solution in which the concentration of substance α has been increased from a sample solution containing substance α or substance β, but is preferably used to produce a concentrated solution in which the concentration of substance α has been increased from a sample solution containing substance α or substance β.

[0017] The pipette tip is used with a pipetting device such as a pipette for aspirating and dispensing liquid. The pipetting device is not particularly limited as long as it is a device that performs the operation of aspirating and discharging a liquid, and examples thereof include pipettes such as micropipettes, macropipettes, measuring pipettes, and Komagome pipettes, and robotic liquid handlers that automate the operation of attaching and detaching pipette tips in addition to the operation of aspirating and discharging. From the viewpoints of safety and prevention of contamination, however, micropipettes, macropipettes, and robotic liquid handlers are preferred.

[0018] The robotic liquid handler may be an automated nucleic acid purification device or an automated protein purification device, and examples of such automated purification devices include the magLEAD system (manufactured by Precision System Science Co., Ltd.), the Purelumn system (manufactured by Precision System Science Co., Ltd.), AssistPlus (manufactured by Integra Biosciences Inc.), and CyBio FeliX (manufactured by Analytik Jena).

[0019] The pipetting device may be manual or electric, but is preferably electric since it allows for accurate pipetting operations. The pipetting device may have multiple channels. From the viewpoint of safety and prevention of contamination, the pipette tip is preferably a disposable pipette tip.

[0020] The pipette tip body has an elongated tubular body, a lower opening at the lower end of the pipette tip body for passing liquid, and an upper opening at the upper end for fitting onto an attachment of a pipetting device. The specifications of the pipette tip body are in accordance with ISO 8655-1:2022 as appropriate.

[0021] Pipetting device attachments are usually standardized conical or frustum-shaped attachments by many manufacturers, and are known to have standard shapes characterized by a specific average diameter and a specific cone angle for each pipette tip size. The shape and size of the upper opening of the pipette tip body are not particularly limited as long as they can fit into such attachments.

[0022] The shape of the tip body is not particularly limited, but preferably has a taper from top to bottom, with the cross section increasing in size from the lower opening to the upper opening.

[0023] The tip body contains a liquid therein. The volume of the chip body is not particularly limited, but is preferably a volume that can handle liquids such as 0.1 μL to 10 μL, 0.5 μL to 10 μL, 2 to 200 μL, 30 to 300 μL, 100 to 1000 μL, 500 to 5000 μL, or 1000 to 10000 μL.

[0024] The material of the tip body is not particularly limited, and any known material can be used. The material of the chip body is preferably non-adsorbent to substance α or substance β and non-reactive to the sample solution. Here, "adsorbent" means the property of being able to adsorb under specific adsorption conditions, and "non-adsorbent" means being non-adsorbent under specific conditions. The material of the chip body is preferably one that can withstand sterilization treatments such as electron beam sterilization, gamma ray sterilization, or autoclave treatment.

[0025] The material of the tip body is preferably a synthetic resin. Examples of synthetic resins include polystyrene, polycarbonate, polyethylene, polypropylene, polyethylene terephthalate, polyetherimide, polyimide, ABS resin, polyvinyl chloride, polyvinylidene chloride, and fluororesin. These may be used alone or in combination of two or more. The synthetic resin is preferably polypropylene because of its high transparency and low drug adsorption. The tip body is preferably a one-piece molded structure made of synthetic resin.

[0026] The synthetic resin may have its surface treated with a hydrophilic compound to reduce drug adsorption. Examples of hydrophilic compounds include phosphorylcholine group-containing compounds such as poly((meth)acryloyloxyethyl phosphorylcholine), which is a homopolymer of 2-methacryloyloxyethyl phosphorylcholine, and copolymers of (meth)acryloyloxyethyl phosphorylcholine and (meth)acrylic monomers.

[0027] The shape and size of the lower end of the tip body are not particularly limited as long as it has a lower opening. An example of the shape of the lower end of the pipette tip is shown in Figure 1. When viewed from the front, the pipette tip may have any of the shapes shown in a to p in Figure 1. For simplicity, the carrier is not shown in Figure 1.

[0028] The entire lower end of the chip body may be a lower opening (e.g., Figures 1a, f, g, j, k, l, m, o, p), or only a portion of it may be a lower opening (e.g., Figures 1b, c, d, e, h, i, n), but it is preferable that the entire lower end of the chip body be a lower opening, as this provides excellent liquid permeability and good carrier dispersibility.

[0029] A filter is disposed in the lower opening at the lower end of the tip body. The lower opening is usually entirely covered with a filter to hold the carrier within the chip body. The shape and size of the lower opening are not particularly limited, but are preferably circular, elliptical, approximately circular, or approximately elliptical.

[0030] The position of the lower opening is not particularly limited as long as it allows liquid to pass through the pipette tip, and may be a position including the lowest end of the pipette tip (e.g., Figures 1a, ck, l, m, o, and p) or a position not including the lowest end (e.g., Figure 1b and n).However, a position including the lowest end of the pipette tip is preferable because the dead volume is small and carriers are less likely to remain near the lowest end of the pipette tip even after repeated aspiration and discharge. The shape of the bottom end of the pipette tip is not particularly limited, and may be an acute angle (e.g., Figures 1a-d, fj, n, o, p) or an obtuse angle (e.g., Figure 1e, k) when viewed from the front of the pipette tip, but is preferably an acute angle.

[0031] The number of the lower openings may be at least one, but may be two or more (for example, FIG. 1d, n).

[0032] At least a portion of the lower opening in which the filter is disposed is provided with a slope. The lower opening in which the filter is disposed has a sloped portion in all or part of it, and preferably the entire lower opening in which the filter is disposed has a sloped portion (e.g., Figures 1a-i, k, p). When a sloped portion is provided in a portion of the lower opening where the filter is arranged (for example, Figure 1j), the portion of the lower opening other than the sloped portion is typically perpendicular to the axis of the pipette tip when viewed from the front.

[0033] At least one inclined portion may be provided, and multiple inclined portions may be provided. When multiple inclined portions are provided, the inclination angles of the multiple inclined portions may be the same (e.g., Figs. 1g, h, n, o) or different (e.g., Fig. 1f). When multiple inclined portions are provided, the multiple inclined portions may be arranged so as to be convex toward the outside of the chip body (e.g., Figures 1f, g, h, n), or so as to be convex toward the inside of the chip body (e.g., Figure 1o), but are preferably arranged so as to be convex toward the outside of the chip body.

[0034] The inclined portion of the pipette tip allows the sample liquid to be smoothly aspirated and discharged, and the carrier in the pipette tip is efficiently dispersed in the sample liquid by aspirating and discharging. Furthermore, dispersion of the carrier by aspirating and discharging can be repeatedly performed. When the pipette tip is provided with the inclined portion, the carrier is dispersed in the sample liquid in the pipette tip during aspiration, creating a state similar to batch chromatography. Therefore, the same advantages as batch chromatography, that is, the carrier is easily dispersed in the sample liquid and substance α or substance β is easily adsorbed onto the carrier, are easily obtained. This advantage is particularly easily obtained when turbulence occurs during aspiration. Carriers accumulated on a filter after discharge may be difficult to disperse even when aspirated. However, when turbulence occurs during aspiration, the carriers are easily dispersed during aspiration, even if they are in an aggregated state immediately after discharge. When the pipette tip is provided with the inclined portion, the carrier accumulates on the filter during dispensing, and the sample liquid is delivered from the top to the bottom of the accumulated carrier, creating a state similar to that of column chromatography. Therefore, the same advantage as column chromatography, namely, substance α or substance β in the sample liquid is likely to be adsorbed by the carrier, is easily achieved. This advantage is particularly likely to be achieved when, between dispensing and the next aspiration, the position of the carrier on the filter shifts due to gravity toward the bottom of the pipette tip, resulting in the carrier only partially covering the filter. When the pipette tip is provided with the inclined portion, repeated aspirating and dispensing of sample liquid results in a state similar to that when batch chromatography and column chromatography are repeatedly performed, making it easier to obtain the benefits of both batch chromatography and column chromatography.

[0035] The inclined portion may be a flat surface (for example, FIGS. 1a to 1k, n, o, and p) or a curved surface (for example, FIGS. 1l and 1m), but is preferably a flat surface.

[0036] When the inclined portion is flat, the inclination angle of the inclined portion is not particularly limited as long as it is greater than 0° with respect to a line perpendicular to the axis of the pipette tip when viewed from the front of the pipette tip. Note that the front view refers to a state in which the pipette tip is viewed from a direction that best shows the characteristics of the shape of the pipette tip when used with a pipetting device. The inclination angle of the inclined portion is an angle greater than 0° and less than 90° formed by the inclined portion and a line perpendicular to the axis of the pipette tip. The axis of the pipette tip refers to a line passing through the centroid of the axial direction of the pipette tip (the longitudinal direction of the pipette tip). Figure 1a' is a diagram illustrating these concepts using Figure 1a. The inclination angle of the inclined portion is preferably greater than 20° and less than 70°, more preferably greater than 40° and less than 70°, relative to a line perpendicular to the axis of the pipette tip when viewed from the front of the pipette tip. When the inclination angle of the inclined portion is within the above range, the carriers can be dispersed more efficiently within the chip body.

[0037] When the inclination angle of the inclined portion is greater than 40° and less than 70° relative to a line perpendicular to the axis of the pipette tip when viewed from the front of the pipette tip, turbulence is likely to occur as the solution is aspirated. Furthermore, as the solution is discharged, the carrier tends to accumulate on the filter so as to uniformly cover the entire surface of the filter. After dispensing, the position of the carrier on the filter tends to shift toward the bottom end of the pipette tip due to gravity between dispensing and the next aspiration, and the carrier tends to cover only a portion of the filter. Furthermore, even when the solution is repeatedly aspirated and discharged, the carrier is unlikely to remain near the bottom end of the pipette tip. Furthermore, the shape of the pipette tip near the bottom end tends to be stable, making it easier to manufacture.

[0038] When the inclined portion is a curved surface, it is preferable that the inclined portion is formed in an arc shape when viewed from the front of the pipette tip (for example, Figure 1l, m), and may be an arc shape that is convex toward the outside of the tip body, an arc shape that is convex toward the inside of the tip body, or an arc shape that combines a convex toward the outside of the tip body and a convex toward the inside of the tip body.

[0039] The position of the lowest end of the tip body is not particularly limited, but if the lowest end of the tip body is formed on the axis of the tip body (for example, Figures 1g, h, m, and p), the solution in the U-shaped tube at the lower end can be easily sucked in and ejected efficiently.

[0040] The surface area of ​​the inclined portion is not particularly limited and may be determined appropriately depending on the capacity of the pipette tip body or the amount of the carrier. For example, when the capacity of the pipette tip body is such that it can handle 1000 μL to 10000 μL of liquid and the volume of the concentrated liquid is 50 to 300 μL, the surface area of ​​the inclined portion is preferably 0.05 to 9 cm. 2 and more preferably 0.1 to 2.0 cm 2 is.

[0041] The pipette tip has a sloped portion at least in part of the lower opening where the filter is located at the bottom end of the tip body, making it easier to disperse the carrier in the pipette tip by suction and discharge, and the simple structure results in a small dead volume.

[0042] The pipette tip holds a carrier within the tip body. The pipette tip may have a barrier filter within the pipette tip body above the carrier to prevent contamination or to prevent the carrier from coming out of the upper opening when the pipette tip is tilted. The position of the barrier filter is not particularly limited as long as it is above the carrier, but it is preferably located near the upper opening of the pipette tip body so as not to come into contact with the aspirated liquid.

[0043] The material of the barrier filter is not particularly limited as long as it is permeable to gases and does not allow the carrier to pass through, and known materials can be used. The material of the barrier filter is preferably one that prevents the passage of aerosols, more preferably polyethylene, and even more preferably sintered high density polyethylene granules.

[0044] The method for manufacturing the pipette tip is not particularly limited, and the pipette tip can be manufactured by a known method. For example, the lower opening may be obtained by injection molding a pipette tip having a shape with a lower opening, or the lower end of the body of an existing tip may be subjected to secondary processing to provide a lower opening.

[0045] Filter The filter is disposed in the lower opening at the bottom end of the chip body to hold the carrier within the chip body. The filter does not allow the carrier to pass through, specifically, it is a filter having pores of a size that does not allow the carrier to pass through. The filter allows the substance α in the sample liquid to pass through, and specifically has pores of a size that allows the substance α to pass through. The filter is preferably one that does not allow the carrier to pass through but allows substances α and β in the sample solution to pass through. Specifically, it is preferable for the filter to have pores that do not allow the carrier to pass through but have pores that are large enough to allow substances α and β to pass through.

[0046] The pore size of the filter is not particularly limited as long as it does not allow the carrier to pass through, that is, the pore size is smaller than the size of the carrier. The pore size of the filter is not particularly limited as long as it allows the substance α to pass through, that is, the pore size is larger than the size of the substance α. The pore size of the filter is preferably such that it does not allow the carrier to pass through but allows the substances α and β to pass through, i.e., the pore size is preferably smaller than the size of the carrier but larger than the size of the substances α and β. The pore size of the filter can be appropriately selected from the range of approximately 100 nm or more and less than 2 mm depending on the size of the carrier, substance α, or substance β used, but is preferably 100 nm to 20 μm, more preferably 500 nm to 10 μm, and even more preferably 500 nm to 5 μm.

[0047] One reason why typical batch chromatography cannot adequately capture a target substance is that the ratio of the amount of carrier to the amount of sample liquid is always constant while the sample liquid is in contact with the carrier. On the other hand, the pipette tip of the present invention is a device that performs a type of batch chromatography. The carrier cannot pass through a filter and therefore remains within the tip body, but the sample liquid can pass through the filter and move back and forth between the inside and outside of the tip body. Therefore, the ratio of the amount of carrier to the amount of sample liquid changes while the sample liquid is in contact with the carrier. Therefore, substance α or substance β can be sufficiently adsorbed onto the carrier.

[0048] For example, when using a sample liquid containing viruses or exosomes, viruses generally have a diameter of about 100 to 200 nm, exosomes have a diameter of about 20 to 200 nm, and the average particle size of the carrier is preferably 0.5 μm to 2 mm. Therefore, the pore size of the filter can be appropriately selected from a range of about 200 nm to 2 mm corresponding to these diameters, but is preferably 250 nm to 20 μm, more preferably 500 nm to 10 μm, and even more preferably 500 nm to 5 μm. Furthermore, for example, when a sample liquid containing a protein is used, the diameter of the protein is generally about 1 to 100 nm, and the average particle size of the carrier is preferably 0.5 μm to 2 mm. Therefore, the pore size of the filter can be appropriately selected in the range of about 100 nm to 2 mm corresponding to these diameters, but is preferably 250 nm to 20 μm, more preferably 500 nm to 10 μm, and even more preferably 500 nm to 5 μm.

[0049] The form of the filter is not particularly limited and may be a porous membrane having an interconnected pore structure, or a woven or nonwoven fabric composed of fibers or ultrafine fibers, but is preferably a porous membrane or nonwoven fabric, more preferably a nonwoven fabric. Ultrafine fibers are fibers with a diameter of 5 μm or less, preferably about 1 μm.

[0050] The material constituting the filter is not particularly limited, but is preferably a material that is less likely to adsorb the substances α and β. The filter is preferably a hydrophilic filter in view of the fact that the substances α and β are less likely to be adsorbed thereto, etc. Examples of such hydrophilic filters include filters made of a hydrophilic material and filters made of a material with insufficient hydrophilicity that have been subjected to a hydrophilization treatment.

[0051] When a hydrophilic filter is used as the filter, bubbles are less likely to get into the liquid in the pipette tip, even when the amount of sample liquid is small and a portion of the filter is not immersed in the sample liquid, and problems are less likely to occur during subsequent aspiration and discharge. Without being bound by theory, this is thought to be because the filter is hydrophilic, so that even when only a portion of the filter comes into contact with the sample liquid, the entire filter is easily wetted, making it difficult for air to pass through the filter.

[0052] Examples of materials for the filter include thermoplastic resins such as polyolefins (e.g., polyethylene, polypropylene), polystyrene, ABS resin, AS resin, EVA resin, polymethylpentene, polyvinyl chloride, polyvinylidene chloride, poly(meth)acrylic acid ester, polyvinyl acetate, polyamide, polyimide, polycarbonate, polyethylene terephthalate, polybutylene terephthalate, polyacetal, polyarylate, polysulfone, and fluororesin; polylactic acid, polyhydroxybutyrate, modified starch resin, polycaprolactone, polybutylene succinate, polybutylene adipate terephthalate, and polybutylene succinate. Examples of suitable materials include biodegradable resins such as carboxylate terephthalate and polyethylene succinate; thermosetting resins such as phenolic resins, urea resins, melamine resins, unsaturated polyester resins, diallyl phthalate resins, epoxy resins, epoxy (meth)acrylate resins, silicon resins, (meth)acrylic urethane resins, and urethane resins; elastomers such as silicone resins, polystyrene elastomers, polyethylene elastomers, polypropylene elastomers, polyurethane elastomers, and fluoroelastomers (e.g., FKM and FFKM); natural materials such as pulp, hemp, cellulose, kenaf, chitin, chitosan, and cotton; and inorganic materials such as glass, silica, and metals. These materials may be used alone or in combination of two or more. Among these, from the viewpoints of heat resistance and processability, the material constituting the filter preferably contains a fluororesin or a fluoroelastomer, more preferably a fluororesin or a fluoroelastomer, and even more preferably a filter composed of a hydrophilically treated fluororesin or a hydrophilically treated fluoroelastomer (particularly a hydrophilically treated FKM).

[0053] The fluororesin is not particularly limited, and examples thereof include polytetrafluoroethylene (PTFE), tetrafluoroethylene-perfluoroalkyl vinyl ether copolymer (PFA), tetrafluoroethylene-hexafluoropropylene copolymer (FEP), ethylene-tetrafluoroethylene copolymer (ETFE), tetrafluoroethylene-hexafluoropropylene-perfluoroalkyl vinyl ether copolymer (EPE), fluoroethylene-vinyl ether copolymer (FEVE), poly(chlorotrifluoroethylene) (PCTFE), ethylene-chlorotrifluoroethylene copolymer (ECTFE), polyvinylidene fluoride (PVDF), polyvinyl fluoride (PVF), vinylidene fluoride-hexafluoropropylene copolymer (VDF-HFP copolymer), and vinylidene fluoride-hexafluoropropylene-tetrafluoroethylene copolymer (VDF-HFP-TFE copolymer). Among these, PTFE and PVDF are preferred, and PTFE is more preferred, as they can more effectively exhibit the effects of the present invention and have excellent chemical resistance.

[0054] Among these, the material constituting the filter is preferably a woven fabric or nonwoven fabric composed of PTFE-containing fibers or ultrafine fibers, because of its good liquid permeability, more preferably a woven fabric or nonwoven fabric composed of PTFE fibers or ultrafine fibers, and even more preferably a nonwoven fabric composed of hydrophilically treated PTFE ultrafine fibers.

[0055] The fluoroelastomer is not particularly limited, and examples thereof include perfluoro(alkyl vinyl ether), perfluoro(alkoxyalkyl vinyl ether), vinylidene fluoride-hexafluoropropylene polymer, vinylidene fluoride-hexafluoropropylene-tetrafluoroethylene polymer, tetrafluoroethylene-propylene polymer, vinylidene fluoride-propylene-tetrafluoroethylene polymer, ethylene-tetrafluoroethylene-perfluoromethyl vinyl ether polymer, vinylidene fluoride-tetrafluoroethylene-perfluoromethyl vinyl ether polymer, and vinylidene fluoride-perfluoromethyl vinyl ether polymer. Among these, ternary FKM is preferred from the viewpoint of excellent heat resistance, chemical resistance, etc., and vinylidene fluoride-hexafluoropropylene-tetrafluoroethylene polymer is more preferred.

[0056] The hydrophilization treatment preferably includes a step of coating the filter with a compound having a hydrophilic group (hereinafter also referred to as "hydrophilic compound"), and more preferably includes a step of immersing the filter in a solution of the hydrophilic compound to coat the filter with the hydrophilic compound, and then crosslinking the hydrophilic compound. Specific examples of such steps include the steps described in WO 2014 / 021167 and JP-B-4-075051. The hydrophilization treatment may be repeated multiple times. For example, the filter may be immersed in a solution of a hydrophilic compound to coat the filter with the hydrophilic compound, and then the hydrophilic compound may be crosslinked. After that, the filter may be further immersed in a solution of a hydrophilic compound that is the same as or different from the hydrophilic compound previously used to coat the filter with the hydrophilic compound.

[0057] The hydrophilic compound is not particularly limited as long as it does not impair the effects of the present invention, and any compound having a straight chain, branched chain, dendrimer, or other shape can be used. Examples of the hydrophilic compound include a hydroxyl group-containing compound, a carboxylic acid group-containing compound, a sulfonic acid group-containing compound, an ether group-containing compound, an epoxy group-containing compound, an amino group-containing compound, an amide group-containing compound, and a phosphorylcholine group-containing compound. The hydrophilic compounds may be used alone or in combination of two or more.

[0058] The hydroxyl group-containing compound is not particularly limited, and examples thereof include polyvinyl alcohol (PVA) and modified products thereof (e.g., ethylene oxide group-modified PVA, carboxyl group-modified PVA, sulfonic acid group-modified PVA, and quaternary ammonium-modified PVA); polysaccharides and derivatives thereof, such as agarose, dextran, chitosan, cellulose, and heparin; collagen; gelatin; copolymers of vinyl alcohol and vinyl group-containing monomers (e.g., vinyl alcohol-vinyl acetate copolymer, ethylene-vinyl alcohol copolymer, and vinyl alcohol-polyvinylpyrrolidone copolymer); (meth)acrylic polyols, fluorine-containing polyols, polyoxyalkylenes (e.g., polyethylene glycol, copolymers of polyethylene glycol and polypropylene glycol [e.g., Pluronic F108, Pluronic F127 (all manufactured by Sigma-Aldrich)]), polyester polyol, diethylene glycol, and other polyols; and hydroxyl group-containing (meth)acrylic compounds such as poly(2-hydroxyethyl (meth)acrylate), 1-hydroxypropan-2-yl (meth)acrylate, 2-hydroxypropan-1-yl (meth)acrylate (also known as 2-hydroxypropyl (meth)acrylate), 2-hydroxy-1-methylethyl (meth)acrylate, hydroxypropyl (meth)acrylate, 2,3-dihydroxypropyl (meth)acrylate, and hydroxyethyl (meth)acrylate.

[0059] The carboxylic acid group-containing compound is not particularly limited, and examples thereof include copolymers of one or more monomers selected from olefin monomers such as ethylene, propylene, and butylene, diene monomers such as butadiene, aromatic group-containing monomers such as styrene, and (meth)acrylic acid ester monomers, with a monomer having a carboxylic acid group [—COOH] such as (meth)acrylic acid; homopolymers of monomers having a carboxylic acid group such as (meth)acrylic acid; and amino acids.

[0060] The sulfonic acid group-containing compound is not particularly limited, but examples thereof include a copolymer of styrene and acrylamido-2-methylpropanesulfonic acid (salt); a ternary copolymer of styrene, n-butyl acrylate, and acrylamido-2-methylpropanesulfonic acid (salt); and a ternary copolymer of styrene, 2-ethylhexyl acrylate, and acrylamido-2-methylpropanesulfonic acid (salt).

[0061] The ether group-containing compound is not particularly limited, but examples thereof include polyethylene glycol and derivatives thereof, fluororesins having an ether group, polyurethane resins having an ether group, and polyphenylene resins having an ether group.

[0062] The epoxy group-containing compound is not particularly limited, but examples thereof include epoxy resins, modified epoxy resins, (meth)acrylic (co)polymers having epoxy groups, polybutadiene resins having epoxy groups, polyurethane resins having epoxy groups, and adducts or condensates of these resins.

[0063] The amino group-containing compound is not particularly limited, but examples thereof include polyethyleneimine, polyvinylamine, polyamidepolyamine, polyamidine, polydimethylaminoethyl methacrylate, and polydimethylaminoethyl acrylate.

[0064] The amide group-containing compound is not particularly limited, but examples thereof include poly(N-isopropyl(meth)acrylamide) and poly(N-vinyl-2-pyrrolidone).

[0065] The phosphorylcholine group-containing compound is not particularly limited, but examples thereof include poly((meth)acryloyloxyethyl phosphorylcholine), which is a homopolymer of 2-methacryloyloxyethyl phosphorylcholine, and a copolymer of (meth)acryloyloxyethyl phosphorylcholine and a (meth)acrylic monomer.

[0066] The weight average molecular weight of the hydrophilic compound is not particularly limited, but is preferably 100 to 1,000,000.

[0067] The time for which the filter is immersed in the solution of the hydrophilic compound is not particularly limited as long as it is a time that allows the filter to be covered with the hydrophilic compound. Although it depends on the concentration of the hydrophilic compound in the solution used, the time is preferably 1 second to 60 minutes, and more preferably 10 seconds to 30 minutes. The immersion temperature and atmosphere are not particularly limited and may be appropriately selected depending on the type of hydrophilic compound, etc.

[0068] When an aqueous solution of the hydrophilic compound is used as the solution, even if an untreated filter is immersed in the aqueous solution of the hydrophilic compound, the hydrophilic compound may not penetrate to the inside of the filter. Therefore, it is preferable to immerse the filter in a water-compatible solvent such as isopropyl alcohol (impregnate the filter with the water-compatible solvent) before immersing the filter in the solution of the hydrophilic compound. When a filter is immersed in a water-compatible solvent, it is preferable to remove the filter from the water-compatible solvent and then immerse it in a solution of a hydrophilic compound to replace the water-compatible solvent with the hydrophilic compound. In this case, the hydrophilic compound may be forced into the filter by applying mechanical strength to the filter. Specifically, the filter may be pressed and rubbed, or a vacuum and pressure impregnation device may be used to reduce pressure or apply pressure, thereby making it easier to impregnate the filter with the hydrophilic compound.

[0069] The water-compatible solvent is not particularly limited, but is preferably a solvent that easily penetrates the filter and easily volatilizes, and specific examples thereof include alcohols such as methyl alcohol, ethyl alcohol, n-propyl alcohol, isopropyl alcohol, n-butyl alcohol, sec-butyl alcohol, tert-butyl alcohol, and isobutyl alcohol; esters such as methyl acetate, ethyl acetate, and butyl acetate; ketones such as acetone and methyl ethyl ketone; ethers such as tetrahydrofuran and dioxane; and aprotic polar solvents such as dimethyl sulfoxide and N,N-dimethylformamide. Among these, methyl alcohol, ethyl alcohol, or isopropyl alcohol is preferred in terms of easy penetration into the filter. The water-compatible solvent may be a liquid obtained by mixing water with at least one selected from the alcohols, esters, ketones, ethers, and aprotic polar solvents. These water-compatible solvents may be used alone or in combination of two or more.

[0070] The time for immersing the filter in the water-compatible solvent is not particularly limited, but is, for example, 1 minute to 24 hours. The immersion temperature and atmosphere are not particularly limited.

[0071] Examples of methods for crosslinking the hydrophilic compound include irradiation crosslinking with ionizing radiation such as an electron beam, thermal crosslinking, and chemical crosslinking using a crosslinking agent. Among these crosslinking methods, chemical crosslinking using a crosslinking agent is preferred because crosslinking is possible even in an aqueous solution and the crosslinking is more reliable.

[0072] The crosslinking agent used in the chemical crosslinking is not particularly limited and may be appropriately selected depending on the type of hydrophilic compound used. Examples of the crosslinking agent include aldehyde compounds such as formaldehyde, glutaraldehyde, and terephthalaldehyde; ketone compounds such as diacetyl and chloropentanedione; compounds having a reactive halogen such as bis(2-chloroethylurea)-2-hydroxy-4,6-dichloro-1,3,5-triazine; compounds having a reactive olefin such as divinyl sulfone; N-methylol compounds; isocyanates; aziridine compounds; carbodiimide compounds; epoxy compounds; halogenated carboxaldehydes such as mucochloric acid; dioxane derivatives such as dihydroxydioxane; inorganic crosslinking agents such as chrome alum, zirconium sulfate, boric acid, borates, and phosphates; diazo compounds such as 1,1-bis(diazoacetyl)-2-phenylethane; compounds containing disuccinimidyl esters; and bifunctional maleic acid imides. These crosslinking agents may be used alone or in combination of two or more.

[0073] The crosslinking agent is preferably used in a large excess amount relative to the amount of the hydrophilic compound in the solution of the hydrophilic compound used.

[0074] The temperature during the crosslinking is preferably 10 to 95°C, more preferably 20 to 60°C, from the viewpoint that a filter having excellent chemical resistance can be easily obtained. The crosslinking time is preferably 1 to 60 minutes from the viewpoints of the durability and productivity of the resulting filter.

[0075] The hydrophilized filter is preferably washed with water or the like for the purpose of removing unreacted hydrophilic compound, etc. This step may be carried out under heating, if necessary.

[0076] The melting point of the material constituting the filter is preferably higher than that of the material constituting the tip body. The melting point of the material constituting the filter is preferably at least 30°C higher than that of the material constituting the tip body, and more preferably at least 100°C higher. By using such a material, for example, the lower opening at the bottom end of the tip body can be melted by heat and the melted lower opening brought into contact with the filter, thereby fusing the filter to the lower opening, thereby making it possible to easily manufacture the pipette tip of the present invention.

[0077] In the pipette tip, the melting point of the material constituting the filter is preferably higher than the melting point of the material constituting the tip body, and the filter is fused to the lower opening. The pipette tip is preferably a pipette tip obtained by melting the lower opening at the lower end of the tip body with heat, bringing the melted lower opening into contact with the filter, and fusing the filter to the lower opening.

[0078] [Carrier] The carrier is not particularly limited as long as it is a carrier that is normally held within the body of a pipette tip. The carrier is preferably a carrier for adsorbing the substance α or the substance β, and such a carrier may be appropriately selected depending on the type of the substance α and the substance β. Details of the substance α and the substance β will be described later. When a purified solution having an increased purity of substance α or a concentrated solution having an increased concentration of substance α is produced from a sample solution containing substance α, the carrier has adsorptivity for substance α and preferably also has non-adsorptivity for impurities. For example, when a purified solution having an increased purity of protein or a concentrated solution having an increased concentration of protein is produced from a sample solution containing the protein, the carrier has adsorptivity for the protein. Here, "adsorbability" means the property of being able to adsorb under specific adsorption conditions, and "non-adsorbability" means the property of not substantially adsorbing under specific conditions.

[0079] Examples of carriers capable of adsorbing proteins include clay, agarose, dextran, hydroxyapatite, silica gel, polystyrene, phenolic resin, acrylic resin, polyolefin (e.g., polyethylene, polypropylene), polyvinyl chloride, and derivatives thereof. For example, agarose derivatives include Sepharose, dextran derivatives include Sephadex, and hydroxyapatite derivatives include hydroxyapatite carbonate. Among these, hydroxyapatite, Sepharose, and agarose are preferred, with hydroxyapatite being more preferred.

[0080] Carriers that are particularly adsorbent to antibodies include, for example, agarose or beads to which a bacterial protein that specifically binds to the antibody has been immobilized, and examples of such carriers include agarose or beads to which Protein A, Protein G, or Protein L has been immobilized. Protein A, Protein G, or Protein L may be recombinant, or may be recombinant products obtained by removing the region that nonspecifically binds to impurities such as albumin and / or the cell wall-binding region from Protein A, Protein G, or Protein L.

[0081] Examples of carriers capable of adsorbing nucleic acids include hydroxyapatite, silica, magnetic silica beads, cellulose, and agarose or beads onto which a substance having structural affinity for complementary strands or nucleic acids has been immobilized. Examples of carriers capable of adsorbing lipids include agarose or beads to which Tim4, which binds to phosphatidylserine, has been immobilized. Examples of carriers capable of adsorbing sugars include ion exchange resins, activated carbon, agarose or beads on which lectins or sugar chain-recognizing antibodies are immobilized, and boronic acid and its derivatives.

[0082] Examples of carriers that have adsorptivity for exosomes include agarose or beads onto which a substance that has affinity for molecules expressed on the surface of exosomes has been immobilized; more specifically, agarose or beads onto which a lipid-binding protein such as Tim4 that binds to phosphatidylserine, a lipid-recognizing antibody, or a lipid-recognizing aptamer has been immobilized; agarose or beads onto which an antibody or aptamer that recognizes an exosome-specific protein such as a protein belonging to the tetraspanin family such as CD9 has been immobilized; and agarose or beads onto which a molecule that recognizes a lectin has been immobilized.

[0083] Examples of carriers that have adsorption properties for viruses include agarose or beads onto which a substance that has affinity for molecules expressed on the surface of the virus has been immobilized (e.g., agarose or beads onto which an antibody or aptamer that recognizes the spike protein of the virus has been immobilized).

[0084] When adsorbing a protein to the carrier, the adsorption may be achieved by using a combination of substances having specific affinity. That is, the carrier may have one of a combination of substances having specific affinity immobilized thereon via a chemical modification group. Examples of the combination of substances having specific affinity include an antibody and an antigen, an antibody and an antibody-binding protein, a GST (glutathione-S-transferase) tag and glutathione, a His tag and a divalent cation, and biotin and avidin.

[0085] Carriers capable of adsorbing proteins include, for example, agarose or derivatives thereof to which antibodies against specific proteins are bound. The carrier may have one or more types of antibodies bound to it. Examples of carriers to which multiple types of antibodies are bound include agarose or derivatives thereof to which multiple antibodies against several proteins (e.g., α1-acid glycoprotein, α1-antitrypsin, α2-macroglobulin, albumin, apolipoprotein AI, apolipoprotein A-II, fibrinogen, haptoglobulin, IgA, IgG, IgM, and transferrin) that are abundantly contained in human serum or plasma are bound.

[0086] When a purified solution with an increased purity of substance α or a concentrated solution with an increased concentration of substance α is produced from a sample solution containing substance β, the carrier has adsorptivity for substance β, preferably for the encapsulation substance (details will be described later), and may have adsorptivity or non-adsorptivity for substance α. For example, when a purified solution with an increased purity of nucleic acid or a concentrated solution with an increased concentration of nucleic acid is produced from a sample solution containing a nucleic acid-containing substance, the carrier is preferably one that has adsorptivity for the nucleic acid-containing substance, preferably for the encapsulation substance, and the carrier may be a carrier that does not adsorptivity for nucleic acid or a carrier that has adsorptivity for nucleic acid. Furthermore, when producing a purified liquid with an increased purity of substance α or a concentrated liquid with an increased concentration of substance α from a sample liquid containing substance β, if a carrier having adsorption properties for substances α and β is used as the carrier, the conditions for contacting the carrier with substance β can be adjusted so that the adsorption properties between the carrier and substance β are higher than the adsorption properties between the carrier and substance α, or so that the carrier adsorbs substance β but does not adsorb substance α.

[0087] Examples of carriers that have adsorptivity for nucleic acid-containing substances, preferably encapsulating substances, more preferably proteins, and have adsorptivity for nucleic acids include hydroxyapatite.

[0088] When producing a purified solution with increased nucleic acid purity or a concentrated solution with increased nucleic acid concentration from a sample solution containing a virus, the carrier preferably has adsorptivity for at least one of proteins, lipids, and sugars that constitute the encapsulation substance, and more preferably has adsorptivity for proteins. In this case, the carrier may be a carrier that does not adsorb nucleic acids, or a carrier that adsorbs nucleic acids.

[0089] When producing a purified solution having an increased purity of substance α (at least one selected from proteins, lipids, and sugars) or a concentrated solution having an increased concentration of substance α (at least one selected from proteins, lipids, and sugars) from a sample solution containing extracellular vesicles such as exosomes, microvesicles, and apoptotic vesicles, the carrier preferably has adsorptivity for at least one selected from proteins, lipids, and sugars that constitute the encapsulation substance, more preferably adsorptivity for proteins. In this case, the carrier may be non-adsorptive for substance α or adsorptive for substance α.

[0090] When producing a purified solution with increased purity of a virus, extracellular vesicles, or a DDS (Drug Delivery System) preparation such as liposomes or lipid nanoparticles (LNPs) from a sample solution containing the virus, extracellular vesicles, or DDS preparation, or a concentrated solution with increased concentration of the virus, extracellular vesicles, or DDS preparation, the carrier preferably has adsorptivity for at least one selected from proteins, lipids, and sugars that constitute the encapsulation substance of the virus, extracellular vesicles, or DDS preparation, more preferably has adsorptivity for lipids, and even more preferably has adsorptivity for phospholipids. When producing a purified solution with increased purity of extracellular vesicles or a concentrated solution with increased concentration of extracellular vesicles from a sample solution containing the extracellular vesicles, the carrier preferably has the ability to adsorb phosphatidylserine, and is particularly preferably agarose or beads on which a lipid-binding protein such as Tim4 that binds to phosphatidylserine has been immobilized.

[0091] Examples of carriers that have adsorptivity for phosphatidylserine, which constitutes the encapsulation substance of exosomes, include agarose or beads onto which a substance having affinity for phosphatidylserine has been immobilized (e.g., agarose or beads onto which a lipid-binding protein such as Tim4 that binds to phosphatidylserine, a lipid-recognizing antibody, or a lipid-recognizing aptamer has been immobilized). Examples of carriers capable of adsorbing proteins that constitute the exosome encapsulation substance include agarose or beads to which antibodies or aptamers that recognize exosome-specific proteins, such as proteins belonging to the tetraspanin family, such as CD9, have been immobilized. Examples of carriers that have adsorptivity for the sugars that constitute the exosome encapsulation substance include agarose or beads to which a molecule that recognizes lectin has been immobilized.

[0092] When producing a purified solution in which the purity of substance α, which is at least one selected from nucleic acids, proteins, lipids, and sugars, has been increased, or a concentrated solution in which the concentration of substance α, which is at least one selected from nucleic acids, proteins, lipids, and sugars, has been increased, from a sample solution containing liposomes, the carrier preferably has adsorptivity for the lipids that make up the encapsulation substance and has non-adsorptivity for substance α, or preferably has adsorptivity for the lipids that make up the encapsulation substance and has non-adsorptivity for nucleic acids, proteins, or sugars.

[0093] Examples of carriers that are non-adsorbent for nucleic acids but adsorbent for proteins include agarose or beads on which a protein-recognizing antibody is immobilized. Examples of carriers that are non-adsorbent for nucleic acids but adsorbent for lipids include agarose or beads to which a lipid-binding protein such as Tim4 or a lipid-recognizing antibody has been immobilized. Examples of carriers that are non-adsorbent for nucleic acids but adsorbent for sugars include agarose or beads on which lectins or sugar chain-recognizing antibodies are immobilized.

[0094] Examples of carriers that are non-adsorbent for proteins but adsorbent for lipids include agarose or beads to which a lipid-binding protein such as Tim4 or a lipid-recognizing antibody has been immobilized. Examples of carriers that are non-adsorbent for proteins but adsorbent for sugars include agarose or beads on which lectins or sugar chain-recognizing antibodies are immobilized.

[0095] Examples of carriers that are non-adsorptive to lipids but adsorbent to nucleic acids include silica and hydroxyapatite. Examples of carriers that are non-adsorbent for lipids but adsorbent for proteins include agarose or beads on which antibodies are immobilized, and hydroxyapatite. Examples of carriers that are non-adsorbent for lipids but adsorbent for sugars include agarose or beads on which lectins or sugar chain-recognizing antibodies are immobilized.

[0096] Examples of carriers that are non-adsorbent for sugars but adsorbent for nucleic acids include silica and hydroxyapatite. Examples of carriers that are non-adsorbent for sugars but adsorbent for proteins include agarose or beads on which antibodies are immobilized, and hydroxyapatite. Examples of carriers that are non-adsorbent for sugars but adsorbent for lipids include agarose or beads to which a lipid-binding protein such as Tim4 or a lipid-recognizing antibody has been immobilized.

[0097] When viruses or extracellular vesicles are adsorbed onto the carrier, antigenic proteins present on the surface of the viruses or extracellular vesicles may be used for adsorption onto the carrier. For example, antigenic proteins present on the surface of exosomes include tetraspanins such as CD9, CD63, and CD81; antigen presentation-related proteins such as MHC1 and MHCII; adhesion molecules such as integrins, ICAM-1, and EpCAM; cytokines / cytokine receptors such as EGFRvIII and TGF-β; and enzymes.

[0098] The carriers may be used alone or in combination of two or more.

[0099] The carrier may be magnetic. The carrier is preferably a carrier having a magnetic material inside the various carriers described above that have adsorptive properties for substance α or substance β, or a carrier having a magnetic material surface modified to directly or indirectly have adsorptive properties for substance α or substance β. Examples of such carriers include carriers having magnetic particles inside Sepharose, carriers in which various biomolecular probes are chemically modified on the surface of dextran-coated magnetic particles, carriers in which various functional groups are introduced into magnetic particles, and carriers in which a magnetic material and polystyrene are coated on polystyrene core particles. Commercially available carriers include the Mag Sepharose series from Cytiva, the Dynabeads series from Thermo Fisher Scientific, the SupraBead series from Recenttec, the SPHERO magnetic particles series from Spherotech, and the Magnetic MicroBeads series from Sigma-Aldrich. The magnetic carrier is preferably a carrier having magnetic particles inside Sepharose.

[0100] The carrier is preferably hydroxyapatite or a magnetic carrier, since it is easily dispersible in a liquid and has high strength.

[0101] The shape of the carrier is not particularly limited, and examples thereof include spherical, particulate, fibrous, rod-like, and plate-like shapes. From the viewpoint of ease of solid-liquid separation and washing, spherical and particulate shapes are preferred.

[0102] The size of the carrier is not particularly limited as long as it is larger than the pores of the filter, and varies depending on the type of substance α, but when the carrier is spherical or particulate, its average particle diameter is preferably 0.5 μm to 2 mm, more preferably 1 μm to 1.5 mm, and even more preferably 1 μm to 1 mm. The average particle diameter can be measured by a light scattering method.

[0103] The density of the carrier is not particularly limited, but is preferably 1 g / cm 3 Higher, preferably 2.5 to 3.5 g / cm 3 is.

[0104] The amount of carrier held within the chip body is not particularly limited and may be selected appropriately depending on the capacity of the chip body, the type of carrier, the type of sample liquid, and the amount of substance α and substance β in the sample liquid, etc. The amount of carrier held within the chip body is preferably less than 100%, more preferably 95% or less, when the amount of carrier when the total horizontal projection area of ​​the carrier is equal to the area of ​​the filter when the carrier is uniformly arranged in a single layer on a filter is taken as 100%.

[0105] [Substance α and substance β] The substance α is a substance contained in the sample liquid and is to be purified or concentrated. The sample liquid may contain one or more types of substance α and substance β, and the concentrated liquid may contain one or more types of substance α.

[0106] The substance α is not particularly limited, and examples thereof include proteins; nucleic acids; lipids; sugars; extracellular vesicles such as exosomes, microvesicles, and apoptotic vesicles; viruses; and DDS (Drug Delivery System) preparations such as liposomes and lipid nanoparticles (LNPs), which may be labeled with a fluorescent dye, biotin, a reporter enzyme, a radioisotope, or the like. The substance α is preferably a biological substance (e.g., protein; nucleic acid; lipid; sugar; extracellular vesicle such as exosome, microvesicle, apoptotic vesicle; virus), more preferably a protein or exosome, and even more preferably an exosome.

[0107] The protein is not particularly limited, and examples thereof include simple proteins such as albumin, globulin, keratin, collagen, and fibroin, and complex proteins such as glycoproteins, phosphoproteins, chromoproteins, and nucleoproteins. Among these, simple proteins are preferred. The protein may be an antibody (immunoglobulin), a contractile protein, an enzyme, a hormonal protein, a structural protein, a storage protein, or a transport protein, and is preferably an antibody (immunoglobulin) or a transport protein.

[0108] The nucleic acid is not particularly limited, and examples thereof include DNA, RNA, microRNA, and mRNA.

[0109] The lipid is not particularly limited, and examples thereof include simple lipids such as glycerides, sterol esters, waxes, and ceramides; complex lipids such as phospholipids and glycolipids; and derived lipids such as fatty acids, terpenoids, steroids, and carotenoids.

[0110] The sugars are not particularly limited, and examples thereof include monosaccharides such as glucose and fructose; disaccharides such as maltose, sucrose and lactose; polysaccharides such as starch, cellulose and glycogen; and sugar chains that are added to proteins or lipids in eukaryotes and exist as components of complexes.

[0111] The substance β is not particularly limited in form as long as it contains the substance α, and is preferably a substance in which the substance α is contained inside an encapsulating substance. The encapsulating substance is, for example, a capsule-like substance that encapsulates at least a portion of the substance α. Substance β exists only if substance α is a protein, nucleic acid, lipid, or sugar, i.e., substance α in substance β is a protein, nucleic acid, lipid, or sugar.

[0112] For example, when a concentrated solution with an increased concentration of exosomes is produced from a sample solution containing exosomes, the exosomes are substance α. When a concentrated solution with an increased concentration of nucleic acids is produced from a sample solution containing exosomes, the nucleic acids are substance α and the exosomes are substance β. Substances α and β are distinguished depending on the target of purification or concentration.

[0113] Examples of the substance α encapsulated in an encapsulating substance include viruses, extracellular vesicles such as exosomes, microvesicles, and apoptotic vesicles, and DDS (Drug Delivery System) preparations such as liposomes and lipid nanoparticles (LNPs). The substance β is preferably a virus or an exosome.

[0114] The virus contains nucleic acid inside an envelope (for example, a capsid or envelope) composed of at least one selected from proteins, lipids, and sugars.

[0115] The extracellular vesicles contain at least one selected from nucleic acids, proteins, lipids, and sugars inside an encapsulating substance composed of at least one selected from proteins, lipids, and sugars. Examples of the extracellular vesicles include exosomes, microvesicles, and apoptotic vesicles, and among these, exosomes are preferred.

[0116] The liposome contains at least one selected from nucleic acids, proteins, lipids, and sugars inside an encapsulating substance (for example, a lipid bilayer membrane) made of lipids. The LNP contains at least one selected from nucleic acids, proteins, lipids, and sugars inside an encapsulating substance composed of lipids (e.g., a lipid monolayer or lipid bilayer composed of ionized lipids, helper lipids, PEG lipids, etc.).

[0117] [Sample solution] The sample liquid is not particularly limited as long as it is a liquid that may contain substance α or substance β. Examples of the sample liquid include biological samples, microbial or cell samples, foods, environmental samples, and liquids obtained by diluting these with a diluent.

[0118] Examples of the biological sample include animal and plant tissues, body fluids, excrement, and swabs, washings, and cultures thereof. More specific examples include blood, plasma, serum, blood culture, urine, saliva, amniotic fluid, pus, cerebrospinal fluid, pleural effusion, pharyngeal swab, nasal swab, nasopharyngeal swab, nasal discharge, sputum, rectal swab, tissue slice, skin, vomit, feces, tympanotomy fluid, alveolar lavage fluid, gastric lavage fluid, intestinal lavage fluid, cervical swab, urethral scraping, organ extract, tissue extract, etc. According to one aspect of the present invention, even when a biological sample containing a large amount of impurities is used, it is possible to concentrate substance α. Therefore, the biological sample is preferably blood, urine, saliva, throat swab, nasal swab, nasopharyngeal swab, nasal discharge, or sputum, and among these, samples derived from mammals, particularly humans, are more preferred.

[0119] The microorganism or cell sample may be, for example, a naturally occurring microorganism or cell, a transformant introduced with a recombinant vector for expressing a target protein, or a hybridoma formed by the fusion of multiple cells, etc. In addition to the microorganism or cell itself, the sample also includes a microorganism or cell lysate and culture supernatant. Examples of the microorganisms or cells include fungi such as yeast and mold, prokaryotic microorganisms such as Escherichia coli, and cultured cells such as plant cells, insect cells, and animal cells, with Escherichia coli and animal cells being preferred.

[0120] The microorganism or cell sample is preferably an animal cell sample, more preferably a culture supernatant of animal cells. The animal cells may be derived from any animal, but are preferably mammalian. The animal cells may be primary cells, established cell lines, or cells derived from iPS cells, ES cells, etc. In this specification, the mammal is preferably a human, cow, dog, cat, pig, minipig, rabbit, hamster, rat, or mouse, more preferably a human.

[0121] Examples of the food include water, alcoholic beverages, soft drinks, processed foods, vegetables, livestock products, seafood, eggs, dairy products, raw meat, raw fish, prepared dishes, etc. When food is used as a sample, not only can part or all of the food be used, but also the surface of the food can be wiped. Furthermore, wipes of food-contact parts or human-contact parts of cooking utensils, etc., or cleaning solutions used to wash such parts can also be used as samples. For samples with a large amount of liquid components, if necessary, some or all of the liquid components can be removed by drying, ultrafiltration, distillation, etc.

[0122] Examples of environmental samples include water, ice, and soil. Examples of water include tap water, seawater, and water collected from rivers, waterfalls, lakes, ponds, etc. Furthermore, samples can also be used that have been wiped off from human contact surfaces such as doorknobs, facility walls, floors, equipment, fixtures, toilets, etc., or cleaning fluids used to wash these. For samples with a high liquid content, some or all of the liquid components may be removed by drying, ultrafiltration, distillation, or the like, as necessary.

[0123] The diluent is not particularly limited and may be, for example, water or a buffer solution. Examples of the buffer solution include those commonly used in biochemical tests, such as Tris buffer and phosphate buffer. The diluent may contain, as appropriate, salts such as sodium chloride, surfactants such as SDS, metal chelating agents such as EDTA, and preservatives such as sodium azide. The diluent may be, for example, a medium for microbial or cell culture, preferably a medium for cell culture, more preferably a medium that does not contain animal-derived serum.

[0124] The method for collecting the sample liquid is not particularly limited, and any known method can be used depending on the type and purpose of the sample liquid, including collection methods using collection tools such as cotton swabs, swabs, platinum loops, droppers, spatulas, spoons, and syringes.

[0125] The amount of the sample liquid is not particularly limited and may be determined according to the capacity of the pipette tip body.

[0126] When the sample solution is a cell culture supernatant, the cell culture supernatant obtained after culturing cells for 12 hours to 7 days is preferred, and the cell culture supernatant obtained after culturing cells for 24 hours to 5 days is more preferred, since it tends to contain fewer components derived from dead cells. The cell culture supernatant may be diluted, for example, 2- to 5-fold with the medium used for cell culture before use.

[0127] [Method of producing concentrated liquid] One aspect of the present invention is a method for producing a concentrated solution with an increased concentration of substance α from a sample solution containing a target substance (substance α) or a substance α-containing substance (substance β) using the pipette tip, the method comprising: step A: aspirating and discharging the sample solution through a lower opening at the bottom end of the pipette tip to adsorb the substance α or substance β onto the carrier; and step B: aspirating and discharging a release liquid to release the substance α from the carrier, thereby obtaining a concentrated solution with an increased concentration of substance α.

[0128] (Process A) Step A is a step of aspirating and discharging the sample liquid through the lower opening at the lower end of the pipette tip, thereby causing the substance α or substance β to be adsorbed onto the carrier. In step A, substance α or substance β in the sample solution diffuses through the sample solution, passes through the filter, reaches the inside of the chip body, and is adsorbed to the carrier in the chip body. This series of steps is repeated multiple times, rather than just once, so that substance α or substance β is more likely to be adsorbed to the carrier. This makes it easy to produce a concentrated solution with a higher concentration of substance α.

[0129] The suction and discharge speed is not particularly limited, and the suction and discharge speeds may be the same or different. During aspiration, the carrier is dispersed in the sample liquid, creating a state similar to that of batch chromatography. Therefore, a fast aspiration speed makes it easier for the carrier to disperse, and it is easier to obtain the same benefits as batch chromatography. Therefore, it is preferable that the aspiration speed is faster than the discharge speed. During discharging, the sample liquid is sent from the top to the bottom of the carrier accumulated on the filter, creating a state similar to that of column chromatography. Therefore, if the discharging speed is slow, substance α or substance β is more likely to be adsorbed onto the carrier, and the same advantages as those of column chromatography are likely to be obtained. Therefore, it is preferable that the discharging speed is slower than the suction speed.

[0130] The suction and discharge may be performed continuously or with an interval between them, but it is preferable to provide an interval between them. By providing an interval between them, it is possible to wait for a certain amount of the carrier dispersed by the suction to accumulate on the filter, and the sample liquid is sent from above to below the carrier where more of it has accumulated, which tends to create a state similar to that of column chromatography, and substance α or substance β is easily adsorbed to the carrier.

[0131] The volume to be aspirated or discharged is preferably 1% or more of the sample liquid, more preferably 10% or more, even more preferably 50% or more, and particularly preferably 70% or more. Suction and discharge are performed preferably at a frequency of 1 time / minute or more, more preferably 1.5 times / minute or more, and even more preferably 2 times / minute or more, for preferably 10 minutes to 24 hours, more preferably 15 minutes to 12 hours, and even more preferably 30 minutes to 1 hour. When carried out under these conditions, the substances α and β can be efficiently adsorbed onto the carrier.

[0132] The instrument or device used for suction and discharge is not particularly limited, and for example, a pipette such as a micropipette, a macropipette, a measuring pipette, or a Komagome pipette can be used, but from the standpoint of safety and prevention of contamination, a micropipette or a macropipette is preferred. The tool or device for performing the pipetting operation may be manual or electric, but is preferably electric since it allows for accurate pipetting.

[0133] (Process B) Step B is a step of aspirating and discharging the release liquid to release the substance α from the carrier, thereby obtaining a concentrated liquid in which the concentration of the substance α is increased. The method for carrying out step B is not particularly limited.

[0134] The release liquid is a liquid that can release the substance α from the carrier. The composition and properties of the release liquid are not limited as long as it can release the substance α from the carrier. When releasing substance α from the carrier, for example, if a substance in which substance α is contained inside an encapsulating substance is used as substance β, the encapsulating substance adsorbed to the carrier may or may not be released from the carrier, but if the encapsulating substance is also released from the carrier, a separation operation between substance α and the encapsulating substance may be required, so it is preferable not to release it from the carrier to avoid such a separation operation. Specifically, it is preferable to release substance α by contacting substance β (e.g., encapsulating substance encapsulating nucleic acid) adsorbed to the carrier with a liquid (e.g., nucleic acid extract) that can release substance α (e.g., nucleic acid) into the liquid.

[0135] When the substance α is adsorbed to a carrier, the adsorptivity of the carrier for the substance α can be changed to release the substance α from the carrier. For example, when a protein is adsorbed to hydroxyapatite, examples of a liquid capable of releasing the protein from the hydroxyapatite include an aqueous solution containing 100 mM or more of a metal chelating agent such as EDTA, a surfactant such as SDS or Triton X-100, or 100 mM or more of phosphoric acid. These may be used alone or in combination of two or more.

[0136] When substance β is a substance in which substance α is housed inside an encapsulation substance, substance α can be released from substance β adsorbed to the carrier into a liquid by denaturing or destroying the encapsulation substance. For example, when viruses or exosomes in which nucleic acid is housed inside proteins are adsorbed to a carrier, nucleic acid can be released from the viruses or exosomes adsorbed to the carrier using the release liquid containing a protein denaturant.

[0137] Examples of the protein denaturant include surfactants such as SDS and Triton X-100; chaotropic agents such as guanidine thiocyanate, guanidine hydrochloride, urea, and NaI; protease such as proteinase K; and organic solvents such as phenol. These may be used alone or in combination with one another. The protein denaturant may also be used in combination with a nuclease inhibitor such as EDTA; or a reducing agent such as 2-mercaptoethanol and DTT. Furthermore, commercially available nucleic acid extraction reagents such as TRIzol Reagent, QIAzol Lysis Reagent, and ISOGEN may also be used as the protein denaturant.

[0138] For example, when a virus, extracellular vesicle, or DDS preparation is adsorbed to agarose or beads (carrier) onto which a substance having affinity for a molecule expressed on the surface of the virus, extracellular vesicle, or DDS preparation has been immobilized, the virus, extracellular vesicle, or DDS preparation can be released from the carrier by changing the binding state between the molecule expressed on the surface of the virus, extracellular vesicle, or DDS preparation and the substance having affinity. For example, when exosomes are adsorbed to agarose or beads (carriers) onto which a lipid-binding protein such as Tim4, a lipid-recognition antibody, or a lipid-recognition aptamer that binds to phosphatidylserine expressed on the surface of exosomes has been immobilized, exosomes can be released from the carrier by changing the binding state between the phosphatidylserine and the lipid-binding protein such as Tim4, the lipid-recognition antibody, or the lipid-recognition aptamer. Examples of liquids that can release exosomes from the carrier include metal chelators such as EDTA at 0.5 mM or higher. By releasing the virus, extracellular vesicles, or DDS preparation from the carrier in this manner, a purified solution containing the virus, extracellular vesicles, or DDS preparation in an intact state and with an increased purity of the virus, extracellular vesicles, or DDS preparation can be produced from a sample solution containing the virus, extracellular vesicles, or DDS preparation.

[0139] The amount of the release liquid used is not particularly limited as long as it is an amount that can sufficiently release substance α from the carrier, but it is usually less than the amount of the sample liquid used so that the concentration of substance α contained in the concentrated liquid can be further increased.

[0140] The release liquid may be brought into contact with the carrier in succession with a plurality of different release liquids, thereby further increasing the concentration of substance α contained in the concentrated liquid. Furthermore, the free liquid after contacting with the carrier may be contacted with the carrier again, thereby further increasing the concentration of substance α contained in the concentrated liquid.

[0141] The volume to be aspirated and discharged is preferably 50% or more of the free liquid, more preferably 60% or more, and even more preferably 70% or more. Suction and discharge are performed preferably at a frequency of 1 time / minute or more, more preferably 1.5 times / minute or more, and even more preferably 2 times / minute or more, for preferably 10 minutes to 24 hours, more preferably 15 minutes to 12 hours, and even more preferably 30 minutes to 1 hour. When carried out under these conditions, the substances α and β can be efficiently liberated from the carrier.

[0142] The instrument or device used for suction and discharge is not particularly limited, and for example, a pipette such as a micropipette, a macropipette, a measuring pipette, or a Komagome pipette can be used, but from the standpoint of safety and prevention of contamination, a micropipette or a macropipette is preferred. The tool or device for performing the pipetting operation may be manual or electric, but is preferably electric since it allows for accurate pipetting.

[0143] (Cleaning process) The method for producing the concentrate preferably includes a step of washing the carrier with a washing liquid after step A and before step B. This step will hereinafter also be referred to as the "washing step." Impurities that are not adsorbed to the carrier tend to remain attached to the walls of the chip body, the filter, the carrier, etc. The washing step allows for more efficient removal of impurities.

[0144] The method for carrying out the washing step is not particularly limited, but is preferably carried out by aspirating and discharging the washing solution, as this is an easy operation and can be adapted to an automated device.

[0145] The washing solution is not particularly limited as long as it does not easily release the substance α from the carrier, and examples thereof include water and a buffer solution. Examples of the buffer solution include buffer solutions commonly used in biochemical tests, such as Tris buffer and phosphate buffer. Depending on the type and amount of impurities, salts such as sodium chloride; surfactants such as SDS; metal chelating agents such as EDTA; and preservatives such as sodium azide can be added to the washing solution as appropriate. For example, when a concentrated solution with an increased concentration of nucleic acids is produced from a sample solution containing viruses, preferably using hydroxyapatite as a carrier, free nucleic acids derived from substances other than viruses can be removed by performing a washing step using a washing solution containing EDTA.

[0146] The volume of the washing solution is not particularly limited and may be determined appropriately depending on the type and amount of impurities, but the volume of the washing solution is preferably 20 times or more, more preferably 100 times or more, and even more preferably 200 times or more the volume of the carrier.

[0147] The washing liquid may be a plurality of different washing liquids used in sequence, which can efficiently remove impurities and further increase the purity of the substance α contained in the concentrated liquid.

[0148] The method for producing a concentrated solution can produce a concentrated solution with an increased concentration of a target substance from a sample solution containing a target substance or a target substance-containing substance simply by aspirating and dispensing, making it suitable for use in a robotic liquid handler that automates not only the aspirating and dispensing of liquid but also the attaching and detaching of pipette tips. The robotic liquid handler may be an automated nucleic acid or protein purification system, and examples of such automated purification systems include the Purelumn system (manufactured by Precision System Science Co., Ltd.), AssistPlus (manufactured by Integra Biosciences Inc.), and CyBio FeliX (manufactured by Analytik Jena).

[0149] Figure 2 is a six-view diagram of the pipette tip according to the first embodiment, in which Figure 2(a) is a front view of the pipette tip, Figure 2(b) is a rear view of the pipette tip, Figure 2(c) is a left side view of the pipette tip, Figure 2(d) is a right side view of the pipette tip, Figure 2(e) is a plan view of the pipette tip, and Figure 2(f) is a bottom view of the pipette tip. Fig. 3 is a perspective view of the pipette tip according to the first embodiment shown in Fig. 2. That is, Fig. 3(a) is a perspective view of the pipette tip as seen from the top side, and Fig. 3(b) is a perspective view of the pipette tip as seen from the bottom side.

[0150] Figure 4 is a six-view diagram of the pipette tip according to the second embodiment, in which Figure 4(a) is a front view of the pipette tip, Figure 4(b) is a rear view of the pipette tip, Figure 4(c) is a left side view of the pipette tip, Figure 4(d) is a right side view of the pipette tip, Figure 4(e) is a plan view of the pipette tip, and Figure 4(f) is a bottom view of the pipette tip. Fig. 5 is a perspective view of the pipette tip according to the second embodiment shown in Fig. 4. That is, Fig. 5(a) is a perspective view of the pipette tip as seen from the top side, and Fig. 5(b) is a perspective view of the pipette tip as seen from the bottom side.

[0151] Figure 6 is a six-view diagram of a pipette tip according to the third embodiment, in which Figure 6(a) is a front view of the pipette tip, Figure 6(b) is a rear view of the pipette tip, Figure 6(c) is a left side view of the pipette tip, Figure 6(d) is a right side view of the pipette tip, Figure 6(e) is a plan view of the pipette tip, and Figure 6(f) is a bottom view of the pipette tip. 7A and 7B are perspective views of the pipette tip according to the third embodiment shown in Fig. 6. That is, Fig. 7A is a perspective view of the pipette tip as seen from the top side, and Fig. 7B is a perspective view of the pipette tip as seen from the bottom side.

[0152] Figure 8 is a six-view diagram of a pipette tip according to the fourth embodiment, in which Figure 8(a) is a front view of the pipette tip, Figure 8(b) is a rear view of the pipette tip, Figure 8(c) is a left side view of the pipette tip, Figure 8(d) is a right side view of the pipette tip, Figure 8(e) is a plan view of the pipette tip, and Figure 8(f) is a bottom view of the pipette tip. 9A and 9B are perspective views of the pipette tip according to the fourth embodiment shown in Fig. 8. That is, Fig. 9A is a perspective view of the pipette tip as seen from the top side, and Fig. 9B is a perspective view of the pipette tip as seen from the bottom side. In the pipette tip according to the fourth embodiment, the lowest end of the tip body is formed on the axis of the tip body. For the sake of simplicity, the carrier is not shown in FIGS. [Example]

[0153] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to these examples.

[0154] <Preparing pipette tips> (Preparing the filter) Nonwoven fabric made of PTFE ultrafine fibers (basis weight: 24 g / m 2 , 300 mm × 20 mm, average pore size 1 μm) was immersed in a 99.7% isopropyl alcohol (IPA) solution (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) at room temperature (25°C) for 30 minutes. Next, the PTFE filter removed from the IPA solution was immersed in 500 mL of an aqueous solution of polyvinyl alcohol (PVA) (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., 160-11485, polymerization degree: 1500, saponification degree: 98%) adjusted to a concentration of 0.1 mass% at room temperature for 30 minutes. The PTFE filter removed from the PVA aqueous solution was then immersed in a mixture of 500 mL of a 5% by mass glutaraldehyde solution (a 25% glutaraldehyde solution manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., diluted with pure water to a concentration of 5% by mass) and 5 mL of a 36% hydrochloric acid aqueous solution (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) at room temperature for 30 minutes. Next, the PTFE filter removed from the mixture was placed in pure water to dissolve the unreacted IPA, PVA and glutaraldehyde. The PTFE filter removed from the pure water was vacuum dried at 80°C for 240 minutes, and then immersed in an MPC (2-methacryloyloxyethyl phosphorylcholine) polymer solution (physical adsorption type MP011 L1 MPC polymer ethanol solution, manufactured by Intelligent Surface Co., Ltd.) at room temperature for 1 minute. The PTFE filter removed from the polymer solution was vacuum dried at 80°C for 120 minutes and then immersed in cleaning water for 180 minutes, and then removed from the solution and vacuum dried at 80°C for 240 minutes to obtain an MPC polymer-coated hydrophilic PTFE filter.

[0155] (Preparation of pipette tips with filters) A pipette tip (manufactured by Thermo Fisher Scientific, Cat. No. 94410813) was cut using an ultrasonic cutter. The inclination angle of the cut surface was set at a specific angle relative to a line perpendicular to the axis of the pipette tip when viewed from the front. A PTFE filter coated with an MPC polymer and treated for hydrophilicity was pressed against the cut surface of the pipette tip and temporarily placed. In this state, the cut surface was pressed downward onto a hot plate heated to 180°C for thermal fusion. The material constituting the pipette tip body was polypropylene, with a melting point of approximately 160°C and a melting point of approximately 320°C for PTFE. The pipette tips shown in Table 1 were fabricated by changing the inclination angle of the cut surface.

[0156] [Table 1]

[0157] In addition, the body of a pipette tip (manufactured by Thermo Fisher Scientific, Cat. No. 94410813) was cut at a position 17 mm from the bottom end so that the cut surface was perpendicular to the axis of the pipette tip (when viewed from the front of the pipette tip, the angle between the cut surface and a line perpendicular to the axis of the pipette tip was 0°), and an MPC polymer-coated hydrophilized PTFE filter was heat-sealed to the cut surface in the same manner as above to produce Comparative Production Example 1. In order to eliminate the influence of the filter area, Production Examples 1 to 7 were produced so that the filter area was the same as that of Comparative Production Example 1 by adjusting the position from the lower end of the cut surface.

[0158] (Experimental Example 1) Using a micropipette, 10 μL of HT carrier particles (Bio-Rad Bio-Gel HT Hydroxyapatite, model number: 130-0150) adjusted to a 12.5% ​​by mass slurry with PBS buffer was added to the top of the pipette tip of Comparative Example 1 and Example 6, creating the pipette tips of Comparative Example 1 and Example 1, respectively.

[0159] An automatic pipettor (Thermo Fisher Scientific, E1-ClipTip electronic pipette, model number: 4670040BT) was connected to the upper end of the pipette tip of Comparative Example 1 and Example 1. Furthermore, the lower end of this pipette tip was placed in a 2 mL U-bottom tube (Eppendorf, No. 0030108450) containing 1 mL of purified water, and positioned so that the solution in the tube could be aspirated. The arrangement of this container (tube) and automatic pipettor mimics an automated device that only includes a pipetting device for aspirating and dispensing solution and a means for moving the pipetting device up and down and back and forth. The automatic pipetter was set to a suction volume of 1 mL, and an attempt was made to aspirate and dispense the solution in manual mode. Whether the solution was aspirated and discharged, and the dispersion of the carrier (the state of the carrier flying up) accompanying the aspirating and dispensing operation were visually observed, and photographs and videos were taken.

[0160] The pipette tip of Example 1 smoothly aspirated and discharged the solution, and the carrier in the pipette tip was efficiently dispersed by aspirating and discharging. Furthermore, dispersion of the carrier by aspirating and discharging could be repeated. With the pipette tip of Comparative Example 1, in order to aspirate all of the solution in the container (tube), the filter surface at the tip of the pipette tip had to be in contact with or close to the U-shaped bottom of the tube, and when the filter surface at the tip of the pipette tip was not in contact with or close to the U-shaped bottom of the tube, aspirating and dispensing was difficult, and aspirating and dispensing small amounts of liquid was particularly impossible.

[0161] (Experimental Example 2) An automatic pipettor (Thermo Fisher Scientific, E1-ClipTip electronic pipette, model number: 4670040BT) was connected to the upper end of the pipette tip from Comparative Example 1 and Example 1. The lower end of this pipette tip was placed in a flat-bottomed, 3.5 cm diameter Petri dish (Nunc, No. 153066) containing 7 mL of purified water, and positioned so that the solution in the Petri dish could be aspirated. The aspirating volume of the automatic pipettor was set to 1 mL, and aspirating and dispensing of the solution was attempted in manual mode. Whether the solution was aspirated and discharged, and the dispersion of the carrier (the state of the carrier flying up) accompanying the aspirating and dispensing operation were visually observed, and photographs and videos were taken.

[0162] The pipette tip of Example 1 smoothly aspirated and discharged the solution, and the carrier in the pipette tip was efficiently dispersed by aspirating and discharging. Furthermore, dispersion of the carrier by aspirating and discharging could be repeated. In the pipette tip of Comparative Example 1, the solution was aspirated and the carrier in the pipette tip was dispersed by the aspirate, but the aspirated solution could not be discharged, and therefore the aspirate and discharge of the solution could not be performed repeatedly.

[0163] (Experimental Example 3) An automatic pipetter (Thermo Fisher Scientific, E1-ClipTip electronic pipette, model number: 4670040BT) was connected to the upper end of the pipette tip from Comparative Example 1 and Example 1. Furthermore, the lower end of this pipette tip was placed in a 2 mL U-bottom tube (Eppendorf, No. 0030108450) containing 1 mL of the BSA-Cy3 solution prepared below, and positioned so that the solution in the tube could be aspirated. Using the custom mode of the automatic pipetter, 1 mL of the solution was aspirated and discharged at speed 3 for 1 hour, thereby adsorbing bovine serum albumin (BSA) labeled with Cy3 fluorescent dye onto the HT carrier particles.

[0164] The BSA-Cy3 solution was prepared as follows. Cy3 fluorescent dye (Amersham, model number: Q13108) was added to a bovine serum albumin solution (Sigma-Aldrich, 100 μg / 10 μL, solvent: HO) and allowed to react at room temperature for 1 hour in the dark. To terminate the reaction, TBS was added and the mixture was left standing at room temperature for 1 hour in the dark. Next, gel filtration (Thermo Fisher Scientific, model number: 89883) was performed to remove excess Cy3 fluorescent dye. The mixture was diluted with PBS (TaKaRa Bio, model number: T900) to prepare a Cy3 fluorescent dye-labeled bovine serum albumin solution (BSA-Cy3 solution) (25 μg / mL).

[0165] After repeated aspiration and dispensing at room temperature for 30 minutes, the liquid in the pipette tip was discharged and the tube was replaced. Specifically, the tube used for adsorption of BSA-Cy3 was removed, and the pipette tip was positioned so that it could aspirate the solution in a new tube containing 1 mL of PBS (manufactured by TaKaRa, model number: T900) as a wash buffer. The HT carrier particles were washed by aspirating and dispensing 1 mL of the solution once at speed 3 using the custom mode of the automatic pipetter.

[0166] After aspiration and dispensing, the liquid in the pipette tip was discharged and the tube was then replaced. Specifically, the tube used for washing was removed, and a new tube containing 50 μL of Elute buffer (5 M guanidine thiocyanate (Fujifilm Wako Pure Chemical Industries, Ltd., model number: 077-04995), 100 mM EDTA aqueous solution (pH 8.0, Nippon Gene Co., Ltd., model number: 311-90075), 100 mM Tris-HCl (pH 6.8): Tris (Fujifilm Wako Pure Chemical Industries, Ltd., model number: 200-07887) pH adjusted with hydrochloric acid (Fujifilm Wako Pure Chemical Industries, Ltd., model number: 080-01066), 3% Triton X-100 (Nacalai, product number: 35501-15), and 1% 2-mercaptoethanol (Sigma-Aldrich Co., Ltd., model number: M3148)) was placed in a position where the pipette tip could be aspirated. Using the custom mode of the automatic pipetter, 0.2 mL of solution was aspirated and dispensed at speed 1 for 5 minutes at room temperature to release the bovine serum albumin labeled with Cy3 fluorescent dye adsorbed to the HT carrier particles from the HT carrier particles, and the resulting liquid was called elute. When the elute buffer was aspirated and discharged, the dispersion of the carrier (the carrier flying up) and the color change of the solution accompanying the aspirating and dispensing operation were visually observed, and photographs and videos were taken.

[0167] In the pipette tip of Example 1, the tip of the pipette tip was in close proximity to the solution surface or in point contact, allowing for smooth aspirating and dispensing of the solution, and the carrier in the pipette tip was efficiently dispersed by aspirating and dispensing. Furthermore, dispersion of the carrier by aspirating and dispensing could be repeated. Furthermore, the pink color of the solution gradually deepened with aspirating and dispensing, confirming that bovine serum albumin labeled with Cy3 fluorescent dye had been released from the HT carrier particles. The fluorescence values ​​of the sample BSA-Cy3 solution and Elute were measured using a Qubit 4 Fluorometer (Thermo Fisher Scientific, model number: Q33226, blue mode (470 nm)). The BSA-Cy3 solution had a fluorescence intensity of 2748, while the Elute had a fluorescence intensity of 28720. In other words, Elute was about 10 times more pink than the sample BSA-Cy3 solution. By using the pipette tip of Example 1, a concentrated solution in which BSA-Cy3 was concentrated about 10 times was produced. With the pipette tip of Comparative Example 1, the tip of the pipette tip was in face-to-face contact with the solution surface, making it impossible to aspirate or eject the solution, and bovine serum albumin labeled with Cy3 fluorescent dye could not be adsorbed reproducibly onto the HT carrier particles. In particular, when eluting with a solution volume of 50 μL, the aspirating and ejecting of the liquid became unstable, resulting in inconsistent results.

[0168] (Experimental Example 4) Using a micropipette, 10 μL of HT carrier particles (Bio-Rad Bio-Gel HT Hydroxyapatite, model number: 130-0150) adjusted to a 12.5% ​​by mass slurry with PBS buffer was added to the top of the pipette tip of each of Manufacturing Examples 1, 2, 3, and 5, to produce the pipette tips of Examples 2, 3, 4, and 5, respectively. An automatic pipetter (E1-ClipTip electronic pipette, model number 4670040BT, manufactured by Thermo Fisher Scientific Co., Ltd.) was connected to the upper end of the pipette tip of Examples 2, 3, 4, and 5. Furthermore, the lower end of this pipette tip was placed in a 2 mL tube with a U-shaped bottom (manufactured by Eppendorf, No. 0030108450) containing 1 mL of ultrapure water, and positioned so that the solution in the tube could be aspirated. Using the custom mode of the automatic pipetter, 1 mL of the solution was aspirated and dispensed at speed 3 for 30 minutes. Whether the solution was aspirated and discharged, and the dispersion of the carrier (the state of the carrier flying up) accompanying the aspirating and dispensing operation were visually observed, and photographs and videos were taken.

[0169] The photographs taken are shown in Figures 10 and 11. The pipette tips of Examples 2, 3, 4, and 5 smoothly aspirated and discharged the solution, and the carrier in the pipette tip was efficiently dispersed by aspirating and discharging. Furthermore, dispersion of the carrier by aspirating and discharging could be repeated.

[0170] With the pipette tip of Example 2 (tilt angle 10°), the carriers rose in the solution as the solution was aspirated, but no swirling of the carriers was observed. As the solution was discharged, the carriers settled in the solution and accumulated on the filter, uniformly covering the entire surface.

[0171] With the pipette tip of Example 3 (tilt angle 20°), the carriers rose in the solution as the solution was aspirated, but no swirling of the carriers was observed. As the solution was discharged, the carriers settled in the solution and accumulated on the filter, uniformly covering the entire surface.

[0172] In the pipette tip of Example 4 (tilt angle 40°), when the carriers rose in the solution as the solution was aspirated, they appeared to swirl, suggesting that turbulence was occurring as the solution was aspirated, but the turbulence was weak. As the solution was discharged, the carriers settled in the solution and accumulated on the filter, uniformly covering the entire surface. After dispensing and before the next aspiration, the position of the carriers on the filter shifted due to gravity toward the bottom of the pipette tip, and the carriers now only covered a portion of the filter.

[0173] In the pipette tip of Example 5 (tilt angle 60°), when the carriers rose in the solution as the solution was aspirated, they were clearly observed swirling strongly, suggesting that turbulence was occurring as the solution was aspirated. As the solution was dispensed, the carriers accumulated on the filter so as to uniformly cover the entire surface of the filter, but the position of the carriers on the filter shifted toward the bottom of the pipette tip due to gravity, and the carriers ended up covering only a portion of the filter.

[0174] A conceptual diagram of the dispersion state of the carriers in the pipette tip of Example 5 is shown in Figure 12. When the carriers rose in the solution as the solution was aspirated, they were clearly observed swirling strongly (State 1), suggesting that turbulence was occurring as the solution was aspirated. As the solution was ejected, the carriers settled in the solution (State 2) and accumulated on the filter so as to uniformly cover the entire surface (State 3). After ejection and before the next aspirate, the position of the carriers on the filter shifted due to gravity toward the bottom of the pipette tip, and the carriers now covered only a portion of the filter (State 4).

[0175] (Experimental Example 5) Using a micropipette, 10 μL of HT carrier particles (Bio-Gel HT Hydroxyapatite, manufactured by Bio-Rad, model number: 130-0150) adjusted to a 12.5% ​​by mass slurry with PBS buffer was added to the top end of the pipette tip of Manufacturing Example 7, creating the pipette tip of Example 6. An automatic pipetter (E1-ClipTip electronic pipette, model number 4670040BT, manufactured by Thermo Fisher Scientific Co., Ltd.) was connected to the upper end of the pipette tip of Examples 1, 4, and 6. Furthermore, the lower end of this pipette tip was placed in a 2 mL U-bottom tube (manufactured by Eppendorf, No. 0030108450) containing 1 mL of BSA-Cy3 solution, and positioned so that the solution in the tube could be aspirated. Using the custom mode of the automatic pipetter, 1 mL of the solution was aspirated and discharged at speed 3 for 30 minutes, thereby allowing bovine serum albumin labeled with Cy3 fluorescent dye to be adsorbed onto the HT carrier particles.

[0176] After aspiration and dispensing, the liquid in the pipette tip was discharged and the tube was replaced. Specifically, the tube used for adsorption of BSA-Cy3 was removed, and the pipette tip was positioned so that the solution in a new tube containing 1 mL of PBS (manufactured by TaKaRa, model number: T900) as a wash buffer could be aspirated. The HT carrier particles were washed by aspirating and dispensing 1 mL of the solution once at speed 3 using the custom mode of the automatic pipetter.

[0177] After washing, the liquid in the pipette tip was discharged and the tube was replaced. Specifically, the tube used for washing was removed, and the pipette tip was positioned so that it could aspirate the solution in a new tube containing 50 μL of purified water. The dispersion of the carriers (the state of the carriers flying up) during the suction and discharge operation was visually observed, and photographs and videos were taken.

[0178] The photographs taken are shown in Figure 13. The pipette tips of Examples 1, 4, and 6 smoothly aspirated and discharged the solution, and the carrier in the pipette tip was efficiently dispersed by aspirating and discharging. Furthermore, dispersion of the carrier by aspirating and discharging could be repeated.

[0179] In the pipette tip of Example 4 (tilt angle 40°), when the carriers rose in the solution as the solution was aspirated, they appeared to swirl, suggesting that turbulence was occurring as the solution was aspirated, but the turbulence was weak. As the solution was dispensed, the carriers settled in the solution and accumulated on the filter, uniformly covering the entire surface. After dispensing and before the next aspiration, the position of the carriers on the filter shifted due to gravity toward the bottom of the pipette tip, and the carriers ended up covering only a portion of the filter.

[0180] In the pipette tip of Example 1 (tilt angle 65°), when the carriers rose in the solution as the solution was aspirated, they were clearly observed swirling strongly, suggesting that turbulence was occurring as the solution was aspirated. As the solution was discharged, the carriers settled in the solution and accumulated on the filter so as to uniformly cover the entire surface of the filter. However, after dispensing and before the next aspiration, the position of the carriers on the filter shifted toward the bottom of the pipette tip due to gravity, and the carriers now only covered a portion of the filter.

[0181] In the pipette tip of Example 6 (70° tilt angle), when the carriers rose in the solution as the solution was aspirated, they were clearly observed swirling strongly, suggesting that turbulence was occurring as the solution was aspirated. As the solution was dispensed, the carriers settled in the solution and accumulated on the filter, evenly covering the entire surface. However, after dispensing and before the next aspiration, the position of the carriers on the filter shifted due to gravity toward the bottom of the pipette tip, resulting in the carriers only partially covering the filter. Some of the carriers near the bottom of the tip did not rise when the solution was aspirated, and remained near the bottom of the tip even after repeated aspirating and dispensing of the solution.

[0182] A conceptual diagram of the dispersion state of the carriers in the pipette tip of Example 6 is shown in Figure 12. When the carriers rose in the solution as the solution was aspirated, they were clearly observed swirling strongly (State 1), suggesting that turbulence was generated as the solution was aspirated. As the solution was dispensed, the carriers settled in the solution (State 2) and accumulated on the filter, uniformly covering the entire surface (State 3). After dispensing and before the next aspiration, the position of the carriers on the filter shifted due to gravity toward the bottom of the pipette tip, and the carriers now only partially covered the filter (State 4). Some of the carriers near the bottom of the tip did not rise when the solution was aspirated, and remained near the bottom of the tip even after repeated aspirating and dispensing of the solution (State 5).

[0183] (Experimental Example 6) (Production Example 8) A pipette tip (Gilson, No. D 10 mL) was cut using an ultrasonic cutter. The inclination angle of the cut surface was 55° relative to a line perpendicular to the axis of the pipette tip when viewed from the front. The pipette tip was cut again in the same manner so that it formed a V-shape when viewed from the front, creating a second cut surface. An MPC polymer-coated hydrophilic PTFE filter was pressed against the cut surface of the pipette tip and temporarily placed. In this state, the cut surface was pressed downward onto a hot plate heated to 180°C and heat-sealed. An MPC polymer-coated hydrophilic PTFE filter was heat-sealed to the second cut surface in the same manner. This was the pipette tip of Production Example 8.

[0184] 50 μL of carrier particles (Streptavidin Mag Sepharose manufactured by Cytiva, model number: 28985738, 10% by mass slurry) were added to the top end of the pipette tip of Preparation Example 8 using a micropipette to create the pipette tip of Example 7.

[0185] An automatic pipetter (Gilson, model number: P10mLM, for 10 mL) was connected to the upper end of the pipette tip of Example 7. Furthermore, the lower end of this pipette tip was placed in a U-bottom 14 mL tube (Falcon, No. 352057) containing 50 μL of purified water, and positioned so that the solution in the tube could be aspirated. Using the custom mode of the automatic pipetter, 200 μL of the solution was aspirated and dispensed at speed 3. Whether the solution was aspirated and discharged, and the dispersion of the carrier (the state of the carrier flying up) accompanying the aspirating and dispensing operation were visually observed, and photographs and videos were taken.

[0186] The photographs taken are shown in Figure 14. The pipette tip of Example 7 smoothly aspirated and discharged the solution, and the carrier in the pipette tip was efficiently dispersed by aspirating and dispensing. Furthermore, dispersion of the carrier by aspirating and dispensing could be repeated. In Experimental Example 6, because the amount of solution used was small, a portion of the filter was not immersed in the solution, but the solution was aspirated and discharged from the filter tip through the portion of the filter that was immersed in the solution. With existing filter tips, if the entire filter is not immersed in the solution, bubbles often enter the liquid in the tip from the portion of the filter that is not immersed in the solution during aspirating, causing problems with subsequent aspirating and dispensing. However, the filter tip of Example 7 did not allow bubbles to enter the liquid in the tip, and no problems occurred with subsequent aspirating and dispensing.

[0187] (Experimental Example 7) (Preparation of cell culture supernatant) Using Capan2, a cultured cell line derived from pancreatic cancer, as an exosome-supplying cell line, we conducted an experiment to concentrate exosomes from cell culture supernatant. It has been known for some time that exosomes are contained in the culture supernatant of adipocytes and other cells, and adipocytes are widely used as exosome-supplying cells. However, in this study, we used Capan2, a cultured cell line derived from pancreatic cancer, as an exosome-supplying cell line.

[0188] Capan2 cells were cultured in a 25 cm2 Greiner CELLSTAR flask (690170) using serum-containing medium (D-MEM medium (Fujifilm Wako Pure Chemical Industries, Ltd., #044-29765), 10% Fetal Bovine Serum (Thermo Fisher Scientific Co., Ltd. (Gibco), #10270106), and 1% Penicillin-Streptomycin (Thermo Fisher Scientific Co., Ltd. (Gibco), #15140122)).

[0189] Fetal bovine serum, commonly used in cell culture, contains bovine exosomes. To prevent bovine exosome contamination of the cell culture supernatant, Capan2 cells were cultured in serum-free medium (D-MEM medium (Fujifilm Wako Pure Chemical Industries, Ltd., #044-29765), 1% penicillin-streptomycin (Thermo Fisher Scientific Co., Ltd. (Gibco), #15140122)) from the middle of the culture period. Specifically, the culture supernatant was removed from a flask of Capan2 cells grown to confluence in serum-containing medium. Subsequently, the cells were washed five times by adding 10 mL of serum-free medium to the flask and immediately removing the medium. Next, 10 mL of serum-free medium was added to the flask and the cells were cultured for 48 hours at 37°C and 5% CO2.

[0190] After 48 hours of culture, the flask was stood upright and 20 mL of serum-free medium was added to bring the total volume to 30 mL. This was used as the input.

[0191] (Chip preparation) In the pipette tips of Preparation Examples 3, 5, and 7, 10 μL of exosome collection carrier particles (Streptoavidin Mag Sepharose, manufactured by Cytiva, product number: 28985738) and MagCapture Exosome Isolation Kit Using a micropipette, 10 μL of a 10% slurry containing PS Ver. 2 (FUJIFILM Wako Pure Chemical Corporation, model number 294-84101) bound to 1 μL of Biotin-labeled Exosome Capture was added to the top of the pipette tip, creating the pipette tips of Examples 8, 9, and 10, respectively.

[0192] An automatic pipetter (Thermo Fisher Scientific, Model No. E1-CLIP TIP, 1250 μL) was connected to the upper end of the pipette tip of Examples 8, 9, and 10. The lower end of this pipette tip was placed in a U-bottom 2 mL tube (Eppendorf, No. 0030108450) containing 1 mL of Capan 2 culture supernatant (Input) supplemented with 2 μL of Exosome Binding Enhancer (500x) included in MagCapture Exosome Isolation Kit PS Ver. 2 (Fujifilm Wako Pure Chemical Industries, Model No. 294-84101), and positioned so that the solution in the tube could be aspirated. Using the custom mode of the automatic pipetter, 1 mL of the solution was aspirated and dispensed at speed 1 for 1 hour, allowing the exosomes in the culture supernatant to adsorb onto the carrier.

[0193] After that, the liquid in the pipette tip was discharged (the discharged solution was considered as "Through"), and the tube was replaced. Specifically, the tube used for adsorption of exosomes was removed, and the pipette tip was positioned so that it could aspirate the solution in a new tube containing 1 mL of Exosome Immobilizing / Washing Buffer included in the MagCapture Exosome Isolation Kit PS Ver.2 (Fujifilm Wako Pure Chemical Corporation, model number: 294-84101). The carrier particles were washed by aspirating and dispensing 1 mL of solution once at speed 1 using the custom mode of the automatic pipettor. The tube was replaced with a new one containing 1 mL of Exosome Immobilizing / Washing Buffer, and this aspirating and dispensing procedure was repeated for a total of three washing procedures.

[0194] After washing, the liquid in the pipette tip was ejected and the tube was replaced. Specifically, the tube used for washing was removed, and a new tube containing 50 μL of elution buffer (20 mM Tris-HCl (pH 7.6): Tris (Fujifilm Wako Pure Chemical Corporation, model number: 200-07887) pH adjusted with hydrochloric acid (Fujifilm Wako Pure Chemical Corporation, model number: 080-01066) diluted with water, 150 mM NaCl (Fujifilm Wako Pure Chemical Corporation, model number: 191-01665), 2 mM EDTA (Nippon Gene, model number: 311-90075), and 1% Triton X-100 (Nacalai, model number: 35501-15) was placed in the pipette tip so that it could aspirate the solution. Using the custom mode of the automatic pipetter, 50 μL of the solution was aspirated and dispensed at speed 1 for 10 minutes at room temperature to elute the exosome components from the carrier particles. The resulting eluate was called Elute.

[0195] In each of the adsorption, washing, and elution steps, dispersion of the carrier (the state of the carrier flying up) during the suction and discharge operations was visually observed, and photographs and videos were taken.

[0196] As a control experiment, exosomes were concentrated using conventional batch chromatography using a 2 mL tube (shown as "Tube" in Figure 15). A 2 mL U-bottom tube (Eppendorf, No. 0030108450) was filled with 10 μL of exosome-capturing carrier particles (Cytiva, Streptoavidin Mag Sepharose, product number: 28985738, 10 μL bound to 1 μL of Biotin-labeled Exosome Capture included in MagCapture Exosome Isolation Kit PS Ver. 2 (Fujifilm Wako Pure Chemical Corporation, product number: 294-84101) (10% slurry)) and Exosome Binding Enhancer (500x) included in MagCapture Exosome Isolation Kit PS Ver. 2 (Fujifilm Wako Pure Chemical Corporation, product number: 294-84101). 1 mL of Capan 2 culture supernatant (Input) containing 2 μL of the eluate was added, and the mixture was mixed by inversion at room temperature for 1 hour using a mixer (TOHO, model number: RM-2M). The carrier particles were then separated using a magnetic bar. The liquid in the tube after separation was designated as Through. The separated carrier particles were washed three times with 1 mL of Exosome Immobilizing / Washing Buffer included in MagCapture Exosome Isolation Kit PS Ver.2 (Fujifilm Wako Pure Chemical Corporation, model number: 294-84101), and then eluted with 50 μL of Elution buffer (20 mM Tris-HCl (pH 7.6): Tris (Fujifilm Wako Pure Chemical Corporation, model number: 200-07887) pH adjusted with hydrochloric acid (Fujifilm Wako Pure Chemical Corporation, model number: 080-01066) and diluted with water), 150 mM NaCl (Fujifilm Wako Pure Chemical Corporation, model number: 191-01665), 2 mM EDTA (Nippon Gene, model number: 311-90075), 1% Exosome components were eluted from the carrier particles using Triton X-100 (Nacalai, 35501-15). The resulting eluate was called Elute.

[0197] Exosomes enriched from the culture supernatant of Capan2 cells were confirmed by Western blotting for the exosome marker CD9. 50 μL of the eluate was mixed with a reducing agent-free sample buffer (Fujifilm Wako Pure Chemical Industries, Ltd., #198-13282), incubated at 100°C for 5 minutes, and then subjected to SDS-PAGE. As a control, 50 μL of the input sample was treated in the same manner.

[0198] SDS-PAGE gels were SuperSep™ Ace 10-20% (Fujifilm Wako Pure Chemical Corporation, #191-15031). After electrophoresis, the samples were transferred from the gel to a PVDF membrane (Trans-Blot Turbo Mini 0.2 μm PVDF Transfer Packs, Bio-Rad, #1704156). The PVDF membrane was blocked with PVDF Blocking Reagent for Can Get Signal (TOYOBO, #NYPBR01) for 1 hour at room temperature. The primary and secondary antibodies used were anti-human CD9 (COSMO BIO, #SHI-EXO-M01) and anti-mouse IgG-HRP (GE, #NA931), respectively, diluted 1:1000 and 1:5000 in Can Get Signal Solution (TOYOBO, #NKB-101). The reaction was carried out at room temperature for 1 hour. Immobilon Forte (Millipore, #WBLUF0100) was used as the detection reagent, and signals were detected using LuminoGraph1 (ATTO).

[0199] In the pipette tip of Example 8 (tilt angle 40°), when the carriers rose in the solution as the solution was aspirated, they appeared to swirl, suggesting that weak turbulence was occurring as the solution was aspirated. As the solution was dispensed, the carriers settled in the solution and accumulated on the filter, uniformly covering the entire surface. After dispensing and before the next aspiration, the position of the carriers on the filter shifted due to gravity toward the bottom of the pipette tip, and the carriers now only partially covered the filter.

[0200] In the pipette tip of Example 9 (tilt angle 60°), when the carriers rose in the solution as the solution was aspirated, they were clearly observed swirling strongly, suggesting that turbulence was occurring as the solution was aspirated. As the solution was discharged, the carriers settled in the solution and accumulated on the filter so as to uniformly cover the entire surface of the filter. However, after discharge and before the next aspiration, the position of the carriers on the filter shifted toward the bottom of the pipette tip due to gravity, and the carriers now only covered a portion of the filter.

[0201] In the pipette tip of Example 10 (70° tilt angle), when the carrier rose in the solution as the solution was aspirated, a strong vortex of the carrier was clearly observed, suggesting that turbulence was occurring as the solution was aspirated. As the solution was dispensed, the carrier settled in the solution and accumulated on the filter, evenly covering the entire surface. However, after dispensing and before the next aspiration, the position of the carrier on the filter shifted due to gravity toward the bottom of the pipette tip, resulting in the carrier only partially covering the filter. Some of the carrier near the bottom of the tip did not rise when the solution was aspirated, and remained near the bottom of the tip even after repeated aspirating and dispensing of the solution.

[0202] The results of Western blotting are shown in Figure 15. The band at approximately 25 kDa is the band for CD9, an exosome-specific marker. In Example 8 (17_40°), Example 9 (18_60°), and Example 10 (19_70°), the through band was thinner than in the control experiment (20_Tube), demonstrating that using the pipette tips of Examples 8, 9, and 10 made it easier for exosomes to adsorb to the carrier compared to the existing batch method. Furthermore, in Example 8 (17_40°), Example 9 (18_60°), and Example 10 (19_70°), the Elute band was darker than in the control experiment (20_Tube), demonstrating that by using the pipette tips of Examples 8, 9, and 10, a concentrated solution with a higher exosome concentration can be obtained compared to the existing batch method.

[0203] In the pipette tips of Example 8 (tilt angle 40°), Example 9 (tilt angle 60°), and Example 10 (tilt angle 70°), the carriers appeared larger than their original size after starting to aspirate and dispense the solution. This is thought to be an aggregate formed when the carriers adsorbed the exosomes in the solution, with multiple carriers larger in particle size than the exosomes aggregating around the exosomes. When multiple carriers with particle sizes larger than substance α or substance β aggregate around substance α or substance β to form agglomerates, the agglomerates tend to clog the column, making it difficult to purify substance α using general column chromatography, especially column tips. However, with the pipette tips of Example 8 (tilt angle 40°), Example 9 (tilt angle 60°), and Example 10 (tilt angle 70°), even when aggregates were formed, exosomes could be purified and concentrated.

[0204] (Experimental Example 8) Using a micropipette, 10 μL of a mixture (10% slurry) of exosome-collecting carrier particles (Streptoavidin Mag Sepharose, manufactured by Cytiva, model number: 28985738) and 1 μL of MagCapture Exosome Isolation Kit PS Ver. 2 (Biotin-labeled Exosome Capture included in Fujifilm Wako Pure Chemical Industries, model number: 294-84101) was added to the top of the pipette tip of Production Example 4 to create the pipette tip of Example 11.

[0205] An automatic pipetter (manufactured by Thermo Fisher Scientific, model number: E1-CLIP TIP electric pipette, 1250 μL capacity) was connected to the upper end of the pipette tip of Examples 9 and 11. Furthermore, the lower end of this pipette tip was placed in a U-bottom 2 mL tube (manufactured by Eppendorf, No. 0030108450) containing 1 μL of Capan 2 culture supernatant, and positioned so that the solution in the tube could be aspirated. When the Capan2 culture supernatant (Input) used in Experimental Example 7 was used as the Capan2 culture supernatant, it was designated "Capan2_Undiluted Solution," and when the Capan2 culture supernatant (Input) used in Experimental Example 7 was diluted 5-fold with serum-free medium, it was designated "Capan2_1 / 5 Diluted Input." Using the custom mode of the automatic pipetter, 1 mL of the solution was aspirated and discharged at speed 1 for 30 minutes, allowing the exosomes in the culture supernatant to adsorb to the carrier.

[0206] Control experiments, washing of exosome-bound carriers, elution, and Western blotting were performed in the same manner as in Experimental Example 7. The control experiment was carried out with N=4, and the pipette tips of Examples 9 and 11 were carried out with N=2. In each of the adsorption, washing, and elution steps, dispersion of the carrier (the state of the carrier flying up) during the suction and discharge operations was visually observed, and photographs and videos were taken.

[0207] In the pipette tip of Example 9 (tilt angle 60°), when the carriers rose in the solution as the solution was aspirated, they were clearly observed swirling strongly, suggesting that turbulence was occurring as the solution was aspirated. As the solution was discharged, the carriers settled in the solution and accumulated on the filter so as to uniformly cover the entire surface of the filter. However, after discharge and before the next aspiration, the position of the carriers on the filter shifted toward the bottom of the pipette tip due to gravity, and the carriers now only covered a portion of the filter.

[0208] In the pipette tip of Example 11 (tilt angle 50°), when the carriers rose in the solution as the solution was aspirated, they were clearly observed swirling strongly, suggesting that turbulence was occurring as the solution was aspirated. As the solution was dispensed, the carriers settled in the solution and accumulated on the filter so as to uniformly cover the entire surface of the filter. However, after dispensing and before the next aspiration, the position of the carriers on the filter shifted toward the bottom of the pipette tip due to gravity, and the carriers now only covered a portion of the filter.

[0209] The results of Western blotting are shown in Figure 16. The band at approximately 25 kDa is the band for CD9, an exosome-specific marker. As shown in Figure 16, in the control experiment (Tube), a faint Through band was detected, but in Example 9 (7_Used 60°, 8_Used 60°) and Example 11 (5_Used 50°, 6_Used 50°), the Through band was below the detection limit, indicating that the majority of exosomes in the culture supernatant were adsorbed to the carrier, with almost none remaining in the culture supernatant. It was revealed that using the pipette tips of Examples 9 and 11 made it easier for exosomes to adsorb to the carrier than with the existing batch method. Furthermore, the Elute bands in the control experiments (Tube 1, Tube 2, Tube 3, and Tube 4) were darker than those in the "Capan 2 1 / 5 Dilution Input" experiment, and a concentrate with an elevated exosome concentration was also obtained in the control experiments (Tube 1, Tube 2, Tube 3, and Tube 4), which are conventional batch methods. On the other hand, bands significantly darker than those in the control experiments (Tube 1, Tube 2, Tube 3, and Tube 4) were detected in Example 9 (Tube 7 Used 60°, Tube 8 Used 60°) and Example 11 (Tube 5 Used 50°, Tube 6 Used 50°). This demonstrates that by using the pipette tips in Example 9 (tilt angle 60°) and Example 11 (tilt angle 50°), a concentrate with a significantly higher exosome concentration can be obtained compared to conventional batch methods, even when using a solution with a low exosome concentration. When the intensity of the detected bands was quantified, bands detected in Examples 9 and 11 were approximately 4.9 and 4.6 times brighter than those in "Capan2_1 / 5 diluted Input," respectively. By using the pipette tips of Example 9 (tilt angle 60°) and Example 11 (tilt angle 50°), it was possible to produce a concentrated solution in which the exosomes in the sample solution were concentrated by more than 4.6 times.

[0210] In Example 9 (tilt angle 60°) and Example 11 (tilt angle 50°), the carriers appeared larger than their original size after the start of suction and discharge of the solution. This is thought to be an aggregate formed when the carriers adsorbed the exosomes in the solution, with multiple carriers larger in particle size than the exosomes aggregating around the exosomes. When multiple carriers with particle sizes larger than substance α or substance β aggregate around substance α or substance β to form agglomerates, the agglomerates tend to clog the column, making it difficult to purify substance α using general column chromatography, especially column tips. However, with the pipette tips of Example 9 (tilt angle 60°) and Example 11 (tilt angle 50°), even when aggregates were formed, exosomes could be purified and concentrated.

[0211] (Experimental Example 9) A pipette tip (Gilson, model number: D10mL) was cut using an ultrasonic cutter. The inclination angle of the cut surface was 50° relative to a line perpendicular to the axis of the pipette tip when viewed from the front. A hydrophilic PTFE filter was placed on a hot plate heated to 180°, and the cut surface of the tip was pressed against the hot plate while being parallel to the hot plate, thereby achieving heat fusion. A 5 μm pore size filter (Millipore, Omnipore, JMWP09025) was used as the hydrophilic PTFE filter, resulting in the pipette tip of Production Example 9. A 10 μm pore size filter (Millipore, Omnipore, JCWP14225) was used as the pipette tip of Production Example 10.

[0212] Using a micropipette, 10 μL of a mixture (10% slurry) of exosome-collecting carrier particles (Streptoavidin Mag Sepharose, manufactured by Cytiva, model number: 28985738) and 1 μL of MagCapture Exosome Isolation Kit PS Ver. 2 (Biotin-labeled Exosome Capture included in Fujifilm Wako Pure Chemical Industries, model number: 294-84101) was added to the top of the pipette tip of Production Example 9 and Production Example 10, respectively, to create the pipette tips of Example 12 and Example 13.

[0213] An automatic pipettor (Thermo Fisher Scientific, Model No. E1-CLIP TIP Electric Pipettor, 1250 μL capacity) was connected to the upper end of the pipette tip from Examples 12 and 13. 1 mL of Capan 2 culture supernatant diluted 1:5 with TBS and 2 μL of 1 mM CaCl2 were placed in a 2 mL U-bottom tube (Eppendorf, No. 0030108450). The lower end of the pipette tip was positioned so that it could aspirate the solution in the tube. Using the custom mode of the automatic pipetter, 1 mL of the solution was aspirated and dispensed at speed 3 for 30 minutes, allowing the exosomes in the culture supernatant to adsorb onto the carrier.

[0214] The exosome-bound carrier was washed three times with 1 mL of MOPS Wash Buffer. Elution was performed using 50 μL of Elute Buffer (20 mM MOPS (Dojindo Laboratories, Ltd., 347-08243), 150 mM NaCl (Fujifilm Wako Pure Chemical Industries, Ltd., model number: 191-01665), 2 mM EDTA (Nippon Gene, No. 311-90075), 1% Triton X-100 (Nacalai, No. 35501-15)). The control experiment, washing of the exosome-bound carrier, elution, and Western blotting were performed under the same conditions as in Experimental Example 7. The control experiment was carried out with N=1, and the pipette tips of Examples 12 and 13 were carried out with N=2.

[0215] In each of the adsorption, washing, and elution steps, dispersion of the carrier (the state of the carrier flying up) during the suction and discharge operations was visually observed. In the pipette tips (50° inclination angle) of Examples 12 and 13, when the carriers rose in the solution as the solution was aspirated, they were clearly observed swirling strongly, suggesting that turbulence was occurring as the solution was aspirated. As the solution was dispensed, the carriers settled in the solution and accumulated on the filter so as to uniformly cover the entire surface of the filter. However, after dispensing and before the next aspiration, the position of the carriers on the filter shifted due to gravity toward the bottom of the pipette tip, and the carriers now covered only a portion of the filter.

[0216] The results of Western blotting are shown in Figure 17. The band at approximately 25 kDa is the band for CD9, an exosome-specific marker. In Example 12 (1_5μm, 2_5μm) and Example 13 (3_10μm, 4_10μm), the through band was thinner than in the control experiment (5_Tube), demonstrating that the use of the pipette tips of Examples 12 and 13 made it easier for exosomes to adsorb to the carrier compared to the existing batch method. Furthermore, in Example 12 (1-5 μm, 2-5 μm) and Example 13 (3-10 μm, 4-10 μm), the elute band was darker than in the control experiment (5-Tube), demonstrating that using the pipette tips of Examples 12 and 13 allowed for the production of concentrated solutions with higher exosome concentrations than the existing batch method. In particular, in Example 12 (1-5 μm, 2-5 μm), which used a filter with a pore size of 5 μm, a concentrated solution with an extremely high exosome concentration was obtained.

[0217] (Experimental Example 10) The body of a pipette tip (Gilson, No. D 10 mL) was cut at a position 20 mm from the bottom end using an ultrasonic cutter. The inclination angle of the cut surface was 55° relative to a line perpendicular to the axis of the pipette tip when viewed from the front. A PTFE filter coated with an MPC polymer and treated for hydrophilicity was pressed against the cut surface of the pipette tip to temporarily set it in place. In this state, the cut surface was pressed downward onto a hot plate heated to 180°C and heat-sealed. This was the pipette tip of Production Example 11.

[0218] Using a micropipette, 100 μL of HT carrier particles (Bio-Rad Bio-Gel HT Hydroxyapatite, model number: 130-0150) adjusted to a 50% slurry with PBS buffer was added to the top of the pipette tip of Example 11 to create the pipette tip of Example 14.

[0219] An automatic pipetter (Gilson, model number: P10mLM, for 10 mL) was connected to the upper end of the pipette tip of Example 14. Furthermore, the lower end of this pipette tip was placed in a 50 mL tube with a U-shaped bottom (Falcon, No. 352070) containing 4 mL of virus solution, and positioned so that the solution in the tube could be aspirated.

[0220] The virus solution used was inactivated influenza virus prepared by treating cultured influenza A virus (109.0 TCID50 / mL) with formalin (Fujifilm Wako Pure Chemical Industries, Ltd., model number: 060-03845). The inactivated virus was diluted 2,000,000 times with 0.1% polyethylene glycol #6000 (Nacalai, model number: 28254-85) / D-PBS(-) (Fujifilm Wako Pure Chemical Industries, Ltd., model number: 045-29795) to prepare an inactivated virus solution. This virus solution was used as the input.

[0221] Using the custom mode of the automatic pipettor, a program was run at room temperature for 1 hour, which involved aspirating 5.5 mL of solution at speed 1, waiting for 5 seconds, dispensing 5.5 mL of solution at speed 1, and waiting for 20 seconds, allowing the virus to adsorb onto the HT carrier particles.

[0222] After repeated aspiration and discharge, the liquid in the pipette tip was discharged and the tube was replaced. Specifically, the tube containing the virus solution was removed (the remaining solution was designated as Through), and a new tube containing 100 μL of lysis buffer (5 M guanidine thiocyanate (Fujifilm Wako Pure Chemical Industries, Ltd., model number: 077-04995), 100 mM EDTA aqueous solution (pH 8.0, Nippon Gene Co., Ltd., model number: 311-90075), 100 mM Tris-HCl (pH 6.8): Tris (Fujifilm Wako Pure Chemical Industries, Ltd., model number: 200-07887) pH adjusted with hydrochloric acid (Fujifilm Wako Pure Chemical Industries, Ltd., model number: 080-01066) and diluted with water), 3% Triton X-100 (Nacalai, 35501-15), and 1% 2-mercaptoethanol (Sigma-Aldrich Co., Ltd., model number: M3148) was placed in a position so that the pipette tip could aspirate the solution.

[0223] Using the custom mode of the automatic pipetter, the following program was run for 10 minutes at room temperature: aspirate 2 mL of solution at speed 1, wait 5 seconds, dispense 2 mL of solution at speed 1, wait 5 seconds, destroy the virus adsorbed to the HT carrier particles, release the encapsulated nucleic acids, and collect the recovered liquid as elute. 100 μL of purified water was added to 100 μL of elute, and a total of 200 μL of the liquid was loaded into an automated nucleic acid extraction device (magLEAD 12gC, Precision System Science), and nucleic acids including viral RNA were extracted using a nucleic acid extraction reagent (MagDEA Dx SV, Precision System Science). For Input and Through, 100 μL of lysis buffer was added to 100 μL of Input and Through, and nucleic acids containing viral RNA were extracted in the same manner as above.

[0224] Using the input, through, and elute samples, quantitative PCR was performed on the influenza A virus M gene, an example of a gene specific to influenza A viruses. Quantitative PCR was performed using primers and probes (MP-39-67For, MP-183-153Rev, and MP-96-75ProbeAs) for detecting the influenza A virus M gene, as described by Nakauchi et al. (Journal of Virological Methods, 2011, One-step real-time reverse transcription-PCR assays for detecting and subtyping pandemic influenza A / H1N1 2009, seasonal influenza A / H1N1, and seasonal influenza A / H3N2 viruses), a commercially available quantitative PCR kit (THUNDERBIRD Probe One-step qRT-PCR Kit, Toyobo Co., Ltd.), and a quantitative PCR system (Applied Biosystems 7500 Fast Real-Time PCR System). Experiments were performed in duplicate.

[0225] In each of the adsorption, washing, and elution steps, dispersion of the carrier (the state of the carrier flying up) during the suction and discharge operations was visually observed. In the pipette tip of Example 14 (tilt angle 55°), when the carriers rose in the solution as the solution was aspirated, they were clearly observed swirling strongly, suggesting that turbulence was occurring as the solution was aspirated. As the solution was discharged, the carriers settled in the solution and accumulated on the filter so as to uniformly cover the entire surface of the filter. However, after dispensing and before the next aspiration, the position of the carriers on the filter shifted due to gravity toward the bottom of the pipette tip, and the carriers now covered only a portion of the filter.

[0226] The results (amplification curves in quantitative PCR) are shown in Figure 18. The vertical axis of the graph represents ΔRn (the intensity of the fluorescent signal generated under specified PCR conditions), and the horizontal axis represents the number of cycles in quantitative PCR. In addition to the ΔRn values ​​when the Input, Through, and Elute samples were used as PCR samples, the ideal value at concentrated concentration is also shown. The ideal value at concentrated concentration was calculated by preparing an influenza A virus solution 40 times more concentrated than the Input, extracting nucleic acids including viral RNA from that solution in the same manner as described above, and using the resulting solution as a PCR sample.

[0227] With Elute, ΔRn increased in fewer cycles than with Input, revealing that it contained a higher concentration of the M gene. In other words, it was possible to produce a concentrated solution (Elute) with an increased concentration of viral RNA, substance α, from a virus solution (sample solution containing substance β). For example, at ΔRn = 0.1, the Ct value of Elute (32.5) was approximately 3.5 times lower than that of Input (36.0), revealing that the viral RNA concentration was approximately 11.3 (23.5) times higher. Furthermore, with Elute, the amplification curve approached the ideal value when concentrated.

[0228] (Experimental Example 11) In recent years, there has been a demand for automation of experimental protocols to minimize variability in experimental data and human error in manual processing of multiple samples. Therefore, we developed a filter-equipped pipette tip compatible with the Precision System Science automated protein purification system (Purelumn system) and performed purification of immunoglobulin G from human serum.

[0229] The body of a pipette tip (1.25 mL, manufactured by Precision System Science) was cut using a rotary sander at a position 17 mm from the bottom end. The inclination angle of the cut surface was adjusted to 60° relative to a line perpendicular to the axis of the pipette tip when viewed from the front. A PTFE filter coated with an MPC polymer and treated for hydrophilicity was pressed against the cut surface of the pipette tip, temporarily placing it in place. In this state, the cut surface was pressed downward onto a hot plate heated to 180°C for thermal fusion. This was the pipette tip of Production Example 12.

[0230] 10 μL of Protein G Sepharose 4 Fast Flow (manufactured by Cytiva, model number: 71708300) was added to the top end of the pipette tip of Preparation Example 12 using a micropipette to prepare the pipette tip of Example 15.

[0231] The pipette tip of Example 15 was placed in the tip placement position of the Purelumn system, and 400 μL of wash buffer (PBS (manufactured by TaKaRa Bio, model number: T900) supplemented with 1 M NaCl (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., model number: 191-01665)) was poured into wells 0, 1, 2, 3, and 4 of the GC cartridge, and human serum (manufactured by Cosmo Bio, #12181201, lot: BJ14005+BJ14012) in 200 μL, and 100 mM glycine (Fujifilm Wako Pure Chemical Industries, Ltd., model number: 077-00735)-HCl (Fujifilm Wako Pure Chemical Industries, Ltd., model number: 080-01066), pH 2.5, in the elute well.

[0232] The protocol involved using the pipette tip of Example 15 to aspirate and discharge once each in well 0 and well 1, followed by 30 minutes of aspirating and dispensing in the sample well to bind immunoglobulin G in human serum to Protein G Sepharose. Subsequently, five aspirates and dispenses were performed in wells 2, 3, and 4, respectively, to wash away nonspecifically bound components from Protein G Sepharose. Finally, aspirates and dispenses for 100 seconds in the Elute well to elute the immunoglobulin G bound to Protein G. After collecting the eluate, 20 μL of neutralization buffer (1 M Tris-HCl, pH 8.0) (manufactured by Nippon Gene Co., Ltd., model number: 314-90065) was added to neutralize the eluate.

[0233] 3 μL of the eluate and 3 μL each of the unpurified human serum (Input) and purified human serum (Through) were mixed with sample buffer (Fujifilm Wako Pure Chemical Corporation, #196-16142) as controls, treated at 100°C for 5 minutes, and then subjected to SDS-PAGE. The SDS-PAGE gel used was SuperSep™ Ace 10-20% (Fujifilm Wako Pure Chemical Corporation, #191-15031). The molecular weight marker used to control the protein molecular weight was Precision Plus Protein Two-Color Standard (Bio-Rad, product number 1610374). After electrophoresis, the gel was silver stained with EzStain Silver (ATTO, product number 2332360) to visualize the proteins in the solution.

[0234] The results are shown in Figure 19. In the human serum before purification (Input), bands corresponding to numerous proteins contained in the serum were confirmed, and among these, protein bands corresponding to immunoglobulin G were present at approximately 50 KDa and 25 KDa. As a result of purification using the pipette tip of Example 15, the 50 KDa and 25 KDa bands were removed from the human serum after purification (Through), and it was confirmed that the 50 KDa and 25 KDa bands were present as the main components in the eluate. A concentrate containing increased concentrations of immunoglobulin G could be prepared from human serum containing immunoglobulin G.

[0235] (Experimental Example 12) Monoclonal antibodies were purified from hybridoma culture medium using the Purelumn system (Precision System Science) and a filter-equipped pipette tip.

[0236] Hybridoma cells producing monoclonal antibodies were CP12 cells (Anal. Chem. 94, 2476-2484, 2022). Hybridoma cells were maintained in a 6-well dish (Falcon, 353046) using serum-containing medium (RPMI-1640 medium (Fujifilm Wako Pure Chemical Industries, Ltd., #183-02023), 10% Fetal Bovine Serum (Thermo Fisher Scientific Co., Ltd. (Gibco), #10270106), and 1% Penicillin-Streptomycin (Thermo Fisher Scientific Co., Ltd. (Gibco), #15140122)) at 37°C and 5% CO2.

[0237] Monoclonal antibodies were produced in a serum-containing medium containing 0.5% fetal bovine serum (Gibco, #10270106) in a 6-well dish (Falcon, #353046). 5 Hybridoma cells were suspended in 2 mL of 0.5% serum medium, and the culture medium was collected after 48 hours and 96 hours. The collected culture medium was centrifuged at 2000 x G for 10 minutes using a centrifuge (Tomy Seiko Co., Ltd., LX-130) to remove hybridoma cells and debris generated during the culture. This was used as the sample for purification (Input). The culture medium after 96 hours was also purified directly from the sample without centrifugation (no centrifugation).

[0238] 20 μL of rProtein A Sepharose Fast Flow (manufactured by Cytiva, model number: 17127901) was added to the top end of the pipette tip of Preparation Example 12 using a micropipette to prepare the pipette tip of Example 16.

[0239] The pipette tip of Example 16 was placed in the tip placement location of the Purelumn system, and 1 mL of Wash Buffer (PBS (manufactured by TaKaRa Bio, model number: T900)) was placed in wells 0, 1, 2, 3, and 4 of the GC Cartridge, 1 mL of culture supernatant sample was placed in the sample well, and 50 μL of 100 mM glycine (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., model number: 077-00735)-HCl (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., model number: 080-01066), pH 2.5, was placed in the elute well.

[0240] The protocol involved using the pipette tip from Example 16 to aspirate and discharge once each in well 0 and well 1, followed by 60 minutes of aspirating and dispensing in the sample well to bind the monoclonal antibody in the culture supernatant sample to rProtein A Sepharose. Subsequently, five aspirating and dispensing cycles were performed in wells 2, 3, and 4, respectively, to wash away nonspecifically bound components from the rProtein A Sepharose. Finally, aspirating and dispensing was performed for 100 seconds in the Elute well to elute the monoclonal antibody bound to rProtein A. After collecting the eluate, 5 μL of neutralization buffer (1 M Tris-HCl, pH 8.0) (Nippon Gene, model number: 314-90065) was added to neutralize the eluate.

[0241] 9 μL of the eluate and 9 μL each of the culture supernatant before purification (Input) and after purification (Through) were mixed with sample buffer (Fujifilm Wako Pure Chemical Industries, Ltd., #196-16142) and treated at 100°C for 5 minutes. Then, they were subjected to SDS-PAGE. The SDS-PAGE gel used was an XV PANTERA LL Gel, 10%, 16-well (DRC, #DLL-225P). The protein molecular weight marker was Precision Plus Protein Two-Color Standard (Bio-Rad, model number 1610374). After electrophoresis, the separated proteins were visualized by Coomassie Brilliant Blue staining using CBB staining solution (Biocraft, model number CBB-1000).

[0242] The results using the centrifuged 48-hour culture medium are shown in Figure 20A. Numerous protein bands contained in the serum-containing medium were observed in the culture supernatant before purification (Input), but after purification using the pipette tip of Example 16, bands derived from the monoclonal antibody were observed in the eluate at approximately 50 KDa and 25 KDa. These bands were present in the Input before purification but had almost disappeared in the Throughout after purification, demonstrating that the monoclonal antibody was specifically purified by the pipette tip operation of Example 16.

[0243] Figure 20B shows the results using the 0-hour, 48-hour, and 96-hour culture medium after centrifugation, as well as the results using the 96-hour culture medium without centrifugation. It was clear that more monoclonal antibody was purified at 96 hours than at 48 hours. This means that it was possible to monitor the amount of monoclonal antibody produced depending on the culture time. Furthermore, even though the 96-hour culture medium without centrifugation contains hybridoma cells and culture debris, it was possible to purify a similar amount of monoclonal antibody to that obtained with centrifugation. These results demonstrate that the pipette tip of Example 16 can be used to monitor the amount of monoclonal antibody produced during hybridoma cell culture without any centrifugation. A concentrated solution containing an increased concentration of monoclonal antibodies could be produced from the hybridoma culture medium.

[0244] (Experimental Example 13) Next, we investigated pretreatment for protein analysis using a filter-equipped pipette tip. Human serum contains numerous proteins, but approximately 12 major proteins account for 95% of the total. High-sensitivity analysis of trace proteins in serum using mass spectrometry or other instruments requires pretreatment to remove these proteins, and commercially available reagents are available. For example, Proteome Purity 12 Human Serum Protein Immunodepletion Resin Solution (R&D, model number: IDR012-020) is a carrier that removes major proteins from human serum or plasma, specifically α1-acid glycoprotein, α1-antitrypsin, α2-macroglobulin, albumin, apolipoprotein AI, apolipoprotein A-II, fibrinogen, haptoglobulin, IgA, IgG, IgM, and transferrin. Automated sample preparation for protein analysis was performed using a filtered pipette tip and the Purelumn system.

[0245] 100 μL of Proteome Purity 12 Human Serum Protein Immunodepletion Resin solution (R&D, model number: IDR012-020) was added to the top of the pipette tip of Preparation Example 12 using a micropipette to create the pipette tip of Example 17.

[0246] The pipette tip of Example 17 was placed in the tip placement location of the Purelumn system, and 100 μL of human serum (Cosmo Bio, #12181201, lot: BJ14005+BJ14012) diluted 100-fold with PBS (TaKaRa Bio, model number: T900) containing 0.5% Triton-X100 (Nacalai Tesque, model number: 35501-15) was placed in the sample well.

[0247] The pipette tip of Example 17 was used to aspirate and discharge the sample well for 30 minutes, allowing the major proteins in human serum to bind to Proteome Purity 12 Human Serum Protein Immunodepletion Resin (R&D, model number: IDR012-020). After aspirating and dispensing, the sample solution was recovered from the sample well.

[0248] As a control for the experiment using the pipette tip of Example 17, major serum proteins were removed using a column. 100 μL of Proteome Purity 12 Human Serum Protein Immunodepletion Resin solution (R&D, model number: IDR012-020) was added to a column (Bio-Rad, model number 7326304) using a micropipette. The column was centrifuged at 1000xG for 10 seconds in a microcentrifuge (TOMY, model number: MX-300) to remove the resin solvent. Afterward, 100 μL of human serum (Cosmo Bio, #12181201, lot: BJ14005+BJ14012) diluted 100-fold with PBS (TaKaRa Bio, model number: T900) containing 0.5% Triton-X100 (Nacalai Tesque, model number: 35501-15) was added. The column was capped and mixed by end-over-end mixing in a BugCrasher GM-01 (TAITEC, Inc.) for 30 minutes at room temperature. The column was centrifuged at 1000xG for 10 seconds in a microcentrifuge (TOMY, model number: MX-300), and the through-liquid was collected.

[0249] 10 μL of the sample solution after 30 minutes of aspirating and dispensing using the pipette tip of Example 17 and 10 μL of the through-liquid after 30 minutes of treatment in the column were mixed with sample buffer (Fujifilm Wako Pure Chemical Industries, Ltd., #196-16142), treated at 100°C for 5 minutes, and then developed by SDS-PAGE. The SDS-PAGE gel used was an XV PANTERA LL Gel, 10%, 16-well (DRC, #DLL-225P). The protein molecular weight marker was Precision Plus Protein Two-Color Standard (Bio-Rad, model number: 1610374). After electrophoresis, the developed gel was stained with Coomassie Brilliant Blue using CBB staining solution (Biocraft, model number CBB-1000) to visualize the proteins separated in the gel.

[0250] The results are shown in Figure 21. Numerous protein bands were identified in the untreated human serum (Input). However, the sample treated with Proteome Purity 12 Human Serum Protein Immunodepletion Resin (R&D, model number: IDR012-020) (column, filter-equipped pipette tip) demonstrated the removal of several abundant proteins. Furthermore, similar protein removal effects were observed when using a column manually and when using an automated system (Purelumn System, Precision System Science) with filter-equipped pipette tips. However, when processing six samples, the automated system with filter-equipped pipette tips enabled processing in two-thirds to less than half the time. We were able to produce a purified solution from human serum that had an increased purity of proteins other than the major proteins in serum (α1-acid glycoprotein, α1-antitrypsin, α2-macroglobulin, albumin, apolipoprotein AI, apolipoprotein A-II, fibrinogen, haptoglobulin, IgA, IgG, IgM, and transferrin).

[0251] The filter-equipped pipette tips and carriers used in the above examples are as follows:

[0252] [Table 2]

Claims

1. a tip body that contains a liquid; a filter disposed in a lower opening at the lower end of the chip body; A pipette tip having the lower opening is entirely covered with a filter, At least a part of the lower opening at the lower end of the tip body where the filter is disposed is A pipette tip having an inclined portion configured so that the filter is inclined relative to a line perpendicular to the axis of the pipette tip when viewed from the front of the pipette tip.

2. 2. The pipette tip according to claim 1, wherein the angle of inclination of the inclined portion is greater than 20 degrees and not greater than 70 degrees relative to a line perpendicular to the axis of the pipette tip when viewed from the front.

3. 3. The pipette tip according to claim 1, wherein a carrier is held within the pipette tip body.

4. 3. The pipette tip according to claim 1, wherein the material constituting the filter is polytetrafluoroethylene (PTFE) or polyvinylidene fluoride (PVDF).

5. 3. The pipette tip according to claim 1, wherein the melting point of the material constituting the filter is higher than that of the material constituting the tip body, and the filter is fused to the lower opening.

6. 3. The pipette tip according to claim 1, for producing a concentrated solution having an increased concentration of substance α from a sample solution containing a target substance (substance α) or a substance containing substance α (substance β).

7. An automated device comprising the pipette tip according to claim 1 or 2.

8. 8. The automated device according to claim 7, comprising a pipetting device for aspirating and dispensing solutions, and means for moving the pipetting device up and down and back and forth.

9. 8. The automated device according to claim 7, wherein the automated device is a robotic liquid handler that automates the operation of attaching and detaching pipette tips in addition to the operation of aspirating and dispensing.

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