Leukocyte capture device

Abstract

The present invention aims to provide a leukocyte capture device that does not require a large device such as a centrifuge and requires a small amount of blood, about 1 μl. The above-mentioned problem is solved by a leukocyte capture device (1) that captures leukocytes by allowing a liquid containing blood to pass through it, wherein a chip (10) has a flat portion (12) and a plurality of protrusions (14) provided on the flat portion (12). The chip (10) is configured so that the blood-containing liquid entering from an inlet (3) passes on the surface of the flat portion (12) in the chip (10) and passes between the protrusions (14) and other adjacent protrusions (14) and is discharged from an outlet (5). The protrusions (14) are arranged in layers on the flat portion (12), each layer includes a plurality of protrusions (14), and is configured so that the blood-containing liquid that has passed through the layer on the inlet (3) side passes through the layer on the outlet (5) side adjacent to the layer, and a capture portion and a bypass portion are formed between the protrusion (14) and the protrusion (14) adjacent thereto, and a capture portion that is part of another layer adjacent to the specific layer is arranged on the outlet (5) side of the bypass portion in a specific layer.

Description

Technical Field

[0001] The present invention relates to a leukocyte capturing device. Background Art

[0002] DNA can be damaged by radiation exposure or the effects of the living environment. Damaged DNA is generally believed to be closely linked to diseases such as cancer. Traditionally, analysis of this DNA damage has involved centrifuging 5 ml of blood to extract white blood cells, staining them, and observing them on a glass slide.

[0003] In addition, a microfluidic path for capturing particles such as leukocytes has been proposed in the past (see patent document 1). Patent document 1 discloses a microfluidic path device with a filtering function, which uses a microfluidic path with a concave capturing portion to capture and separate only solids above a certain size from a solid-liquid mixture. In patent document 1, the purpose is to accommodate one or more solids above a certain size in one capturing portion and to completely capture solids above a certain size before the terminal of a separation portion equipped with multiple capturing portions. It is not considered to capture only one solid in one capturing portion in order to easily observe the captured solids such as leukocytes. In addition, in order to prevent the solids temporarily captured by the capturing portion from floating again and flowing out of the capturing portion, it is necessary to make the solid-liquid mixture always flow from the inlet to the outlet of the microfluidic path.

[0004] Prior art literature

[0005] Patent Literature

[0006] Patent Document 1: Japanese Patent Application Laid-Open No. 2009-109232 Summary of the Invention

[0007] Problems to be solved by the invention

[0008] However, conventional methods require up to 5 ml of blood, making them invasive. Furthermore, they require large equipment such as centrifuges, making them difficult to use for point-of-care testing (POCT), such as on-site analysis.

[0009] In addition, when the purpose is to separate and carry solid components of a specific size such as white blood cells from a solid-liquid mixture such as a blood-containing liquid through a capture portion and perform on-site analysis, the concave capture portion of the microchannel described in Patent Document 1 has low capture efficiency of solid components such as white blood cells.

[0010] The present invention aims to solve the above-mentioned problems. Specifically, the present invention aims to provide a leukocyte capture device that has a higher capture efficiency than conventional leukocyte capture devices, does not require large equipment such as centrifuges, and requires only a small amount of blood, approximately 1 μl.

[0011] Technical means to solve problems

[0012] The present invention includes the following (1) to (4).

[0013] (1) A leukocyte capturing device comprising a chip for allowing a liquid containing blood to pass therethrough to capture leukocytes contained in the liquid containing blood,

[0014] The chip has a planar portion and a plurality of protrusions provided on the planar portion, and is configured such that the blood-containing liquid entering from the inlet passes along the surface of the planar portion of the chip and between the protrusions and other adjacent protrusions, and is discharged from the outlet;

[0015] The protrusions are provided in layers on the planar portion, each layer including a plurality of the protrusions, and the structure is such that the blood-containing liquid that has passed through an inlet-side layer passes through an outlet-side layer adjacent to the layer.

[0016] In each layer, a capture portion having a width of 2 μm to 7.5 μm and a bypass portion having a width of 8 μm to 20 μm are formed between the convex portion and another adjacent convex portion.

[0017] The inlet side portions of the two protrusions constituting the capture portion are chamfered so that the widths of the inlet side portions gradually narrow toward the depth of the capture portion; and

[0018] The capturing portion, which is a part of another layer adjacent to the specific layer, is arranged to face the outlet side of all or part of the bypass portion in the specific layer.

[0019] (2) The leukocyte capturing device according to (1) above, wherein a width between the specific layer and another layer adjacent thereto is 8 μm to 30 μm.

[0020] (3) The leukocyte capturing device according to (1) or (2) above, wherein a ratio of a width of the bypass portion to a width of the capturing portion is greater than 1 and not more than 3.

[0021] (4) A leukocyte capture device according to any one of (1) to (3) above, wherein a portion of the end surface on the inlet side of the convex portion other than the capture portion extends parallel to the layer direction, and an end surface of the convex portion constituting the bypass portion extends in a direction perpendicular to the layer direction.

[0022] Effects of the Invention

[0023] According to the present invention, a leukocyte capture device having a higher capture efficiency than the prior art can be provided, which does not require a large device such as a centrifuge and requires a small amount of blood, approximately 1 μl. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a schematic diagram of the chip surface in the leukocyte capture device of the present invention in a preferred embodiment.

[0025] Figure 2 yes Figure 1 Magnified view of part A in FIG.

[0026] Figure 3 yes Figure 1 BB line cross section in.

[0027] Figure 4 These are magnified photographs of the surfaces of chips used in Examples and Comparative Examples.

[0028] Figure 5 This is an enlarged photograph of the chip observed with a fluorescence microscope, showing the state of leukocyte capture in Example 1 (fluorescence dimmed).

[0029] Figure 6 This is an enlarged photograph of the chip observed with a fluorescence microscope, showing the state of leukocyte capture in Example 2 (fluorescence dimmed).

[0030] Figure 7 This is a schematic diagram of the capture section and bypass section of a conventional microfluidic device.

[0031] Figure 8 This is a diagram showing the boundaries of the capture unit used for capture determination.

[0032] Figure 9 This is an enlarged photograph of the chip observed with a fluorescence microscope, showing the state of leukocyte capture in a comparative evaluation experiment. DETAILED DESCRIPTION

[0033] The leukocyte capturing device of the present invention will be described.

[0034] The leukocyte capture device of the present invention comprises a chip for allowing a blood-containing liquid to pass therethrough to capture the leukocytes contained in the blood-containing liquid, the chip comprising a planar portion and a plurality of convex portions provided on the planar portion, the chip being configured so that the blood-containing liquid entering from an inlet passes over the surface of the planar portion in the chip and between the convex portions and other adjacent convex portions, and is discharged from an outlet; the convex portions are provided in layers on the planar portion, each layer comprising a plurality of convex portions, and the chip being configured so that the blood-containing liquid that has passed through the layer on the inlet side passes through the chip. a layer passing through the outlet side adjacent to the layer; a capture portion with a width set to 2μm to 7.5μm between the above-mentioned convex portion and another above-mentioned convex portion adjacent thereto, and a bypass portion with a width set to 8μm to 20μm are formed in each layer; the width of the entry side portion of the two above-mentioned convex portions constituting the above-mentioned capture portion is chamfered in a manner that gradually narrows toward the depth of the above-mentioned capture portion; and the above-mentioned capture portion is arranged opposite to the outlet side of all or part of the above-mentioned bypass portion in a specific layer, and the capture portion is made as a part of another layer adjacent to the specific layer.

[0035] The leukocyte capturing device of the present invention will be described using the accompanying drawings.

[0036] Figure 1 This is a schematic diagram showing the leukocyte capturing device 1 of the present invention. Figure 2 yes Figure 1 Magnified view of part A in FIG. Figure 3 yes Figure 1 BB line cross section in.

[0037] Figure 1 The leukocyte capture device 1 of the present invention illustrated in FIG includes a chip 10, an inlet 3 for supplying a blood-containing liquid to the chip 10, and an outlet 5 for discharging the blood-containing liquid that has passed through the chip 10. The structure of the leukocyte capture device of the present invention is not limited to Figure 1 The structures exemplified in, for example, Figure 1 The entire leukocyte capture device 1 of the present invention may also be covered by a housing.

[0038] like Figures 1 to 3 As shown, the chip 10 in the leukocyte capture device 1 of the present invention includes a planar portion 12 and a plurality of protrusions 14 provided on the planar portion 12 .

[0039] In such a leukocyte capture device 1 of the present invention, the blood-containing liquid entering from the inlet 3 flows toward the outlet 5 by a pump or hydrostatic pressure, electroosmotic flow, etc. During this process, it flows on the surface of the planar portion 12 of the chip 10 and flows between the protrusion 14 and another protrusion 14 adjacent to it, and the leukocytes are sandwiched between specific protrusions 14 and captured.

[0040] Here, the blood-containing liquid is not particularly limited as long as it contains human blood, and may be, for example, a mixture of human blood added to a phosphate buffer, an anticoagulant, a staining solution, etc. Alternatively, the blood-containing liquid may be human blood itself.

[0041] In addition, if Figure 1 As shown, the convex portions 14 are provided on the flat portion 12 in a layered manner.

[0042] exist Figure 1 In the diagram, the layer closest to the inlet 3 is designated as the first layer, and the layer adjacent to the first layer on the outlet side (downstream side) is designated as the second layer. Furthermore, a certain layer is designated as the Pth layer, the layer adjacent to the Pth layer on the outlet side (downstream side) is designated as the P+1th layer, and the layer adjacent to the P+1th layer on the outlet side (downstream side) is designated as the P+2th layer.

[0043] In addition, each layer includes a plurality of protrusions 14 . Figure 1 , each layer includes seven convex portions 14, but the number of convex portions 14 included in each layer is not particularly limited. Furthermore, the number of layers is not particularly limited either.

[0044] The blood-containing liquid that enters the leukocyte capture device 1 of the present invention from the inlet 3 flows on the surface of the planar portion 12, first passing through the flow paths between the protrusions 14 in the first layer, then through the flow paths between the protrusions 14 in the second layer. The liquid then flows through the flow paths between the protrusions 14 in the Pth layer, and then through the flow paths between the protrusions 14 in the P+1th layer.

[0045] In addition, if Figure 2 As shown, in each layer, a capture portion 21 having a width L1 between a convex portion 14 and an adjacent convex portion 14 (a width of a flow path) set to 2 μm to 7.5 μm, and a bypass portion 23 having a width L2 set to 8 μm to 20 μm are formed.

[0046] exist Figure 2 In the example of FIG. 1 , in each of the P-th layer, the P+1-th layer, and the P+2-th layer, the capturing portion 21 and the bypass portion 23 are alternately formed as flow paths between the plurality of convex portions 14. However, in the leukocyte capturing device of the present invention, the capturing portion and the bypass portion formed in each layer may not be as alternate as in FIG. Figure 2 For example, a plurality of capture portions may exist continuously within a layer.

[0047] Furthermore, on the outlet side of the bypass portion 23 in a specific layer, a capture portion 21 is arranged as a part of another layer adjacent to the specific layer. Figure 2In the example of FIG. 1 , the capture section 21 in the P+1th layer is arranged on the outlet side (downstream side) of the bypass section 23 in the Pth layer.

[0048] like Figure 2 As illustrated, the bypass portion 23 in the P-th layer and the capture portion 21 in the P+1-th layer are preferably arranged in a direction perpendicular to the layer direction. More specifically, when a straight line perpendicular to the layer direction is drawn, the bypass portion 23 in the P-th layer and the capture portion 21 in the P+1-th layer are preferably arranged so that the straight line passes through the bypass portion 23 in the P-th layer and the capture portion 21 in the P+1-th layer (i.e., the straight line does not contact the protrusion 14).

[0049] exist Figure 1 、 Figure 2 In the example shown, white blood cells contained in the blood-containing liquid that flows from the inlet side (upstream) and reaches the P-th layer cannot, in principle, pass through the capture section 21. Therefore, at least some of the white blood cells are captured by the capture section 21 of the P-th layer. If white blood cells are captured, the capture section 21 is blocked. Meanwhile, components other than white blood cells (red blood cells, platelets, etc.) pass through the capture section 21 of the P-th layer and reach the P+1-th layer. Furthermore, all components contained in the blood-containing liquid that reaches the P-th layer can pass through the bypass section 23. Therefore, white blood cells not captured by the capture section 21 of the P-th layer pass through the bypass section 23 of the P-th layer and reach the P+1-th layer, where at least some of them are captured by the capture section 21 of the P+1-th layer. Here, since the capture section 21 of the P+1-th layer is located on the outlet side (downstream side) of the bypass section 23 of the P-th layer, white blood cells that pass through the bypass section 23 of the P-th layer are easily captured by the capture section 21 of the P+1-th layer.

[0050] In such Figure 1 、 Figure 2 In the plan view shown, the protrusion 14 is preferably rectangular or substantially rectangular (a shape based on a rectangle with some of its four corners linearly cut off and chamfered, or a shape in which at least some of the four corners of the rectangle are cut off and rounded). The area of ​​the chamfered portion cut off linearly and the chamfered portion cut off for rounding is preferably smaller than the area (projected area) of the captured leukocytes.

[0051] In addition, if Figure 2 As shown in the example, the entrance side of the two protrusions 14 constituting the capturing portion 21 is chamfered so that the portions thereof gradually narrow toward the depth of the capturing portion 21. This is because this makes it easier for white blood cells to be captured by the capturing portion. Furthermore, once captured by the capturing portion, white blood cells deform and become embedded in the chamfered portion, making it difficult for them to escape from the capturing portion.

[0052] The angle formed by the straight lines formed by the chamfers is preferably 30 to 60 degrees relative to the direction perpendicular to the layer direction (the direction from the inlet to the outlet). When the chamfers are, for example, spoon-shaped or circular rather than linear, the average angle formed by their tangents is preferably 30 to 60 degrees. If this angle is less than 30 degrees, the rate at which leukocytes flow into the bypass section 23 tends to increase, reducing the capture efficiency. Furthermore, if this angle is greater than 60 degrees, the probability of multiple leukocytes being captured by a single capture section 21 tends to increase.

[0053] As for chamfering, as long as the entry side portion gradually narrows toward the depth of the capture portion 21, the entry side portions of both of the two convex portions constituting the capture portion may be chamfered or only one of the entry side portions may be chamfered. When the entry side portions of both sides are chamfered, the chamfering angles may be the same or different.

[0054] When the projection 14 is rectangular or substantially rectangular, additional white blood cells that have reached the capturing portion 21 that has already captured microparticles move along the end surface of the projection 14 toward the layer, passing through the bypass portion 23 toward the adjacent layer on the downstream side, and are more likely to be captured by the capturing portion 21 in the downstream layer. As a result, the present inventors have discovered that the efficiency of capturing white blood cells is improved.

[0055] Especially if Figure 2 As shown in the case, when the inlet side portions of the two protrusions 14 constituting the capture portion 21 are chamfered in a manner that continuously and gradually narrows toward the depth of the capture portion 21 (preferably linearly), and the portion of the inlet side end face of the protrusion 14 other than the capture portion 21 extends parallel to the layer direction, and the bypass portion 23 extends in a direction perpendicular to the layer direction, the effect becomes significant, the capture efficiency of white blood cells is further improved, and therefore it is preferred.

[0056] When the projection 14 is not rectangular or substantially rectangular (eg, circular or elliptical), since its outer shape includes an R, there is a possibility that white blood cells may move along the R instead of moving to the capture portion 21 in the adjacent layer on the downstream side.

[0057] The width L1 of the trapping portion 21 is 2 μm to 7.5 μm, preferably 3 μm to 6 μm, and more preferably 4 μm to 5 μm.

[0058] The width L2 of the bypass portion 23 is 8 μm to 20 μm, preferably 8.5 μm to 15 μm, and more preferably 9 μm to 10 μm.

[0059] It should be noted that the width L1 and the width L2 refer to the shortest distance between a convex portion 14 and an adjacent convex portion 14 in each layer.

[0060] Furthermore, the ratio of the width L2 of the bypass portion 23 to the width L1 of the capturing portion 21 (L2 / L1) is preferably greater than 1 and not more than 3, and more preferably 1.5 to 2.5. This is because the flow into the bypass portion 23 can be appropriately suppressed, making it easier for white blood cells to be captured by the capturing portion.

[0061] Furthermore, the width L3 between the P-th layer and the P+1-th layer is preferably 8 μm to 30 μm, and more preferably 9 μm to 10 μm.

[0062] It should be noted that the width L3 refers to the shortest distance between the Pth layer and the P+1th layer.

[0063] Furthermore, the maximum width L4 of the chamfered portion on the entrance side of the capturing portion 21 is preferably 10 μm to 35 μm, and more preferably 15 μm to 25 μm.

[0064] Figure 3 The height h of the convex portion 14 shown is preferably 8 μm to 30 μm, more preferably 9 μm to 15 μm.

[0065] The chip size and material are not particularly limited. For example, it can be made of resins such as silicone rubber, acrylic resin, polycarbonate, cyclic olefin polymer, cyclic olefin copolymer, polystyrene, polyethylene, and polyethylene terephthalate, and is preferably formed by attaching the resin to a substrate such as glass.

[0066] Example

[0067] <Fabrication of Leukocyte Capture Device>

[0068] Leukocyte capture devices with six types of chips (the widths of the bypass and capture sections were the values ​​shown in Table 1) were fabricated according to the following procedure. It should be noted that the widths of the specific layer and the adjacent layers in all leukocyte capture devices ( Figure 2 The width L3) in the figure is 10 μm.

[0069] First, a photosensitive resin (SU-83050, manufactured by Nippon Kayaku Co., Ltd.) was uniformly applied to the surface of a plate-shaped silicone wafer using a spinner.

[0070] Next, the photosensitive resin is irradiated with ultraviolet rays through a specific mask.

[0071] Next, the photosensitive resin on the silicone wafer exposed to ultraviolet rays was baked at 95°C.

[0072] Next, the portion not irradiated with ultraviolet rays was removed using a developer (SU-8 Developer, manufactured by Nippon Kayaku Co., Ltd.) to prepare a mold.

[0073] Next, silicone rubber (SILPOT184, manufactured by Dow Corning) was poured into the mold.

[0074] Next, the silicone rubber was vulcanized at 100° C. for 0.5 hours.

[0075] Next, the silicone rubber is peeled off from the silicone wafer, thereby forming a chip having a flow path formed therein.

[0076] Next, holes were punched at positions that would serve as an inlet and an outlet to create a fluid introduction portion, thereby completing the leukocyte capture device.

[0077] <Join>

[0078] Using a light source (L12530-01, manufactured by Hamamatsu Photonics KK), both sides of the glass substrate on which the chip formed with the flow path was formed were irradiated with vacuum ultraviolet light for 15 seconds.

[0079] The chip was observed under a fluorescence microscope, and the magnified photograph is shown in Figure 4 .

[0080] <Experiment>

[0081] Peripheral blood obtained from an adult male was diluted 2-fold with PBS (phosphate buffered saline, manufactured by Wako Pure Chemical Industries, Ltd.).

[0082] Next, 1 μl to 2 μl of diluted blood was dropped into the inlet of the chip, and the liquid was delivered by hydrostatic pressure.

[0083] After standing for about 1 hour, unnecessary blood was removed and PBS was added dropwise to transfer the liquid, thereby removing substances other than the captured leukocytes.

[0084] Next, the PBS was removed, and DNA binding staining solution (DAPI) was added dropwise, and the cells were allowed to stand for 30 minutes.

[0085] The chips of each of the six leukocyte capture devices were then observed using a fluorescence microscope to confirm leukocyte capture. The results are shown in Table 1. In Table 1, "○" indicates that single leukocyte capture was confirmed in at least one capture area, while "×" indicates that single leukocyte capture was not confirmed in any capture area.

[0086] In addition, the chips of Example 1 and Example 2 were observed using a fluorescence microscope, and the magnified photographs of the obtained dimmed fluorescence are shown in FIG. Figure 5 and Figure 6 . Figure 5 The magnification and Figure 4The same situation. Figure 6 This is an image of the entire device. Figure 5 and Figure 6 The white dots in the image represent white blood cells. Figure 5 and Figure 6 It was confirmed that single-cell capture of leukocytes was achieved in Examples 1 and 2. Observation was performed using a fluorescence microscope in the same manner as in Examples 1 and 2 for Examples 3 and 4, and it was confirmed that single-cell capture of leukocytes was achieved.

[0087] On the other hand, in the case of Comparative Examples 1 and 2 in which the widths of the capturing portion and the bypass portion were too wide, leukocytes could not be sufficiently captured.

[0088] [Table 1]

[0089] Width of bypass section (μm) Width of capture portion (μm) Capture results Example 1 10 5 ○ Example 2 10 4 ○ Example 3 10 7.5 ○ Example 4 15 5 ○ Comparative Example 1 50 25 × Comparative Example 2 40 20 ×

[0090] <Comparative Evaluation>

[0091] To compare the leukocyte capture efficiency of the leukocyte capture device of the present invention with that of the prior art, the leukocyte capture device of the present invention and the prior art microfluidic channel device having a concave capture portion described in Patent Document 1 were prepared and comparative evaluation experiments were conducted. Figure 7 A schematic diagram showing the capture section and bypass section of a microfluidic device.

[0092] As indicators for evaluating the capture efficiency, two indicators represented by [Formula 1] and [Formula 2] are used.

[0093] Single capture capture unit rate (%) = [number of single capture capture units] / [number of capture units within the observation range] × 100 [Formula 1]

[0094] Single captured leukocyte rate (%) = [number of single captured leukocytes] / [number of leukocytes present in the observation range] × 100 [Formula 2]

[0095] The number of capture units within the observation range indicates the number of capture units within the observation range set for the leukocyte capture device or microfluidic device being compared and evaluated. The number of single capture units indicates the number of capture units that captured only one leukocyte within the observation range. The number of single captured leukocytes indicates the number of leukocytes captured by a single capture unit within the observation range, which is equivalent to the number of single capture units. The number of leukocytes present within the observation range indicates the number of all leukocytes present within the observation range, including not only leukocytes captured by a single capture unit, but also multiple leukocytes captured by a single capture unit and leukocytes present in the flow path outside of the capture unit.

[0096] In addition, whether the leukocytes are captured by the capturing portion can be determined as follows: Figure 8 The capturing portion of the leukocyte capturing device of the present invention shown in (a) and Figure 8 In the capture section of the conventional microfluidic device shown in (b), if a portion of a leukocyte enters the minimum rectangle surrounding a group of convex portions constituting one capture section, as indicated by the red lines in each figure, the leukocyte is determined to be captured.

[0097] The reasons for adopting these indicators as evaluation indicators of capture efficiency are as follows.

[0098] In fluorescence image analysis, such as DNA damage assessment, which is a hypothetical use case for the leukocyte capture device of the present invention, if automated image analysis is considered using an image processing program, it is desirable that the number of leukocytes captured by each capture unit, a single leukocyte, should be at least as high as necessary for analysis within a single field of view of a fluorescence microscope or the like used for analysis. Furthermore, considering the ease of creating an image processing program, it is desirable that no leukocytes remain in the flow path outside of the capture unit upon completion of separation and arraying.

[0099] That is, in order to evaluate the two preferred properties shown above, the two indicators of single capture portion rate and single capture leukocyte rate are used as evaluation indicators of capture efficiency, wherein the single capture portion rate is the proportion of capture portions that capture only one cell among the capture portions within the observation range, and the single capture leukocyte rate is the proportion of cells captured within the observation range that are captured by the capture portion only one cell.

[0100] <Fabrication of Leukocyte Capture Device and Microfluidic Device>

[0101] A chip comprising the leukocyte capture device of the present invention was fabricated as Example 5 by the same procedures as in Examples 1 to 4 described above. A chip comprising a microfluidic device having a concave capture portion was also fabricated by the same procedures as in Comparative Example 3. The dimensions of the capture portion and bypass portion of each chip are shown in Table 2. Figure 2 L1, Figure 7 L1' represents the width of the capture portion, Figure 2 L2, Figure 7 L2' represents the width of the bypass portion, Figure 2 L3, Figure 7 L3' represents the distance between layers, Figure 2 L4, Figure 7 L4' respectively denotes the maximum width of the entry-side portion of the capture portion.

[0102] [Table 2]

[0103]

[0104] <Experiment>

[0105] The experiment was carried out by the same procedure as in Examples 1 to 4 described above.

[0106] For both chips, the chips were observed using a fluorescence microscope. Within the observation range containing the same number of capture sections, the number of capture sections present within the observation range, the number of leukocytes present within the observation range, and the number of single captured leukocytes (number of single captured capture sections) were counted. The capture efficiency was evaluated using Formulas 1 and 2. The capture efficiency of each chip is shown in Table 3. Figure 9 (a) shows the state of leukocytes captured within the observation target range of the chip of the leukocyte capturing device of the present invention, Figure 9 (b) shows the state of leukocytes captured within the observation area of ​​a conventional microfluidic device chip. The individual particles emitting fluorescence in each figure are leukocytes.

[0107] [Table 3]

[0108]

[0109] As shown in Table 3, this comparative evaluation experiment confirmed that Example 5 showed a 68% higher single capture rate and a 53% higher single leukocyte capture rate than Comparative Example 3. This indicates that the leukocyte capture device of the present invention has a significantly higher capture efficiency for solid components such as leukocytes than conventional methods.

[0110] The reason for this difference can be speculated as follows: by chamfering the inlet side of the two convex parts constituting the capture part of the present invention in such a way that the width of the entry side part gradually narrows toward the depth of the above-mentioned capture part, compared with the concave structure of the capture part in the prior art, the static pressure of the fluid is used to produce the effect of clamping the cells deep into the capture part from both sides, making it less likely that temporarily captured cells will detach or multiple cells will be captured in one capture part.

[0111] This application claims priority based on Japanese patent application No. 2020-163378, filed on September 29, 2020, the disclosure of which is incorporated herein in its entirety.

[0112] Reference numerals

[0113] 1. Leukocyte Capture Device of the Present Invention

[0114] 3 Entrance

[0115] 5 Exit

[0116] 10 chips

[0117] 12 Flat surface

[0118] 14 convex part

[0119] 21 Capture Department

[0120] 23 Bypass

Claims

1. A leukocyte capture device, characterized in that: The leukocyte capture device includes a chip that allows a liquid containing blood to pass therethrough to capture leukocytes contained in the liquid containing blood. The chip has a planar portion and a plurality of convex portions provided on the planar portion, and the chip is configured so that the blood-containing liquid entering from the inlet passes on the surface of the planar portion in the chip and between the convex portion and another convex portion adjacent thereto and is discharged from the outlet; The protrusions are provided in layers on the planar portion, each layer including a plurality of the protrusions, and the structure is such that the blood-containing liquid that has passed through an inlet-side layer passes through an outlet-side layer adjacent to the layer; In each layer, a capture portion having a width set to 2 μm to 7.5 μm between the convex portion and another adjacent convex portion and a bypass portion having a width set to 8 μm to 20 μm are formed; The two convex portions constituting the capture portion are chamfered so that the width of the inlet side portion thereof gradually narrows toward the depth of the capture portion; and The capturing portion, which is a part of another layer adjacent to the specific layer, is arranged to face the outlet side of all or part of the bypass portion in the specific layer.

2. The leukocyte capture device according to claim 1, wherein: The width between a specific layer and another adjacent layer is 8 μm to 30 μm.

3. The leukocyte capture device according to claim 1 or 2, wherein: A ratio of a width of the bypass portion to a width of the capture portion is greater than 1 and is equal to or less than 3.

4. The leukocyte capture device according to claim 1 or 2, wherein: A portion of an end surface on the inlet side of the convex portion excluding the capturing portion extends parallel to the layer direction, and an end surface of the convex portion constituting the bypass portion extends in a direction perpendicular to the layer direction.

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