Aperture array filter, particle arrangement device and particle capture method

Abstract

In this pore array filter (1), a plurality of pores (3) are formed in a filter body (2), adjacent pores (3) are separated from each other by a pore partition wall (6), two or more through-pores (8) are formed in a pore bottom (4), the minimum width of an opening of each through-pore (8) is 1 [mu] m to 3.5 [mu] m, and the ratio of the total opening area of the through-pores is 0.5% to 3.5%. When the center of a minimum circumscribed circle (G1) surrounding all the through holes (8) of each hole bottom (4) is moved to the center of any one of the plurality of through holes (8), the moved minimum circumscribed circle (G2) at least partially overlaps all the plurality of through holes (8).

Description

Technical Field

[0001] The present invention relates to a hole array filter, a particle arrangement device and a particle capturing method for capturing particles such as cells and beads.

[0002] This application claims priority based on Japanese Patent Application No. 2022-181802 filed in Japan on November 14, 2022, and the contents of which are incorporated herein by reference. Background Art

[0003] In recent years, especially in the field of innovative drugs, the target of cell analysis has been subdivided from the cell group level to the single cell level, and the following method has been used: using a cell screening device (particle array device), cells are captured one by one in the fine and large number of holes of a hole array filter, and then a screening test is performed on a large number of cells at one time to select cells with target characteristics. As a method for screening cells, for example, the following method is used: cells captured in multiple holes are brought into contact with a liquid in which a reagent such as a capture agent that binds to a specific antibody is dispersed, and cells that secrete secretions that bind to the capture agent are found and recovered from the holes.

[0004] The particle arrangement device disclosed in Patent Document 1 has a hole array filter made of a resist, in which a plurality of holes each having a through hole at the bottom are arranged and formed. The opening shape of each hole is similar to a cell, and is circular or elliptical. The embodiment of Patent Document 1 discloses a hole array filter having an opening diameter of 100 μm, a hole depth of 30 to 100 μm, and an opening diameter of the through hole of 2 to 5 μm.

[0005] The particle arrangement device disclosed in Patent Document 2 has a hole array filter made of a silicon wafer, in which a plurality of holes are arranged and formed by etching, and a SiNx membrane having a through hole is provided at the bottom of each hole. The opening shape of each hole is circular. The hole array filter is made of a silicon wafer, so in the embodiment of Patent Document 2, the thickness of the filter body is 380 μm, the opening diameter of the hole is about 100 μm, and the distance between the centers of the holes is about 150 μm.

[0006] The particle array device disclosed in Patent Document 3 has a hole array filter for secretion analysis, which includes two layers: a hole substrate having through holes at the bottom for accommodating cells, and a substrate for accommodating cells for detecting secretions from the cells. The aforementioned hole substrate is made using a resist. In the embodiment of Patent Document 3, the holes are circular, the opening diameter is 12 μm, which is a size close to that of cells, and the distance between the centers of the holes is about 100 μm.

[0007] Prior art literature

[0008] Patent Literature

[0009] Patent Document 1: U.S. Patent No. 10370630

[0010] Patent Document 2: U.S. Patent No. 9638636

[0011] Patent Document 3: Japanese Patent Application Publication No. 2019-213566 Summary of the invention

[0012] Problems to be solved by the invention

[0013] However, in the above-mentioned hole array filter, when a sample liquid obtained by dispersing cells, beads, etc. in a dispersion medium is passed through the hole array filter, it is preferable to capture cells, etc. in a so-called single cell manner, that is, to capture cells, etc. in as many holes as possible and to accommodate only one cell in one hole. If cells, etc. can be captured in multiple holes by passing the liquid once and the single cell rate is high, target cells, etc. can be separated one by one and captured with a high probability.

[0014] On the other hand, when cells are captured and then cultured, or when cells are recovered from each well using a capillary tube, it is easier to operate when the well opening diameter is relatively large. This is because if cells are cultured in a well with a small opening diameter, there is a possibility that divided cells overflow from the well and capture is impaired. In addition, when the well opening diameter is larger than the outer diameter of the tip of the capillary tube, the tip of the capillary tube can be directly inserted into the well, making it easier to recover cells, etc.

[0015] However, if the opening diameter of the hole is simply increased, the number of holes per unit area of ​​the hole array filter decreases. For example, if the width of the wall separating the holes is constant and the hole opening diameter is compared to the case of 20 μm and the case of 50 μm, the number of holes per unit area becomes 1 / 4. If the opening diameter of each through hole formed in the bottom of the hole is the same in the case of 20 μm and the case of 50 μm, the ratio of the through hole area per unit area of ​​the filter, that is, the opening rate of the through hole, is also reduced to 1 / 4, and the overall liquid permeability of the hole array filter is deteriorated, and the efficiency of the liquid permeation operation is reduced.

[0016] If the opening diameter of a through hole formed in each well is increased in order to improve the fluid permeability of the well array filter, there is a concern that cells etc. may pass through the through hole. Therefore, although it is possible to increase the number of through holes in each well, it has been found that simply increasing the number of through holes will reduce the single cell rate even when the opening diameter of the wells remains the same.

[0017] The inventors of the present application have analyzed this phenomenon in detail and found that in order to ensure the fluid permeability of the hole array filter and improve the single cell rate, it is better to form the through holes in a manner to obtain the following effects. That is, (1) when a cell or the like is captured in any hole, the opening area of ​​the through holes in the hole is reduced by the cell or the like, thereby reducing the total flow rate, and the second cell or the like is not guided to the flow in the same hole, but the cell or the like is guided to the adjacent empty hole to be captured; (2) however, when a cell or the like is captured in a hole, if all the through holes are blocked and the flow rate is extremely reduced, the fluid permeability of the hole array filter as a whole will be deteriorated.

[0018] The inventors of the present application have studied the conditions of the through holes for achieving the above-mentioned effect. As a result, it was found that the above-mentioned effect can be obtained under the following conditions: that is, two or more through holes are formed at the bottom of the hole, the minimum width of the upper surface opening of the through holes is 1 μm or more and 3.5 μm or less, the ratio of the total opening area of ​​the through holes to the area of ​​the hole formation region of the hole array filter is 0.5% or more and 3.5% or less, and even if the center of the minimum circumscribed circle of all the through holes in the same hole is moved to the center of any one of the plurality of through holes, the minimum circumscribed circle satisfies a positional relationship of at least partially overlapping with respect to all the plurality of through holes.

[0019] The present invention is made in view of the above-mentioned situation, and its object is to provide a hole array filter, a particle arrangement device and a particle capture method that can capture cells and other particles one by one in as many holes as possible, has a high single cell rate and good liquid permeability, and can arrange and stably capture a large number of cells.

[0020] Means for solving problems

[0021] [Mode 1] The hole array filter of Mode 1 of the present invention is characterized in that it has a flat filter body, a plurality of wells opened in a hole forming area on the upper surface of the filter body are formed in the filter body, the adjacent wells are separated from each other by well partitions, bottoms are formed at the lower ends of the wells, two or more through holes reaching the lower surface of the filter body are formed at the well bottoms, the minimum width of the upper surface openings of the through holes is not less than 1 μm and not more than 3.5 μm, the ratio of the total opening area of ​​the through holes to the area of ​​the hole forming area is not less than 0.5% and not more than 3.5%, and a minimum circumscribed circle surrounding all the through holes formed at the bottoms of the holes is assumed, and even if the center of the minimum circumscribed circle is moved to the center of any one of the through holes, the minimum circumscribed circle satisfies a positional relationship of at least partial overlap relative to all the through holes.

[0022] According to the hole array filter of method 1, the minimum width of the through hole is 1 μm to 3.5 μm, and the ratio of the total opening area of ​​the through holes to the area of ​​the hole forming region is 0.5% to 3.5%, so it is possible to suppress the passage of particles such as cells or beads to be captured through the through holes while ensuring the flow rate of the hole array filter as a whole. In addition, by making the multiple through holes formed at the bottom of each hole satisfy the above-mentioned positional relationship, when one of the above-mentioned particles is captured in the hole, the probability that the particle at least partially overlaps with all the above-mentioned multiple through holes is high. Therefore, after the first particle is captured, the flow rate of the dispersion medium passing through all the through holes in the hole is moderately reduced, and the second and subsequent particles are suppressed from entering the same hole with the flow, and the probability of cells, etc. being guided to adjacent empty holes and captured increases. As a result, the probability of capturing particles one by one in a large number of holes is high, the single cell rate is high, and the permeability is well maintained without reduction, and the separation of particles can be stably performed through each hole.

[0023] [Method 2] Alternatively, in Method 1, two or more through holes are formed in the bottom of each of the holes on an imaginary line segment, or three or more through holes are formed in the bottom of each of the holes at the vertices of an imaginary triangle and on the sides or inside of the triangle, or four or more through holes are formed in the bottom of each of the holes at the vertices of an imaginary quadrilateral and on the sides or inside of the quadrilateral.

[0024] In this case, when a particle such as a cell or bead is captured in a hole, the ratio of the particle at least partially overlapping with all the plurality of through holes is high. Therefore, after the first particle is captured, the flow rate of the dispersion medium passing through all the through holes in the hole is appropriately reduced, and the second and subsequent particles are inhibited from entering the hole, so that the particles can be separated more stably through each hole.

[0025] [Mode 3] Alternatively, in Mode 1 or 2, the opening shape of the through hole may be circular, elliptical, or a shape formed by connecting a plurality of circular or elliptical shapes.

[0026] In this case, by making the opening shape of the aforementioned through hole circular, elliptical, or a shape formed by connecting multiple circles or ellipses, when particles such as cells or beads are placed on the aforementioned through hole, the proportion of at least partial overlap between the aforementioned particles and all the aforementioned multiple through holes is high, which can ensure an increase in the flow difference before and after the particles are captured in the hole, thereby improving the aforementioned effect.

[0027] [Mode 4] In any one of Modes 1 to 3, the through hole may have an opening shape of a polygon or a polygon with rounded corners. The polygon may be a triangle, a quadrangle, or a pentagon.

[0028] In this case, when the opening shape of the through hole is a polygon or a polygon with rounded corners, the minimum width of the upper surface opening of the through hole becomes smaller than the flow path cross-sectional area of ​​the through hole, so that the particles such as cells or beads to be captured can be further suppressed from passing through the through hole. In addition, since the hole is a polygon, it is easy to identify the particles and the hole during automatic image analysis.

[0029] [Aspect 5] In any one of aspects 1 to 4, at least the inner surface of the pore may be coated with a polymer that inhibits cell adhesion. The polymer may be, for example, MPC polymer, PEG, PVA, PMEA, or a mixture thereof.

[0030] In this case, since the polymer coated on the inner surface of the aforementioned hole inhibits cell adhesion, it becomes easy to remove cells or particles from the hole. As for the coating of the aforementioned polymer, the aforementioned polymer can be coated not only on the inner surface of the aforementioned hole, but also on the upper surface of the aforementioned hole partition wall, the inner surface of the aforementioned through hole, and the lower surface of the aforementioned filter body. In this case, it is possible to suppress the problem of cells and the like adhering to the upper surface of the aforementioned hole partition wall, or cells and the like adhering to the lower surface of the aforementioned filter body.

[0031] [Mode 6] In any one of Modes 1 to 5, the maximum inscribed circle diameter of the through hole may be 4 μm or less.

[0032] In this case, it is possible to effectively suppress particles such as cells and beads from passing through the through-holes.

[0033] [Mode 7] In any one of Modes 1 to 6, the hole partition walls and the hole bottoms may be formed of different materials, and the hole bottoms may be formed of a material having a higher strength than the hole partition walls.

[0034] In this case, since the hole bottom is made of a stronger material than the hole partition wall, the overall strength of the hole array filter can be enhanced while mitigating the shrinkage of the hole wall during molding, thereby reducing warping and deformation of the hole array filter.

[0035] [Method 8] It can also be that in any one of methods 1 to 7, the aforementioned hole partition walls and the aforementioned hole bottoms are formed of materials different from each other, the aforementioned hole bottoms are divided into tile shapes corresponding to the shapes of the openings of each of the aforementioned holes and gaps are formed between adjacent aforementioned hole bottoms, and the material forming the aforementioned hole partition walls penetrates into these gaps and solidifies.

[0036] In this case, after the bottom of the aforementioned hole is divided into a state of floor tiles with gaps between them, the aforementioned hole partition walls can be formed and the material forming the aforementioned hole partition walls can be infiltrated and solidified in the aforementioned gaps. Therefore, compared with the case where the hole bottom is formed by a single plate, the warping and deformation caused by curing shrinkage in the hole array filter can be reduced.

[0037] [Mode 9] In any one of Modes 1 to 8, the thickness of the bottom of the hole may be 1 nm to 2 μm.

[0038] In this case, since the bottom of the hole is thin, it is easy to observe the captured object in the hole through the bottom of the hole using an inverted microscope, and the autofluorescence is small according to the amount of thinning, so the adverse effect of autofluorescence on observation can also be reduced.

[0039] [Mode 10] The particle arrangement device involved in Mode 10 of the present invention comprises: a hole array filter as described in any one of the aforementioned modes 1 to 9, and a device body that supports the aforementioned hole array filter and has a sample flow path from the opening side of the aforementioned hole of the aforementioned hole array filter through the aforementioned through hole toward the bottom side of the aforementioned hole.

[0040] According to the particle arrangement device involved in mode 10, it is possible to suppress the passage of particles such as cells or beads to be captured through the through holes while ensuring the flow rate of the hole array filter as a whole. In addition, by making the multiple through holes formed at the bottom of each hole satisfy the aforementioned positional relationship, when one of the aforementioned particles is captured in the hole, the probability that the particle at least partially overlaps with respect to all the aforementioned multiple through holes is high. Therefore, after the first particle is captured, the flow rate of the dispersion medium passing through all the through holes in the hole is moderately reduced, and the particles after the second are suppressed from entering the hole with the flow, and the probability of cells, etc. being guided to adjacent empty holes and captured increases. As a result, the probability of capturing particles one by one in a large number of holes is high, the single cell rate is high, and the fluid permeability is good, and the separation of particles can be stably performed through each hole.

[0041] [Mode 11] The particle capture method according to Mode 11 of the present invention comprises: a step of passing a liquid containing an organic solvent that does not dissolve or deteriorate the hole array filter and the device body through the sample flow path of the particle array device of Mode 10; a step of passing an aqueous solution through the sample flow path to replace the liquid containing the organic solvent with the aqueous solution; and a step of passing an aqueous solution containing particles to be captured by the holes through the sample flow path after replacement with the aqueous solution to capture the particles in the holes. The liquid containing an organic solvent is not limited, and for example, ethanol, an aqueous solution containing 30 to 80% of an organic solvent such as ethanol, and preferably a 70% ethanol aqueous solution.

[0042] According to the particle capture method involved in method 11, the liquid containing the organic solvent is passed through the sample flow path, and then the sample flow path is replaced with an aqueous solution. Then, an aqueous solution containing particles such as cells, components attached to cells, beads of secretions, or components for attaching cells, beads of secretions, etc. is passed through, thereby reducing the flow resistance of the hole array filter, and bubbles are less likely to remain in the holes, making it easy to capture the cells or particles one by one in each hole.

[0043] [Mode 12] In any one of modes 1 to 11, even if the center of the minimum circumscribed circle is moved to the center of any one of the plurality of through holes, the minimum circumscribed circle at least partially overlaps with respect to all the plurality of through holes, and the maximum width of the repeating portion with the smallest repeating area among all the repeating portions is 0.7% or more of the radius of the minimum circumscribed circle. The maximum width is set to a value along a straight line connecting the centers of the minimum circumscribed circles before and after the movement. The maximum width may also be any one of 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, and 30% or more of the radius of the minimum circumscribed circle. When the shape of the through hole is circular and the maximum width of the repeating portion is 0.7% of the radius of the minimum circumscribed circle, the repeating area of ​​the repeating portion with the smallest repeating area among all the repeating portions is equivalent to 0.025% of the opening area of ​​the through hole. Similarly, when the repeating area of ​​the repeating portion is 0.05%, 0.10%, 0.25%, 0.50%, and 1.00% of the opening area of ​​the through hole, the maximum width is respectively equivalent to 1.12%, 1.80%, 3.32%, 5.20%, and 8.26% of the radius of the minimum circumscribed circle. Even when the shape of the through hole is any shape other than a circle, the repeating area of ​​the repeating portion with the smallest repeating area among all the repeating portions may be 0.025%, 0.05%, 0.10%, 0.25%, 0.50%, or 1.00% of the opening area of ​​the through hole.

[0044] Effects of the Invention

[0045] As described above, according to the present invention, it is possible to suppress the passage of particles such as cells or beads to be captured through the through holes, while ensuring the flow rate as a whole of the hole array filter, and by making the multiple through holes formed at the bottom of each hole satisfy the aforementioned positional relationship, so that when one of the aforementioned particles is captured in the hole, the probability that the particle at least partially overlaps with respect to all the aforementioned multiple through holes is high. Therefore, after the first particle is captured, the flow rate of the dispersion medium passing through all the through holes in the hole is moderately reduced, and the second and subsequent particles are suppressed from entering the hole with the flow, and the probability that cells, etc. are guided to adjacent empty holes and captured increases. As a result, the probability of capturing particles one by one in a large number of holes is high, the single cell rate is high, and the liquid permeability is good, and the separation of particles can be stably performed through each hole. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] [ Figure 1 ] is an enlarged plan view of the hole array filter of the first embodiment of the present invention.

[0047] [ Figure 2 ] is an enlarged cross-sectional view of the hole array filter of the first embodiment.

[0048] [ Figure 3 ] (a) and (b) are planar enlarged views of the holes of the hole array filter of the first embodiment, and (c) is a planar enlarged view of the holes of the hole array filter of the comparative example.

[0049] [ Figure 4 ] (a) to (c) are enlarged plan views of the holes of the hole array filter of the second to fourth embodiments.

[0050] [ Figure 5 ] (a) to (c) are enlarged plan views of the holes of the hole array filter of the 5th to 7th embodiments.

[0051] [ Figure 6 ] (a) and (b) are planar enlarged views of the holes of the hole array filter of the 8th and 9th embodiments, and (c) is a planar enlarged view of the holes of the hole array filter of other comparative examples.

[0052] [ Figure 7 ] (a) and (b) are planar enlarged views of the holes of the hole array filter of the 10th embodiment and the 11th embodiment, and (c) is a planar enlarged view of the holes of the hole array filter of other comparative examples.

[0053] [ Figure 8 ] (a) to (c) are enlarged plan views of the holes of the hole array filter of other comparative examples.

[0054] [ Fig. 9 ] (a) and (b) are planar enlarged views of the holes of the hole array filter of the 12th and 13th embodiments, and (c) is a planar enlarged view of the holes of the hole array filter of other comparative examples.

[0055] [ Fig.10 ] (a) and (b) are planar enlarged views of the holes of the hole array filter of the 14th embodiment and the 15th embodiment, and (c) is a planar enlarged view of the holes of the hole array filter of other comparative examples.

[0056] [ Fig.11 ] is an enlarged plan view of the hole array filter of the 16th embodiment.

[0057] [ Fig.12 ] is an enlarged plan view of the hole array filter of the 17th embodiment.

[0058] [ Fig.13 ](a) to (f) are enlarged cross-sectional views showing a method for manufacturing a hole array filter according to an embodiment of the present invention.

[0059] [ Fig.14 ] is a plan view showing a particle arrangement device according to one embodiment of the present invention.

[0060] [ Fig.15 ] is a longitudinal cross-sectional view showing the particle arrangement device of this embodiment.

[0061] [ Fig.16 ] is a diagram for illustrating the conditions for establishing an embodiment of the present invention.

[0062] [ Fig.17 ] is a diagram for illustrating the conditions for establishing an embodiment of the present invention.

[0063] [ Fig.18 ] is a chart used to illustrate the conditions for the establishment of an embodiment of the present invention.

[0064] [ Fig.19 ] is a diagram for illustrating the conditions for establishing an embodiment of the present invention.

[0065] [ Fig. 20 ] is a chart used to illustrate the conditions for the establishment of an embodiment of the present invention.

[0066] [ Fig.21 ] is a table showing the effects of an embodiment of the present invention.

[0067] [ Fig. 22 ] is a chart showing the effects of an embodiment of the present invention.

[0068] [ Fig.23 ] is a table showing the effects of an embodiment of the present invention.

[0069] [ Fig.24 ] is a chart showing the effects of an embodiment of the present invention. DETAILED DESCRIPTION

[0070] Hereinafter, embodiments of the present invention will be described using the drawings.

[0071] [First embodiment]

[0072] Figure 1 and Figure 2 The first embodiment of the hole array filter is shown in an enlarged plan view and an enlarged cross-sectional view. The hole array filter 1 has a flat filter body 2 with a certain thickness, and a large number of holes 3 are formed in the hole forming area on the upper surface of the filter body 2. The so-called hole forming area means an area used as a filter of the filter body 2, and the filter body 2 may also have a support area without holes formed around the hole forming area for supporting the filter.

[0073] The adjacent holes 3 are separated from each other by the hole partition walls 6. Figure 2 As shown, a flat hole bottom 4 is formed at the lower end of each hole 3 to block the lower end of the hole 3. The opening of the hole 3 can be circular or elliptical, but in a preferred embodiment, it is formed as a polygon or a polygon with rounded corners. In particular, in this embodiment, the hole 3 is formed as a regular hexagon, and each hole 3 and the hole partition 6 are arranged in a honeycomb shape. At least the hole bottom 4 is formed of a transparent material so that particles such as cells C in the hole 3 can be observed through the hole bottom 4 using an inverted microscope. The hole partition 6 can be formed of a transparent material, but it can also be formed of a non-transparent material.

[0074] The equivalent circle diameter of the opening of hole 3 is not limited, but is preferably set to be 5 μm or more and 200 μm or less. The so-called equivalent circle diameter refers to the diameter of a circle having an area equal to that of the opening of hole 3. When the equivalent circle diameter of the opening of hole 3 is 5 μm or more, particles such as cells or beads can be captured, and when it is 200 μm or less, particles such as cells or beads can be easily captured one by one or in a suitable constant number in each hole 3. The equivalent circle diameter of the opening of hole 3 is more preferably 10 μm or more and 100 μm or less, and further preferably 15 μm or more and 60 μm or less, so that cells can be captured one by one in hole 3.

[0075] It should be noted that the so-called beads are particles formed of resins, etc., to which components and secretions of cells are attached for analysis. Beads with components for capturing and detecting specific secretions of cells bound to the surface can be placed in the well 3 together with living cells to identify living cells that secrete specific secretions; or beads with components for capturing constituent components of cells bound to the surface and cells can be placed in the well 3, the cells can be destroyed and the contents can be captured on the beads and recovered outside the well 3, and the contents captured by the beads can be studied. In addition, components such as drugs that are released under external stimulation such as light can be bound to the surface of the beads, and cells and beads can be added to the well 3 together, and the drugs can be released under light irradiation to observe the drug response of the cells. In addition, magnetic beads can sometimes be used, and the beads can be attracted by a magnet arranged below the well array filter 1 to capture them in the well 3.

[0076] In each of the hole bottoms 4, a plurality of through holes 8 are formed that reach the lower surface of the filter body 2. In this embodiment, the number of through holes 8 in each hole 3 is two. Figure 3As shown, the through holes 8 are formed in the center of the hole bottom 4 with a spacing W between the centers and vertically penetrate the hole bottom 4. In the present invention, the number of through holes 8 is not limited, and may be 3 or more than 4. When a plurality of through holes 8 are formed in the hole bottom 4, there is an advantage that cells C are not easy to pass through the through holes 8 even when sufficient flow is ensured. Moreover, at least a part of the through holes 8 is formed at a position off-center of the hole bottom 4, so that all the through holes 8 are less likely to be blocked by cells C, and after the cells C are captured, it is easy to make the liquid in the hole 3 flow appropriately through the through holes 8.

[0077] The minimum width of the upper surface opening of the through hole 8 is set to be 1 μm or more and 3.5 μm or less. At the same time, the ratio of the total opening area of ​​the through holes 8 to the area of ​​the hole forming region is set to be 0.5% or more and 3.5% or less. In this way, it is possible to suppress the passage of particles such as cells or beads to be captured through the through holes while ensuring the flow rate of the hole array filter as a whole.

[0078] The minimum width of the upper surface opening of the through hole 8 refers to the width of the narrowest portion of the opening of the through hole 8 when the through hole 8 is viewed from above, for example, the diameter when the through hole 8 is circular, the short diameter when the through hole 8 is elliptical, and the length of the short side when the through hole 8 is rectangular. The minimum width of the upper surface opening of the through hole 8 is more preferably 2 μm or more and 3.5 μm or less, and further preferably 2 μm or more and 2.75 μm or less.

[0079] The total opening area of ​​the through holes 8 is the total value of the opening areas (= flow path cross-sectional area) of all the through holes 8 formed in the hole forming region. When the ratio of the total opening area of ​​the through holes 8 is 0.50% or more, the resistance to the flow of liquid through the through holes 8 becomes moderately small, so it is easy to pass liquid through the hole array filter 1 by the operation of the pipette. When the ratio of the total opening area of ​​the through holes 8 is 3.5% or less, when passing liquid through the hole array filter 1 by the operation of the pipette, the liquid sample does not flow excessively, and the operability is excellent. The ratio of the total opening area of ​​the through holes 8 is more preferably 0.75% or more and 3.20% or less.

[0080] In this embodiment, if Figure 3 As shown in (a) and (b) of FIG. 1 , it is assumed that the minimum circumscribed circle G1 surrounding all the through holes 8 formed in each hole bottom 4 is moved to the center C1 or C2 of any one of the plurality of through holes 8, and the minimum circumscribed circle G2 after the movement still satisfies the positional relationship of at least partially overlapping with respect to all the through holes 8. For example, Figure 3(a) shows a case where the center of the minimum circumscribed circle G1 is moved to the center C2 of one through hole 8, and the left through hole 8 having the center C1 overlaps with the minimum circumscribed circle G2 after the movement at an overlapping portion J. Although not shown, when the center of the minimum circumscribed circle G1 is moved to the center C1 of another through hole 8, the right through hole 8 having the center C2 overlaps with the minimum circumscribed circle G2 after the movement at an overlapping portion J of the same shape.

[0081] Figure 3 In the embodiment shown in (b), the distance between the centers C1-C2 of the through holes 8 is kept constant. Figure 3 In case (a), when the diameter D of the through hole 8 is reduced and the center of the minimum circumscribed circle G1 is moved to the center C2 of one through hole 8, the through hole 8 having the center C1 is connected to the moved minimum circumscribed circle G2 at an overlapping portion J.

[0082] Figure 3 The comparative example shown in (c) is Figure 3 In the case of (b) in which the diameter D of the through hole 8 is further reduced, the through hole 8 having the center C1 and the minimum circumscribed circle G2 after the shift do not overlap and are separated because the diameter D of the through hole 8 is small. Therefore, the relationship required by the present invention is not satisfied.

[0083] like Figure 3 In this way, when the two through holes 8 are circular and have the same diameter, the above relationship is satisfied when the diameter D of the through hole 8 and the distance W between the centers of the through holes 8 satisfy the following formula, and an overlapping portion J is generated.

[0084] D>W / 2

[0085] like Figure 3 As in (a) and (b) in the figure, by making the diameter D of the through hole 8 and the distance W between the centers of the through holes 8 satisfy the above formula, when one particle such as a cell C is captured in the hole 3, the probability that the particle such as a cell C at least partially overlaps with all the multiple through holes 8 is high. Therefore, after the first particle such as a cell C is captured, the flow rate of the dispersion medium passing through all the through holes 8 in the hole 3 is appropriately reduced, and the second and subsequent particles such as a cell C are suppressed from entering the hole 3 with the flow, and the probability that the particles such as a cell C are guided to the adjacent empty hole 3 and captured increases. As a result, the probability of capturing particles such as a cell C one by one in a large number of holes 3 becomes high.

[0086] The maximum width of the repeating portion J with the smallest repeating area among all the repeating portions J may also be greater than 0.7% of the radius of the minimum circumscribed circle G1. The maximum width is set to the value of the straight line connecting the centers C1 and C2 of the minimum circumscribed circles G1 and G2 before and after the movement. The maximum width may also be any one of greater than 0.5%, greater than 1%, greater than 2%, greater than 5%, greater than 10%, greater than 15%, greater than 20%, and greater than 30% of the radius of the minimum circumscribed circle G1. The shape of the through hole 8 is as follows: Figure 3 In the case where the maximum width of the repeating portion J is a circle and is 0.7% of the radius of the minimum circumscribed circle G1, the repeating area of ​​the repeating portion J with the smallest repeating area among all the repeating portions J is equivalent to 0.025% of the opening area of ​​the through hole 8. Similarly, when the repeating area of ​​the repeating portion J is 0.05%, 0.10%, 0.25%, 0.50%, and 1.00% of the opening area of ​​the through hole 8, the maximum width of the repeating portion J with the smallest repeating area is equivalent to 1.12%, 1.80%, 3.32%, 5.20%, and 8.26% of the radius of the minimum circumscribed circle G1, respectively. Even if the shape of the through hole 8 is any shape other than a circle, the repeating area of ​​the repeating portion J with the smallest repeating area among all the repeating portions J may be 0.025%, 0.05%, 0.10%, 0.25%, 0.50%, or 1.00% of the opening area of ​​the through hole 8. The description of this paragraph is used in all the following embodiments.

[0087] on the other hand, Figure 3 In the comparative example shown in (c), since the diameter D of the through hole 8 and the distance W between the centers of the through holes 8 do not satisfy the formula (1), and the through hole 8 having the center C1 does not overlap with the minimum circumscribed circle G2 after the movement, when a cell C or other particle is captured in the hole 3, the probability that the cell C or other particle does not overlap with any of the through holes 8 is high. Therefore, after the first cell C or other particle is captured, the flow rate of the dispersion medium passing through the through hole 8 that does not overlap with the cell C or other particle is still large, and the second and subsequent cell C or other particles are likely to enter the hole 3 along with the flow. As a result, the single cell rate is reduced.

[0088] The diameter of the minimum circumscribed circle G1 is not limited, but is preferably 3 μm to 30 μm, and more preferably 5 μm to 20 μm. The minimum width of the portion separating adjacent through holes 8 is not limited, but is preferably 0.5 μm to 10 μm, and is particularly preferred when it is 1 μm to 3 μm because the through holes 8 are contained inside the small minimum circumscribed circle G1.

[0089] The maximum thickness B of the hole partition wall 6 in the horizontal direction (see Figure 1) is not limited, and is preferably 1 μm to 20 μm. By making the maximum thickness B greater than 1 μm, the strength of the hole partition wall 6 can be made appropriate, for example, when a pipette is pressed against the upper surface of the hole partition wall 6 to absorb cells C, the hole partition wall 6 can be prevented from collapsing. In addition, by making the maximum thickness B less than 20 μm, it is possible to suppress the cells, beads, and particles waiting to be captured from remaining on the hole partition wall 6, and the density of the hole 3 can also be increased. In addition, when the pipette is pressed against, appropriate flexibility can be brought to the hole partition wall 6, and damage to the front end of the pipette can be suppressed. The maximum thickness B of the hole partition wall 6 is preferably 2 μm to 20 μm, and more preferably 2 μm to 12 μm.

[0090] The ratio of the total opening area of ​​the holes 3 to the area of ​​the hole forming region is not limited, but is preferably set to 40% or more. When the ratio is 40% or more, the proportion of particles such as cells supplied to the hole array filter 1 that are captured in the holes 3, that is, the particle capture rate, can be sufficiently increased. The ratio of the total opening area of ​​the holes 3 to the area of ​​the hole forming region is preferably 40% or more and 95% or less, and more preferably 40% or more and 75% or less. The high ratio is related to the thinness of the hole partition wall 6.

[0091] The ratio of the depth of the hole 3 to the maximum thickness B of the hole partition wall 6 in the horizontal direction is not limited, and the depth of the hole 3 is preferably more than twice the thickness B of the hole partition wall 6. When the depth of the hole 3 is more than twice the thickness B of the hole partition wall 6, it is possible to suppress the particles such as cells C captured in the hole 3 from escaping to the outside of the hole 3 due to the interference of the liquid flow, so that once the particles are captured, they can be stably maintained in the hole 3. More preferably, the depth of the hole 3 is more than 2 times and less than 40 times the thickness B of the hole partition wall 6, and further preferably more than 2 times and less than 15 times.

[0092] The top view shape of the through hole 8 is circular in this embodiment, but the present invention is not limited to a circle, and may be an ellipse, a triangle, a quadrangle, a pentagon, a hexagon or other polygon, a polygon with rounded corners, a rectangle, a star, a slit, a dumbbell, an H-shape, or any other irregular shape. Modifications are described below.

[0093] The top view shape of the hole 3 is not limited in the present invention, and may be circular or elliptical. If it is a polygon or a polygon with rounded corners, it is easy to identify particles and holes 3 during automatic image analysis. Among the polygons, it is preferably any one of a triangle, a quadrangle or a hexagon as shown in the figure. When it is a triangle, a quadrangle or a hexagon, holes 3 of the same shape can be regularly arranged to increase the arrangement density of the holes 3. Among them, when it is an equilateral triangle, a regular quadrangle, or a regular hexagon, the arrangement density of the holes 3 can be increased by a geometrically simple arrangement, so it is preferred. About the modified example, it is described below.

[0094] The hole partition wall 6 and the hole bottom 4 may be formed of the same material, but preferably of different materials, and the hole bottom 4 may be formed of a material having higher strength than the hole partition wall 6. In this case, the overall strength of the hole array filter 1 can be improved, and the shrinkage of the hole bottom 4 during molding can be alleviated, thereby reducing the warping and deformation of the hole array filter 1. In order to focus on the entire observation range when observing the particles captured in the hole 3 using an inverted microscope, it is important that there is no warping in the hole array filter 1.

[0095] The material of the hole partition wall 6 and the hole bottom 4 is not limited, and various photoresists can be used to realize the microstructure. It is preferred that the hole bottom 4 be made of a material with higher strength than the hole partition wall 6 by changing the type and composition of the photoresist and / or changing the exposure conditions. When the hole partition wall 6 and the hole bottom 4 are formed of photoresist, for example, the partition wall resist 6A (see Fig.13 ) is formed by a photoresist with a relatively small amount of multifunctional epoxy, forming a bottom resist 4A of the hole bottom 4 (see Fig.13 ) is formed by a photoresist having a relatively large amount of multifunctional epoxy, which can make the strength of the material of the hole bottom 4 higher than the strength of the material of the hole partition 6. In this case, the autofluorescence of the material of the hole partition 6 becomes relatively weaker, and the autofluorescence of the material of the hole bottom 4 becomes relatively stronger, but since the thickness of the hole bottom 4 is small, it will not have a negative impact on the observation based on the inverted microscope.

[0096] In the present invention, the method for forming the hole bottom 4 and the hole partition 6 is not limited. In order to relieve the stress of the hole array filter 1, it is preferably made into the following structure: the hole partition 6 and the hole bottom 4 are formed separately from each other, the hole bottom 4 is divided into a floor tile shape corresponding to the shape of the opening of each hole 3, and gaps are formed between adjacent hole bottoms 4. The material forming the hole partition 6 intrudes into these gaps and solidifies. An example of a specific manufacturing method is described below. By forming the hole array filter 1 with a structure in which the hole bottom 4 is divided into such a floor tile shape and the hole partition 6 intrudes into the gap of the hole bottom 4, it is possible to further suppress the warping and deformation in the hole array filter 1 caused by the solidification shrinkage of the hole bottom 4.

[0097] In the present invention, although it is not necessary, a polymer that inhibits cell adhesion can be pre-coated on at least a portion of the inner surface of the hole 3. In this case, since the polymer coated on the inner surface of the hole 3 inhibits the adhesion of the cell C, it becomes easy to remove the cell C or particles from the hole 3. As such a polymer, for example, an MPC polymer (2-methacryloyloxyethyl phosphorylcholine polymer), PEG (polyethylene glycol), PVA (polyvinyl alcohol), PMEA (poly (2-methoxyethyl acrylate)), and a mixture thereof can be used. The coating position of the polymer can also be not only the inner surface of the hole 3, but also the upper surface of the hole partition wall 6, the inner surface of the through hole 8, and the lower surface of the filter body 2. The aforementioned polymer is also coated. In this case, the problem of cells and the like adhering to the upper surface of the hole partition wall 6 or cells and the like adhering to the lower surface of the filter body 2 can be avoided.

[0098] In the present invention, although not limited, the thickness of the well bottom 4 may be 1 nm or more and 2 μm or less. In this case, since the well bottom 4 is sufficiently thin, it is easy to observe the captured particles such as cells C in the well 3 through the well bottom 4 using an inverted microscope, and the autofluorescence of the well bottom 4 is suppressed to a small extent according to the amount of thinning of the well bottom 4, and the adverse effect on observation caused by the autofluorescence can also be reduced. The thickness of the well bottom 4 is more preferably 100 nm or more and 2 μm or less, and further preferably 500 nm or more and 1.5 μm or less.

[0099] According to the hole array filter 1 including the above-mentioned structure, since the minimum width of the through hole 8 and the ratio of the total opening area of ​​the through holes 8 to the area of ​​the hole forming region are appropriate, it is possible to suppress the passage of the particles such as the cells C to be captured through the through holes 8 while ensuring the flow rate of the hole array filter 1 as a whole. In addition, by making the plurality of through holes 8 formed in the bottom 4 of each hole satisfy the above-mentioned positional relationship (formula (1)), when a particle such as a cell C is captured in the hole 3, there is a high probability that the particle at least partially overlaps with all the plurality of through holes 8.

[0100] Therefore, if Figure 2 As shown, after the first particle is captured, the flow rate of the dispersion medium passing through all the through holes 8 in the hole 3 is appropriately reduced, which inhibits the second and subsequent particles from entering the same hole 3 along with the flow. On the contrary, the probability of cells C and the like being guided to the adjacent empty hole 3 and captured increases. As a result, the probability of capturing particles one by one in a large number of holes 3 is high, that is, the capture rate and the single cell rate are both high, and the liquid permeability is good, and the excellent effect of being able to efficiently and stably separate particles through each hole is achieved.

[0101] In addition, in this embodiment, since the hole partition wall 6 between the holes 3 is sufficiently thin, particles such as cells and beads are not easily retained on the hole partition wall 6, and the probability of multiple particles entering one hole 3 can be reduced by appropriately setting the opening diameter, and particles can be stably captured in the hole 3 having a sufficient depth compared to the opening diameter. In addition, since the hole 3 is polygonal, it is easy to identify particles and the hole 3 during automatic image analysis, and since the hole 3 can be arranged at a high density, it has the advantage of being able to screen a large number of particles at one time.

[0102] [Second Embodiment of Hole Array Filter]

[0103] Figure 4 A modified example of the shape of the through-holes 8 is shown when the number of the through-holes 8 in each hole bottom 4 of the hole array filter 1 is two.

[0104] Figure 4 (a) is the second embodiment, in which the through holes 8 are formed into rectangles and are arranged in parallel in such a way that two sides of each other are arranged on the same line. The corners of the rectangle are rounded, but they can also be unrounded. The minimum circumscribed circle G1 passes through the two vertices on the outside of each through hole 8 and circumscribes each through hole 8. The centers C1 and C2 of the through holes 8 are defined as the centers of the two points where the opening edge of the through hole 8 and the diameter S of the minimum circumscribed circle G1 extending in the directions opposite to each other of the through holes 8 intersect. The same is true in other embodiments.

[0105] In the second embodiment, it is also envisioned that the minimum circumscribed circle G1 surrounding all the through holes 8 formed in the bottom 4 of each hole is moved to the center C1 or C2 of any one of the through holes 8. The minimum circumscribed circle G2 after the move satisfies a positional relationship of at least partial overlap relative to all the through holes 8. Figure 4 (a) shows the case where the center of the minimum circumscribed circle G1 is moved to the center C2 of one through hole 8, and the left through hole 8 having the center C1 overlaps with the minimum circumscribed circle G2 after the movement at a repeating portion J. Thus, the same effect as the first embodiment can be obtained, and since the through hole 8 is rectangular, the diameter of the maximum inscribed circle is smaller in terms of the opening area than in the case of a circular through hole, and the possibility of the cell C etc. passing through is reduced. The other configurations of the second embodiment are the same as those of the first embodiment, and the above description is referred to.

[0106] [Third Embodiment of Hole Array Filter]

[0107] Figure 4(b) is a third embodiment, in which the through holes 8 are formed into an ellipse or an oblong shape, and are arranged in parallel in such a way that their short diameters (may also be long diameters) are arranged on the same line. The minimum circumscribed circle G1 passes through a point on the outside of each through hole 8 and circumscribes each through hole 8. The centers C1 and C2 of the through holes 8 are the centers of the two points where the opening edge of the through hole 8 and the diameter S of the minimum circumscribed circle G1 extending in the direction opposite to each other of the through holes 8 intersect.

[0108] In the third embodiment, it is also envisioned that the minimum circumscribed circle G1 surrounding all the through holes 8 formed in the bottom 4 of each hole is moved to the center C1 or C2 of any one of the through holes 8. The minimum circumscribed circle G2 after the move satisfies a positional relationship of at least partial overlap relative to all the through holes 8. Figure 4 (b) shows the case where the center of the minimum circumscribed circle G1 is moved to the center C2 of one through hole 8, and the left through hole 8 having the center C1 overlaps with the minimum circumscribed circle G2 after the movement at the overlapping portion J. Thus, the same effect as the first embodiment can be obtained, and since the through hole 8 is an ellipse, the diameter of the maximum inscribed circle is smaller in terms of the opening area than in the case of a circle, and the possibility of the cell C etc. passing through is reduced. The other configurations of the third embodiment are the same as those of the first embodiment, and the above description is used.

[0109] [Fourth embodiment of hole array filter]

[0110] Figure 4 (c) is the fourth embodiment, in which the through hole 8 is formed into a triangle, preferably an isosceles triangle, and the bases are arranged parallel to each other. The corners of the triangle are rounded, but they may not be rounded. The minimum circumscribed circle G1 passes through the vertex of the outside of each through hole 8 and circumscribes each through hole 8. The centers C1 and C2 of the through hole 8 are the centers of the two points where the opening edge of the through hole 8 and the diameter S of the minimum circumscribed circle G1 extending in the direction opposite to each other of the through holes 8 intersect.

[0111] In the fourth embodiment, it is also envisioned that the minimum circumscribed circle G1 surrounding all the through holes 8 formed in the bottom 4 of each hole is moved to the center C1 or C2 of any one of the through holes 8. The minimum circumscribed circle G2 after the move satisfies a positional relationship of at least partial overlap relative to all the through holes 8. Figure 4(c) in the figure shows a case where the center of the minimum circumscribed circle G1 is moved to the center C2 of one through hole 8, and the left through hole 8 having the center C1 overlaps with the minimum circumscribed circle G2 after the movement at a repeating portion J. Thus, the same effect as in the first embodiment can be obtained, and since the through hole 8 is a triangle, the diameter of the maximum inscribed circle is smaller in terms of the opening area than in the case of a circle, and the possibility of the passage of cells C etc. is reduced. In addition, the configuration is performed in such a way that the bases of the triangles are opposite, so it is easy to increase the area of ​​the repeating portion J, and the flow path blockage rate when cells C etc. are placed can be increased. The other configurations of the fourth embodiment are the same as those of the first embodiment, and the above description is used.

[0112] [Fifth Embodiment of Hole Array Filter]

[0113] Figure 5 A modification example is shown in which the shape of the through-holes 8 is made elongated when the number of the through-holes 8 in each hole bottom 4 of the hole array filter 1 is two.

[0114] Figure 5 (a) is the fifth embodiment, in which the through holes 8 are formed into elongated rectangles and are arranged in parallel in such a way that the two short sides are arranged on the same line. The corners of the rectangle are rounded, but they may not be rounded. The minimum circumscribed circle G1 passes through the two vertices on the outside of each through hole 8 and circumscribes each through hole 8. The centers C1 and C2 of the through holes 8 are the centers of the two points where the opening edge of the through hole 8 and the diameter S of the minimum circumscribed circle G1 extending in the directions opposite to each other of the through holes 8 intersect.

[0115] In the fifth embodiment, it is also envisioned that the minimum circumscribed circle G1 surrounding all the through holes 8 formed in the bottom 4 of each hole is moved to the center C1 or C2 of any one of the through holes 8. The minimum circumscribed circle G2 after the move satisfies a positional relationship of at least partial overlap relative to all the through holes 8. Figure 5 (a) shows the case where the center of the minimum circumscribed circle G1 is moved to the center C2 of one through hole 8, and the left through hole 8 having the center C1 overlaps with the minimum circumscribed circle G2 after the movement at a repeating portion J. Thus, the same effect as the first embodiment can be obtained, and since the through hole 8 is an elongated rectangle, the diameter of the maximum inscribed circle is smaller in terms of the opening area than in the case of a circle, and the possibility of the cell C etc. passing through is reduced. In addition, the configuration is performed in such a way that the long sides of the rectangle are opposite to each other, so it is easy to increase the area of ​​the repeating portion J, and the flow path blockage rate when the cell C etc. is placed can be increased. The other configurations of the fifth embodiment are the same as those of the first embodiment, and the above description is used.

[0116] [Sixth embodiment of hole array filter]

[0117] Figure 5 (b) is the sixth embodiment, in which the through holes 8 are formed into a semicircle and arranged opposite to each other with their chords parallel to each other. The corners of the semicircle are rounded, but they may not be rounded. The minimum circumscribed circle G1 passes through the vertex of the outer side of each through hole 8 and circumscribes each through hole 8. The centers C1 and C2 of the through holes 8 are the centers of the two points where the opening edge of the through hole 8 and the diameter S of the minimum circumscribed circle G1 extending in the direction in which the through holes 8 are opposite to each other intersect.

[0118] In the sixth embodiment, it is also envisioned that the minimum circumscribed circle G1 surrounding all the through holes 8 formed in the bottom 4 of each hole is moved to the center C1 or C2 of any one of the through holes 8. The minimum circumscribed circle G2 after the move satisfies a positional relationship of at least partial overlap relative to all the through holes 8. Figure 5 (b) shows the case where the center of the minimum circumscribed circle G1 is moved to the center C2 of one through hole 8, and the left through hole 8 having the center C1 overlaps with the minimum circumscribed circle G2 after the movement at a repeating portion J. Thus, the same effect as the first embodiment can be obtained, and since the through hole 8 is semicircular, the diameter of the maximum inscribed circle is smaller in terms of the opening area than in the case of a circle, and the possibility of the cell C etc. passing through is reduced. In addition, the arrangement is performed in such a way that the chords of the semicircle are opposite to each other, so it is easy to increase the area of ​​the repeating portion J, and the flow path blockage rate when the cell C etc. is placed can be increased. The other configurations of the sixth embodiment are the same as those of the first embodiment, and the above description is used.

[0119] [Seventh embodiment of hole array filter]

[0120] Figure 5 (c) is the seventh embodiment, in which the through holes 8 are formed into a semicircle, and are arranged opposite to each other with their chords parallel to each other, and the chords are formed into an arc shape, forming a so-called banana shape. The corners of the semicircle are rounded, but they can also be unrounded. The minimum circumscribed circle G1 passes through the vertices of the outside of each through hole 8 and circumscribes each through hole 8. The centers C1 and C2 of the through holes 8 are the centers of the two points where the opening edge of the through hole 8 and the diameter S of the minimum circumscribed circle G1 extending in the direction opposite to each other of the through holes 8 intersect.

[0121] In the seventh embodiment, it is also envisioned that the minimum circumscribed circle G1 surrounding all the through holes 8 formed in the bottom 4 of each hole is moved to the center C1 or C2 of any one of the through holes 8. The minimum circumscribed circle G2 after the move satisfies a positional relationship of at least partial overlap relative to all the through holes 8. Figure 5(c) in the figure shows a case where the center of the minimum circumscribed circle G1 is moved to the center C2 of one through hole 8, and the left through hole 8 having the center C1 overlaps with the minimum circumscribed circle G2 after the movement at a repeating portion J. Thus, the same effect as in the first embodiment can be obtained, and since the through hole 8 is banana-shaped, the diameter of the maximum inscribed circle is smaller in terms of the opening area than in the case of a circular shape, and the possibility of the cell C etc. passing through is reduced. In addition, the concave sides of the banana shape are arranged so as to be opposite to each other, so that it is easy to increase the area of ​​the repeating portion J, which can increase the flow path blockage rate when the cell C etc. is placed, and can also increase the area where the cell C can be placed. The other configurations of the seventh embodiment are the same as those of the first embodiment, and the above description is used.

[0122] [Eighth Embodiment of Hole Array Filter]

[0123] Figure 6 The case where a total of three circular through holes 8 are formed in the center of the hole bottom 4 is shown.

[0124] Figure 6 (a) is the eighth embodiment, in which a total of three circular through holes 8 are formed in the center of the hole bottom 4. The through holes 8 can be formed at the vertices of an equilateral triangle, or at the vertices of an isosceles triangle, a right triangle, or a scalene triangle other than an equilateral triangle. When arranged in a triangle, the center of the hole bottom 4 is preferably located inside the triangle, preferably at the center of gravity. The other configurations of the eighth embodiment are the same as those of the first embodiment, and the aforementioned description is used.

[0125] In the eighth embodiment, it is also envisioned that the minimum circumscribed circle G1 surrounding all the through holes 8 formed in the bottom 4 of each hole is moved to the center of any one of the three through holes 8. The minimum circumscribed circle G2 after the move satisfies a positional relationship of at least partial overlap relative to all the through holes 8. Figure 6 (a) shows a case where the center of the minimum circumscribed circle G1 is moved to the center C2 of one through hole 8, and the two through holes 8 on the right side having the center C1 overlap with the minimum circumscribed circle G2 after the movement at the overlapping portion J. Thus, the same effect as that of the first embodiment can be obtained. In addition, there is an advantage that a relatively large area for mounting cells C can be ensured in the center of the three through holes 8.

[0126] If the diameter D of the through hole 8 of the eighth embodiment is reduced, Figure 6 When the center of the minimum circumscribed circle G1 is moved to the center C2 of one through hole 8 as in (b) in FIG. 1 , the two through holes 8 on the right side having the center C1 are connected to the minimum circumscribed circle G2 after the movement at the overlapping portion J. If the minimum circumscribed circle G1 is further reduced, the result is as follows: Figure 6When the center of the minimum circumscribed circle G1 is moved to the center C2 of one through hole 8 as in (c) in FIG. 1 , the two through holes 8 on the right side having the center C1 no longer overlap with the minimum circumscribed circle G2 after the movement. Therefore, Figure 6 (c) in the figure is a comparative example.

[0127] [Ninth embodiment of hole array filter]

[0128] Figure 7 The case where a total of four through holes 8 are formed in the center of the hole bottom 4 is shown.

[0129] Figure 7 (a) is the 9th embodiment, in which a total of 4 through holes 8 are formed in the center of the hole bottom 4. Each through hole 8 can be formed at the vertex position of a square, or at the vertex position of a rectangle, trapezoid, or scalene quadrilateral other than a square. When arranged in a quadrilateral, the center of the hole bottom 4 is preferably located inside the quadrilateral, preferably at the center of gravity. In addition, three through holes 8 can be arranged at the vertex positions of a triangle, and the fourth through hole 8 can be arranged on the inside of the triangle or on any one of the three sides. In this case, the triangle can be an equilateral triangle, or an isosceles triangle other than an equilateral triangle, a right triangle, or a scalene triangle. When arranged in a triangle, the center of the hole bottom 4 is preferably located inside the triangle, preferably at the center of gravity. The other configurations of the 9th embodiment are the same as those of the 1st embodiment, and the aforementioned description is used.

[0130] In the ninth embodiment, it is also envisioned that the minimum circumscribed circle G1 surrounding all the through holes 8 formed in the bottom 4 of each hole is moved to the center of any one of the four through holes 8. The minimum circumscribed circle G2 after the move satisfies a positional relationship of at least partial overlap relative to all the through holes 8. Figure 7 (a) shows a case where the center of the minimum circumscribed circle G1 is moved to the center C2 of the through hole 8 on the right, and the three through holes 8 on the left overlap with the minimum circumscribed circle G2 after the movement at the overlapping portion J. Thus, the same effect as that of the first embodiment can be obtained. In addition, there is an advantage that a relatively large area for mounting cells C can be ensured in the center of the three through holes 8.

[0131] If the diameter D of the through hole 8 of the ninth embodiment is reduced, Figure 7 When the center of the minimum circumscribed circle G1 is moved to the center C2 of one through hole 8 as in (b) in FIG. 1 , the two through holes 8 on the right side having the center C1 are connected to the minimum circumscribed circle G2 after the movement at the overlapping portion J. If the minimum circumscribed circle G1 is further reduced, as shown in FIG. Figure 7When the center of the minimum circumscribed circle G1 is moved to the center C2 of a through hole 8 as in (c) in FIG. 1 , the through hole 8 on the left side having the center C1 and the minimum circumscribed circle G2 after the movement no longer overlap. Figure 7 (c) in the figure is a comparative example.

[0132] [Other Comparative Examples of Hole Array Filters]

[0133] Figure 8 This is an example in which a total of seven through holes 8 are formed in the center of the hole bottom 4 .

[0134] Figure 8 In (a) to (c), six through holes 8 are formed at the vertices of the regular hexagon, and the seventh through hole 8 is formed at the center of the regular hexagon. If the through holes 8 have the same opening diameter, then Figure 8 When the center of the minimum circumscribed circle G1 is moved to the center C2 of one through hole 8, the through hole 8 on the left side having the center C1 cannot overlap with the minimum circumscribed circle G2 after the movement. Figure 8 (a) to (c) are all comparative examples.

[0135] [Tenth embodiment of hole array filter]

[0136] Fig. 9 The 10th embodiment of the hole array filter 1 is shown. In this example, a through hole 8 having a planar shape (dumbbell shape) formed by connecting two circular through holes 8 with a slit 8A is formed in the center of the hole bottom 4. In the case of a through hole 8 having such a shape, when the maximum inscribed circle diameter of the through hole 8 is less than 4 μm, the opening area (cross-sectional area of ​​the flow path) of the through hole 8 can be ensured to be larger according to the amount of the slit 8A, and the flow rate of the through hole 8A can be increased while preventing particles such as cells and beads from passing through the through hole 8A. In this case, it is preferred that the center of the hole bottom 4 is located on the through hole 8A. The other configurations of the 10th embodiment are the same as those of the first embodiment, and the above description is used.

[0137] In the tenth embodiment, it is also envisioned that the minimum circumscribed circle G1 surrounding all the through holes 8 formed in the bottom 4 of each hole is moved to the center of any one of the through holes 8. The minimum circumscribed circle G2 after the move satisfies a positional relationship of at least partial overlap relative to all the through holes 8. Fig. 9 (a) shows a case where the center of the minimum circumscribed circle G1 is moved to the center C2 of the through hole 8 on the right side, and the through hole 8 on the left side overlaps with the minimum circumscribed circle G2 after the movement at an overlapping portion J. Thus, the same effect as the first embodiment can be obtained.

[0138] If the diameter D of the through hole 8 of the tenth embodiment is reduced, Fig. 9 When the center of the minimum circumscribed circle G1 is moved to the center C2 of one through hole 8 as in (b) in FIG. 1 , the two through holes 8 on the right side having the center C1 are connected to the minimum circumscribed circle G2 after the movement at the overlapping portion J. If the minimum circumscribed circle G1 is further reduced, as shown in FIG. Fig. 9 When the center of the minimum circumscribed circle G1 is moved to the center C2 of a through hole 8 as in (c) in FIG. 1 , the through hole 8 on the left side having the center C1 and the minimum circumscribed circle G2 after the movement no longer overlap. Fig. 9 (c) in the figure is a comparative example.

[0139] [11th embodiment of hole array filter]

[0140] Fig.10 (a) and (b) in the figure show the eleventh embodiment of the hole array filter 1. In this example, three slits 8A extending radially from the center and through holes 8 formed at the ends of the slits 8A are formed in the center of the hole bottom 4. In the case of the through holes 8 connected by such slits 8A, even if the maximum inscribed circle diameter is small, it is possible to ensure a large opening area (flow path cross-sectional area) corresponding to the amount of the slits 8A. In this case, it is preferred that the center of the hole bottom 4 is located at the center of the radial slits 8A. The other configurations of the eleventh embodiment are the same as those of the first embodiment, and the above description is used.

[0141] In the 11th embodiment, it is also envisioned that the minimum circumscribed circle G1 surrounding all the through holes 8 formed in the bottom 4 of each hole is moved to the center of any one of the through holes 8. The minimum circumscribed circle G2 after the move satisfies a positional relationship of at least partial overlap relative to all the through holes 8. Fig.10 (a) and (b) show the case where the center of the minimum circumscribed circle G1 is moved to the center C2 of the through hole 8 on the right side, and the through hole 8 on the left side overlaps with the minimum circumscribed circle G2 after the movement at an overlapping portion J. Thus, the same effect as the first embodiment can be obtained.

[0142] If the diameter D of the through hole 8 of the eleventh embodiment is further reduced, Fig.10 When the center of the minimum circumscribed circle G1 is moved to the center C2 of a through hole 8 as in (c) in FIG. 1 , the through hole 8 on the left side having the center C1 and the minimum circumscribed circle G2 after the movement no longer overlap. Fig.10 (c) in the figure is a comparative example.

[0143] [Twelfth embodiment of hole array filter]

[0144] Fig.11The 12th embodiment of the hole array filter 1 is shown, in which the hole partition walls 6 are formed in a square grid shape, and the hole bottoms 4 are formed in square shapes. The hole partition walls 6 are not limited to the square grid shape, but may be a rhombus grid shape or a rectangular grid shape. Preferably, the shapes of the holes 3 are the same as each other. The holes 3 may also be arranged differently from each other in a row. The through holes 8 may be the same as any one of the first to eleventh embodiments. The other structures of the 12th embodiment may be the same as those of the first embodiment, and the above description is used. In the 12th embodiment, the same effects as those in the first to eleventh embodiments can also be obtained.

[0145] [Thirteenth embodiment of hole array filter]

[0146] Fig.12 The 13th embodiment of the hole array filter 1 is shown, and in this example, it is characterized in that the hole partition wall 6 is formed into a so-called truss shape in which equilateral triangles are alternately arranged in reverse, and the hole bottom 4 is each formed into an equilateral triangle. The hole partition wall 6 is not limited to an equilateral triangle, and can also be an isosceles triangle, a right triangle shape, or an unequal triangle. Preferably, the shapes of the holes 3 are the same as each other. The through hole 8 can be the same as any one of the first to eleventh embodiments. The other structures of the 13th embodiment can be the same as those of the first embodiment, and the above description is cited. In this 13th embodiment, the same effects as those in the first to twelfth embodiments can also be obtained.

[0147] [One embodiment of a method for manufacturing a hole array filter]

[0148] Fig.13 (a) to (f) are cross-sectional views showing an example of a method for manufacturing the hole array filter 1. First, Fig.13 As shown in (a) of FIG. 1 , a sacrificial film 12 insoluble in a resist solvent and a developer is formed on a substrate 10 formed of a Si wafer or the like. The sacrificial film 12 can be formed of, for example, a styrene-based elastomer, dextrin, or dextran.

[0149] Then, if Fig.13 As shown in (b) in FIG. 1 , a negative bottom resist 4A is formed on the sacrificial film 12 with a certain thickness required as the bottom 4 of the hole. The bottom resist 4A is exposed to light in a predetermined pattern by photolithography, and further developed, as shown in FIG. Fig.13As shown in (c) in the figure, the hole bottoms 4 having the through holes 8 are formed into a polygonal floor tile shape, and are arranged with gaps 14 between them. The width of the gaps 14 is set to be slightly smaller than the thickness of the hole partition 6. In addition, the gaps 14 between the hole bottoms 4 can also be set to be partially bridged by thin beams (bridges). In this case, the beams can be used for positioning so that the arrangement of the hole bottoms 4 is not disordered.

[0150] Then, if Fig.13 As shown in (d) in the figure, a partition wall resist 6A covering the hole bottom 4 is formed with a thickness required for the hole partition wall 6. In this case, for example, it is preferred that the partition wall resist 6A is formed by a photoresist having a relatively small amount of multifunctional epoxy, and the bottom resist 4A is formed by a photoresist having a relatively large amount of multifunctional epoxy, so that the strength of the material of the hole bottom 4 is relatively higher than the strength of the material of the hole partition wall 6. In this case, as a secondary effect, although the autofluorescence of the material of the hole partition wall 6 is relatively weak and the autofluorescence of the material of the hole bottom 4 is relatively strong, since the thickness of the hole bottom 4 is small, it will not have an adverse effect on observation using an inverted microscope. The partition wall resist 6A is exposed to light in a prescribed pattern using a photolithography method, and further developed, thereby Fig.13 As shown in (e) in FIG. 1 , the hole partition walls 6 are formed. Thus, the partition wall resist 6A intrudes into the interior of the gap 14, and the inside of the gap 14 is insolubilized by exposure, thereby connecting the hole bottoms 4 to each other.

[0151] Next, the hole bottom 4 and the hole partition 6 are heated as a whole to, for example, 180°C to further thermally cure the resist, and then the sacrificial film 12 is dissolved with a solvent that will not deteriorate the resist, and the substrate 10 is peeled off from the hole bottom 4, thereby completing the hole array filter 1 as shown in the above-mentioned first to thirteenth embodiments.

[0152] According to the manufacturing method, the hole array filter 1 having holes 3 with high shape accuracy can be efficiently manufactured. In addition, according to the manufacturing method, after the hole bottom 4 is divided into a tile-like state with gaps 14 formed between each other, the hole partition walls 6 are formed and the material forming the hole partition walls 6 is infiltrated into the gaps 14 and solidified, thereby reducing the warping and deformation caused in the hole array filter 1 due to the solidification shrinkage of the hole bottom 4.

[0153] [Embodiments of Particle Array Device]

[0154] Fig.14 and Fig.15The present invention shows a plan view and a longitudinal cross-sectional view of an embodiment of a particle arrangement device 40. The particle arrangement device 40 has a rectangular bottom plate portion 42 and a wall portion 44 formed in the central portion of the bottom plate portion 42. The wall portion 44 has a square frame-shaped peripheral wall portion rising from the bottom plate portion 42, and two partitions rising and formed parallel to each other on the inner side of the peripheral wall portion, and the inner side of the peripheral wall portion is divided into a first chamber 46, a second chamber 48, and a third chamber 50 by the partition. A cylindrical opening portion 52 is formed at the bottom of the first chamber 46, and a cylindrical opening portion 54 is also formed at the bottom of the third chamber 50.

[0155] The bottom of the second chamber 48 is open. Fig.15 As shown, the hole array filter 1 opens a tiny flow path 56 from the bottom plate portion 42 and is fixed to the lower end of the wall portion 44. A certain gap is also opened between the bottom of the first chamber 46 and the bottom plate portion 42, and between the bottom of the third chamber 50 and the bottom plate portion 42 to form a flow path 56. Thus, by placing a liquid sample containing particles such as cells C and beads into the second chamber 48, the liquid is made to flow out from the opening 52 or the opening 54, so that the cells C, beads and other particles can be captured in the holes 3 of the hole array filter 1. That is, a sample flow path is formed that reaches the flow path 56 via the second chamber 48, the hole array filter 1, and the through hole 8. In addition, a liquid reagent can also be added from the opening 52 or the opening 54 to react with the particles such as cells C and beads in the second chamber 48.

[0156] [Embodiment of Particle Capture Method]

[0157] One embodiment of the particle capture method involved in the present invention comprises: a process of passing a liquid containing an organic solvent that does not dissolve or deteriorate the hole array filter 1 and the device body 40 through a sample flow path of the hole array filter 1 of the particle arrangement device 40, that is, a sample flow path from the opening side of the hole 3 through the through hole 8 to the bottom side of the hole 3; a process of passing an aqueous solution through the aforementioned sample flow path to replace the aforementioned liquid containing the organic solvent with the aforementioned aqueous solution; and a process of passing an aqueous solution containing particles to be captured by the hole 3 through the aforementioned sample flow path to capture the particles in the hole 3.

[0158] Specifically, a liquid such as an organic solvent such as ethanol that is water-soluble and has low interfacial tension, or an aqueous solution containing 30 to 80% of an organic solvent such as ethanol is supplied to the second chamber 48 of the particle arrangement device 40, so that the liquid containing the organic solvent is passed through the hole array filter 1. As the aforementioned liquid, for example, a 70% ethanol aqueous solution is also suitable. Thus, the through hole 8 is also filled with the organic solvent. Next, an aqueous solution such as a buffer solution such as a phosphate buffer solution (PBS) is added to the second chamber 48, and the liquid containing the organic solvent is sucked from the opening 52 and / or the opening 54. This operation is repeated multiple times to replace the liquid containing the organic solvent in the second chamber 48, the through hole 8, and the flow path 56 with the aqueous solution of the phosphate buffer solution.

[0159] Next, a sample liquid in which particles such as cells C and / or beads are dispersed in a phosphate buffer or the like is supplied to the second chamber 48, and the phosphate buffer is aspirated from the opening 52 and / or the opening 54, thereby capturing cells C and / or beads in each hole 3 one by one or in an almost constant number.

[0160] Next, a reagent that can bind to the target cell C or beads attached with cell secretions is added from the second chamber 48 or the openings 52, 54. After the target is labeled, the target is identified using an inverted microscope, and the target cell C or beads are captured from the hole 3 using a pipette, capillary, etc.

[0161] According to the above particle capture method, a liquid containing an organic solvent is passed through the sample flow path passing through the hole array filter 1, and after further replacement with an aqueous solution, an aqueous solution containing particles such as cells, components or secretions attached to cells, or beads for attaching components or secretions to cells is passed through, thereby reducing the resistance to the flow of liquid, and making it difficult for bubbles to remain in the holes 3, making it easy to capture particles such as cells C or beads one by one or in a predetermined number in each hole 3. Therefore, the effect of the hole array filter 1 of the first to thirteenth embodiments is complementary, and there is an advantage that a large number of particles can be screened efficiently and stably.

[0162] Although the embodiments of the present invention have been described above, the present invention is not limited to these embodiments, and additions, deletions, and changes to the constituent elements may be made within the scope described in the claims.

[0163] Example

[0164] Next, examples of the present invention will be described.

[0165] [Experiment 1]

[0166] like Fig.16As shown, the conditions for the establishment of an embodiment of the present invention are obtained when two sides of two identical rectangular through holes 8 are arranged on straight lines. The spacing between the through holes 8 is set to a (μm), the length of the horizontal side of the rectangular through hole 8 is set to b (μm), the length of the vertical side is set to c (μm), 1 / 2 of the distance from the upper left vertex of the through hole 8 on the left to the lower right vertex of the through hole 8 on the right is set to R (μm), and the length from the center of one through hole 8 to the other through hole 8 is set to r (μm). The distance R (μm) becomes the radius of the minimum circumscribed circle. The length r (μm) is the minimum contact radius for the moved minimum circumscribed circle to contact the through hole 8 on the right after the center of the minimum circumscribed circle is moved to the center of the through hole 8 on the left.

[0167] The radius R (μm) of the minimum circumscribed circle is determined as follows using the spacing amount a (μm), the length of the horizontal side b (μm), and the length of the vertical side c (μm).

[0168] [Mathematical formula 1]

[0169]

[0170] The minimum contact radius r (μm) is calculated as follows using the spacing amount a (μm) and the length of the lateral side b (μm).

[0171] [Mathematical formula 2]

[0172]

[0173] Therefore, as long as the spacing a (μm) of the through hole 8, the length b (μm) of the horizontal side of the rectangular through hole 8, and the length c (μm) of the vertical side are set in a manner that satisfies R>r, the conditions of the embodiment of the present invention can be met. Specific examples of the case where the conditions of the embodiment are met (embodiment) and the case where the conditions are not met (comparative example) are shown below.

[0174] [Examples of satisfied situations]

[0175] The spacing a of the through hole 8 is 1.00 (μm), the length b of the horizontal side of the rectangular through hole 8 is 2.50 (μm), the length c of the vertical side is 6.02 (μm), and the diameter 2R of the minimum circumscribed circle is 8.50 (μm).

[0176] [Examples of unsatisfactory situations]

[0177] The spacing a of the through hole 8 is 3.00 (μm), the length b of the horizontal side of the rectangular through hole 8 is 2.50 (μm), the length c of the vertical side is 2.87 (μm), and the diameter 2R of the minimum circumscribed circle is 8.50 (μm).

[0178] [Experiment 2]

[0179] like Fig.17 As shown, the conditions for establishing the embodiment of the present invention are obtained when three identical through holes 8 are arranged in such a way that the centers of the circular through holes 8 are located at the vertices of an equilateral triangle. The spacing between the centers of the through holes 8 is set to a (μm), the diameter of the through hole 8 is set to r (μm), the radius of the minimum circumscribed circle is set to A (μm), and the length from the center of one through hole 8 to the point where it touches another through hole 8 is set to B (μm). The length B (μm) is the minimum contact radius after the center of the minimum circumscribed circle is moved to the center of the through hole 8 on the lower left so that the moved minimum circumscribed circle touches the through hole 8 on the upper side.

[0180] The radius A (μm) of the minimum circumscribed circle is expressed by the following formula.

[0181] [Mathematical formula 3]

[0182]

[0183] The minimum contact radius B (μm) is expressed by the following formula.

[0184] [Formula 4]

[0185]

[0186] Therefore, the width of the repeating portion J is expressed by the following formula.

[0187] [Formula 5]

[0188]

[0189] Fig.18 The AB value is shown when the diameter r of the through hole 8 is changed to 2.25, 2.50, 2.75, and 3.00 μm respectively, and the spacing a (μm) between the centers of the through holes 8 is changed, that is, the width (μm) of the smallest circumscribed circle overlapping the through hole 8 on the upper side after moving to the center of the lower left through hole 8.

[0190] Fig.18 The AB value becomes a negative number, indicating that after moving to the center of the through hole 8 on the lower left, the minimum circumscribed circle does not overlap with the through hole 8 on the upper side, which is recorded as "No" in the figure. Fig.18 The AB value becomes a positive number, indicating that after moving to the center of the through hole 8 on the lower left, the smallest circumscribed circle overlaps with the through hole 8 on the upper side, which is marked as "yes" in the figure. Fig.18As shown, for example, when the diameter r (μm) of the through hole 8 is set to 2.25, the case where the spacing amount a (μm) is greater than 3.00 μm and less than 5.32 μm becomes an embodiment of the present invention, and the case where the spacing amount a (μm) is greater than 5.32 does not meet the requirements of the present invention and becomes a comparative example.

[0191] [Experiment 3]

[0192] like Fig.19 As shown, the conditions for establishing the embodiment in which four identical circular through holes 8 are arranged at the vertices of a rectangle are obtained. The spacing between the centers of the through holes 8 in the left and right directions in the figure is set to a (μm), the spacing between the centers of the through holes 8 in the up and down directions is set to b (μm), the diameter of the through holes 8 is set to r (μm), the radius of the minimum circumscribed circle that contacts all the through holes 8 is set to A (μm), and the distance from the center of the lower right through hole 8 to the point where it contacts the upper left through hole 8 is set to B (μm). The distance B (μm) is the minimum contact radius of the minimum circumscribed circle after the center of the minimum circumscribed circle is moved to the center of the lower right through hole 8 so that the moved minimum circumscribed circle contacts the upper left through hole 8.

[0193] The radius A (μm) of the minimum circumscribed circle is determined as follows, assuming that the horizontal spacing is a (μm), the vertical spacing is b (μm), and the diameter of the through hole 8 is r (μm).

[0194] [Mathematical formula 6]

[0195]

[0196] The distance B (μm) from the center of the lower right through-hole 8 to the point where it contacts the upper left through-hole 8 is expressed by the following formula.

[0197] [Formula 7]

[0198]

[0199] If A>B, the conditions of the present invention are met, and thus the following formula can be obtained. Fig.19 As long as the configuration satisfies this formula, the effect of the present invention can be obtained.

[0200] [Mathematical formula 8]

[0201]

[0202] Fig. 20, for each case where the diameter r (μm) of the through hole 8 is set to 1.50, 1.75, 2.00, 2.25, 2.50, 2.75, 3.00, 3.25, and 3.50 μm, the range of "yes" that satisfies the establishment condition of the embodiment of the present invention is shown when the spacing amount a (μm) in the left-right direction of the center of the through hole 8 and the spacing amount b (μm) in the up-down direction of the center of the through hole 8 are changed. Fig. 20 As shown, it can be seen that even when the through-holes 8 are arranged at the vertices of the rectangle, the establishment conditions of the embodiment of the present invention can be easily determined. Fig.19 In the arrangement of, examples of the case where the conditions of the embodiment are satisfied (embodiment) and the case where the conditions are not satisfied (comparative example) are shown below.

[0203] [Examples of satisfied situations]

[0204] The spacing a between the centers of the through holes 8 in the left-right direction is 4.35 μm, the spacing b between the centers of the through holes 8 in the up-down direction is 4.35 μm, the diameter r of the through holes 8 is 3.35 μm, and the diameter (2A) of the smallest circumscribed circle connected to all the through holes 8 is 9.50 μm.

[0205] [Examples of unsatisfactory situations]

[0206] The spacing a between the centers of the through holes 8 in the left-right direction is 4.42 μm, the spacing b between the centers of the through holes 8 in the up-down direction is 4.54 μm, the diameter r of the through holes 8 is 3.16 μm, and the diameter (2A) of the smallest circumscribed circle connected to all the through holes 8 is 9.50 μm.

[0207] In addition, the following shows Fig.19 As described above, the following are specific examples of the case where the conditions of the embodiment are satisfied (embodiment) and the case where the conditions are not satisfied (comparative example) when four rectangular through holes 8 are arranged with their vertical and horizontal sides aligned on the same line.

[0208] [Examples of satisfied situations]

[0209] Length of the horizontal side of the rectangular through hole 8: 2.50 μm, length of the vertical side of the through hole 8: 3.25 μm, spacing between the through holes 8 in the left-right direction: 1.00 μm, spacing between the through holes 8 in the up-down direction: 1.00 μm, diameter of the smallest circumscribed circle connecting all the through holes 8: 9.50 μm

[0210] [Examples of unsatisfactory situations]

[0211] Length of the horizontal side of the rectangular through hole 8: 2.00 μm, length of the vertical side of the through hole 8: 2.51 μm, spacing between the through holes 8 in the left-right direction: 1.00 μm, spacing between the through holes 8 in the up-down direction: 3.05 μm, diameter of the smallest circumscribed circle connected to all the through holes 8: 9.50 μm

[0212] [Experiment 4]

[0213] A pore array filter 1 having through holes 8 of various numbers, shapes and sizes formed in the pore bottom 4 was actually manufactured and assembled in a particle array device 40 to study the single cell rate after passing a sample liquid in which cells C were dispersed and the permeability of ethanol.

[0214] By Fig.13 The hole array filters of Examples 1 to 6 and Comparative Examples were actually manufactured by the method described in one embodiment of the method for manufacturing a hole array filter. The opening of the hole 3 was set to a regular hexagon, and the opening diameter between two parallel sides was set to 50 μm. The partition resist 6A was formed by a negative photoresist with a relatively small amount of multifunctional epoxy, and the bottom resist 4A was formed by a negative photoresist with a relatively large amount of multifunctional epoxy.

[0215] Fig.21 The figure shows: SEM images (scanning electron microscope images) of the through holes 8 of the hole array filters of the manufactured embodiments 1 to 6 and the comparative example; the top view shape of each through hole 8; the number of through holes 8 in each hole 3; the configuration of the through holes 8; the minor diameter (μm) and major diameter (μm) of the through holes 8; the distance between the centers of the through holes 8 (μm); whether the minimum circumscribed circle after the movement overlaps with all the through holes 8 (through hole overlap) regardless of which through hole 8 the minimum circumscribed circle moves to. In terms of through hole overlap, ○ indicates overlap, and × indicates non-overlap.

[0216] like Fig.21 As shown, in Examples 1 to 6, no matter which through-hole 8 the minimum circumscribed circle is moved to, the minimum circumscribed circle after the movement overlaps with all through-holes 8. On the other hand, in the example in which the through-holes 8 are arranged at the vertex positions of a regular hexagon, when the minimum circumscribed circle is moved to the center of the through-hole 8 on the outside, the minimum circumscribed circle does not overlap with the through-hole 8 on the opposite side.

[0217] Next, the hole array filter of Examples 1 to 6 and the comparative example is assembled in the particle array device 40 with a hole forming area of ​​17 mm×17 mm, and after passing the liquid with ethanol, the liquid is passed with phosphate buffer (PBS) three times. Next, 1 ml of phosphate buffer in which only the specified cells are dispersed in a specified number of seeded cells is added to the second chamber 48, and the liquid is passed through the hole array filter. PBMC (peripheral blood mononuclear cells) are used as cells. The number of holes in the hole array filter is 90,000, and the number of cells is about 15 to 90% relative to the number of holes.

[0218] Next, the cells in the well 3 are stained with a DAPI aqueous solution, and the cells in the well 3 are observed through the bottom plate 42 using an inverted fluorescence microscope. The number of wells A in which more than one cell C is captured in all the wells and the number of wells B in which only one cell C is captured are counted in the field of view of the microscope, and B / A is taken as the single cell rate. The cell occupancy rate (%) relative to the number of wells is the ratio of the number of seeded cells to the total number of wells in the well array filter (number of seeded cells / total number of wells). The results are shown in Fig. 22 .

[0219] Fig. 22 The values ​​of "Poisson distribution" in the graph represent the cell occupancy rate (%) and single cell rate (%) relative to the number of wells calculated from the ratio of the number of seeded cells to the number of wells.

[0220] like Fig. 22 As shown, in the hole array filters of Examples 1 to 6, a higher single cell rate (%) can be obtained than the single cell rate (%) of the Poisson distribution obtained by statistical probability calculation. This means that after the first cell is captured in hole 3, the second cell becomes difficult to enter, and the cell flows into other adjacent holes 3, indicating that the shape of the hole 3 and the setting of the through hole 8 are appropriate. The single cell rate of Examples 1, 3, and 5 in which the center-to-center distance of the through hole 8 is 4.00 μm is high. The single cell rate (%) of the comparative example is only at the same level as that of the Poisson distribution, which proves the effect of the present invention.

[0221] [Experiment 5]

[0222] The flow time of ethanol was measured using the hole array filters of Examples 1 to 6 and the comparative example. Each hole array filter was assembled on the particle arrangement device 40, and the hole forming area of ​​the hole array filter was set to a square of 17 mm × 17 mm. 0.5 ml of ethanol was injected into the second chamber 48, and the flow time (seconds) until the entire amount of ethanol passed through the hole array filter and flowed into the flow path 56 was measured. In addition, the ratio of the total opening area of ​​the through holes 8 in the hole forming area and the opening rate (%) were also calculated. These results are shown in Fig.23 and Fig.24 .

[0223] In Examples 1 to 6, the flow time of ethanol is less than 99 seconds, which is within the allowable range in actual use. In the comparative example, since there are 7 through holes 8, the opening rate (%) is high, but Examples 1, 2, and 4 are not inferior to the comparative example. In actual use, it is easier to use if the flow time of ethanol is less than 60 seconds, so it can be seen that the opening rate of the through hole 8 is expected to be 0.5% or more. When the hole 3 is hexagonal and the opening diameter between two parallel sides is 10μm, the thickness of the hole partition 6 is 2μm, and a circular through hole with a diameter of 2μm is provided, the opening rate of the through hole 8 is 3%. Therefore, it can be seen that the opening rate is expected to be in the range of 0.5 to 3%. According to Figures 21 to 24 The results shown show that Examples 1, 2, and 5 simultaneously achieved high single cell rates and fluid permeability, and Examples 1 and 5 were particularly good.

[0224] Industrial Applicability

[0225] According to the present invention, it is possible to ensure the flow rate of the hole array filter as a whole while suppressing the passage of particles such as cells or beads to be captured through the through holes, and by making the multiple through holes formed at the bottom of each hole satisfy the aforementioned positional relationship, so that when one of the aforementioned particles is captured in the hole, the probability that the particle at least partially overlaps with respect to all the aforementioned multiple through holes is high. Therefore, after the first particle is captured, the flow rate of the dispersion medium passing through all the through holes in the hole is moderately reduced, and the particles after the second are suppressed from entering the hole with the flow, and the probability of cells and the like being guided to adjacent empty holes and captured increases. As a result, the probability of capturing particles one by one in a large number of holes is high, the single cell rate is high, and the liquid permeability is good, and the separation of particles can be stably performed through each hole. Therefore, it can be used in industry.

[0226] Description of Reference Numerals

[0227] 1 Hole array filter 2 Filter body

[0228] 3 holes 4 holes bottom

[0229] 4A Bottom resist 6 Hole wall

[0230] 6A Partition resist 8 Through hole

[0231] 10 substrate 12 sacrificial film

[0232] 14 Gap 40 Particle Arrangement Device

[0233] 41 device body 42 bottom plate

[0234] 44 Wall 46 Room 1

[0235] 48 Room 2 50 Room 3

[0236] 52 opening 54 opening

[0237] 56 Flow path A opening diameter

[0238] B septum thickness C cell

[0239] P Pipette W Spacing Width

[0240] D Diameter G1 Minimum circumscribed circle

[0241] G2 The minimum circumscribed circle J after the move Repeated part

Claims

1. A hole array filter, characterized in that A flat plate-shaped filter body is provided, wherein a plurality of holes are formed in the filter body and open in a hole forming region on an upper surface of the filter body, wherein adjacent holes are separated from each other by hole partition walls. A hole bottom is formed at the lower end of each of the holes, and two or more through holes reaching the lower surface of the filter body are formed at the hole bottom. The minimum width of the upper surface opening of the through hole is not less than 1 μm and not more than 3.5 μm, The ratio of the total opening area of ​​the through holes to the area of ​​the hole forming region is 0.5% to 3.5%, Consider a minimum circumscribed circle surrounding all of the multiple through holes formed at the bottom of each hole. Even if the center of the minimum circumscribed circle is moved to the center of any one of the multiple through holes, the minimum circumscribed circle satisfies a positional relationship of at least partial overlap relative to all of the multiple through holes.

2. The hole array filter according to claim 1, characterized in that The through holes formed at the bottom of each hole are formed in two or more numbers on an imaginary line segment, or the through holes formed at the bottom of each hole are formed in three or more numbers at the vertices of an imaginary triangle and on the sides or inside of the triangle, or the through holes formed at the bottom of each hole are formed in four or more numbers at the vertices of an imaginary quadrilateral and on the sides or inside of the quadrilateral.

3. The hole array filter according to claim 1 or 2, characterized in that: The opening shape of the through hole is circular, elliptical, or a shape formed by connecting a plurality of circular or elliptical shapes.

4. The hole array filter according to claim 1 or 2, characterized in that: The opening shape of the through hole is a polygon or a polygon with rounded corners.

5. The hole array filter according to claim 1 or 2, characterized in that: At least the inner surface of the pore is coated with a polymer that inhibits cell adhesion.

6. A particle arrangement device, characterized in that: have: The pore array filter according to claim 1 or 2; and The device body supports the hole array filter and has a sample flow path extending from the opening side of the holes of the hole array filter to the bottom surface side of the holes through the through holes.

7. A particle capture method comprising: A step of passing a liquid containing an organic solvent that does not dissolve or deteriorate the pore array filter and the device body through the sample flow path of the particle array device according to claim 6; a step of passing an aqueous solution through the sample flow path to replace a liquid containing an organic solvent with the aqueous solution; and A particle capturing step of passing an aqueous solution containing particles to be captured by the pores through the sample flow path to capture the particles in the pores.

Images