Particle capture device

Abstract

The selective capture of smaller particles from among the particles contained in a fluid. [Solution] The particle capture device according to the embodiment is a particle capture device that captures particles contained in a fluid flowing through a channel by dielectrophoresis, and comprises an electrode pair and a capture membrane. A voltage is applied to the electrode pair. The capture membrane is provided on the electrode pair and along at least one surface of the channel. The capture membrane has connected wells for particle capture. The connected wells have a plurality of first wells connected to each other.

Description

Technical Field

[0001] The embodiments disclosed in this specification and the drawings relate to a particle capture device.

Background Art

[0002] Conventionally, there is known a particle capture device that uses dielectrophoresis to capture particles such as cells contained in a fluid in a microwell or the like according to their size. Such a particle capture device is used, for example, when separately arraying and detecting small blood cells such as lymphocytes and red blood cells and circulating tumor cells (CTC), or when separating blood cells of various sizes.

[0003] When capturing particles in a well using dielectrophoresis, the dielectrophoretic force per unit area increases as the size of the well decreases. Therefore, simply reducing the size of the well causes non-specific capture and makes it impossible to selectively capture small-sized particles.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] One of the problems to be solved by the embodiments disclosed in this specification and the drawings is to selectively capture small-sized particles among the particles contained in a fluid. However, the problems to be solved by the embodiments disclosed in this specification and the drawings are not limited to the above problems. It is also possible to regard the problems corresponding to the respective effects of each configuration shown in the embodiments described later as other problems.

Means for Solving the Problems

[0006] The particle capture device according to this embodiment is a particle capture device that captures particles contained in a fluid flowing through a channel by dielectrophoresis, and comprises an electrode pair and a capture membrane. A voltage is applied to the electrode pair. The capture membrane is provided on the electrode pair and along at least one surface of the channel. The capture membrane has connected wells for particle capture. The connected wells have a plurality of first wells connected to each other. [Brief explanation of the drawing]

[0007] [Figure 1] A perspective view of a particle capture device according to one embodiment. [Figure 2] A plan view of a particle capture device according to one embodiment. [Figure 3] A schematic plan view of a connected well according to one embodiment. [Figure 4] A cross-sectional view illustrating an example of operation of a particle capture device according to one embodiment. [Figure 5] Enlarged view showing an example of operation of a particle capture device according to one embodiment. [Figure 6] A diagram showing an example of a target particle in a particle capture device according to one embodiment. [Figure 7] This figure shows the simulation results of the electric fields formed in various wells of a particle capture device according to one embodiment and a comparative example. [Figure 8] A graph showing the magnitude of the gradient of the electric field strength formed on each first well in a connected well according to one embodiment and an unconnected well according to a comparative example. [Figure 9] A figure showing the results of a particle capture experiment using a particle capture device according to one embodiment, specifically for capturing a second target particle. [Figure 10] This figure shows the results of a particle capture experiment involving a second target particle in a comparative example particle capture device. [Figure 11] This figure shows the results of a capture experiment of a first target particle in a connected well according to one embodiment and in an unconnected well according to a comparative example. [Figure 12]Graph showing the results of the capture experiment of the first target particles in the connected wells according to one embodiment and the non - connected wells according to the comparative example. [Figure 13] Figure showing the results of another capture experiment of the first target particles in the connected wells according to one embodiment. [Figure 14] Figure showing the results of another capture experiment of the first target particles in the non - connected wells according to the comparative example. [Figure 15] Figure showing the state of the first target particles on the connection wells in the connected wells according to one embodiment. [Figure 16] Figure showing an example of the electric field on the connection wells in the connected wells according to one embodiment. [Figure 17] Figure showing the results of the separation and capture experiment of the first target particles and the second target particles in the particle capture device according to one embodiment. [Figure 18] Graph showing the results of the separation and capture experiment of the first target particles and the second target particles in the particle capture device according to one embodiment. [Figure 19] [[ID=2l]]Schematic plan view of the connected wells according to Modification Example 1 of one embodiment. [Figure 20] Plan view of the particle capture device according to Modification Example 2 of one embodiment. [Figure 21] Schematic diagram of the particle capture device according to Modification Example 3 of one embodiment. [Figure 22] Plan view of the particle capture device according to Modification Example 4 of one embodiment. [Figure 23] Plan view of the particle capture device according to Modification Example 5 of one embodiment.

Embodiments for Carrying Out the Invention

[0008] Hereinafter, embodiments of the particle capture device will be described with reference to the drawings. In the following description, components having substantially the same functions and configurations will be denoted by the same reference numerals, and duplicate descriptions will be made only when necessary.

[0009] <Particle Capture Device> A particle capture device 1 according to one embodiment will be described with reference to Figures 1 and 2. Figure 1 is a perspective view of the particle capture device 1 according to one embodiment. Figure 2 is a plan view of the particle capture device 1 according to one embodiment. Note that the cover member 5 is omitted in Figure 2.

[0010] Particle capture device 1 is a device that captures particles contained in a fluid by dielectrophoresis. More specifically, particle capture device 1 is equipped with a fluid channel as a flow path through which a fluid containing particles of different sizes flows, and is a device that separates and captures particles in the fluid by size using dielectrophoresis and then arrays them. In Figures 1 and 2, the symbol FD represents the direction in which the fluid flows. The fluid is, for example, the blood of a cancer patient, or a leukocyte fraction purified from blood. The particles contained in the fluid are living cells such as lymphocytes, red blood cells, and circulating tumor cells (CTCs). In other words, particle capture device 1 can be applied to, for example, the separate capture of small blood cells such as lymphocytes and red blood cells from larger cells such as circulating tumor cells.

[0011] In the following explanation, the upstream side in the fluid flow direction FD may simply be referred to as the "upstream side." Similarly, the downstream side in the fluid flow direction FD may simply be referred to as the "downstream side." Furthermore, in the following explanation, when referring to the "size" of a particle, "size" refers to the diameter of the particle unless otherwise specified.

[0012] As shown in Figure 1, the particle capture device 1 according to this embodiment includes, for example, a bottom plate 2, an electrode pair 3, a capture membrane 4, and a cover member 5.

[0013] The base plate 2 is an insulator such as a glass plate. An electrode pair 3 is provided on the upper surface of the base plate 2. The electrode pair 3 is, for example, a thin-film conductor. The material of the electrode pair 3 is, for example, ITO (indium tin oxide).

[0014] The electrode pair 3 is, for example, a pair of comb-shaped electrodes having electrodes 31 and 32. A voltage is applied to the electrode pair 3. In this embodiment, an alternating current (AC) voltage is applied to the electrode pair 3. More specifically, electrodes 31 and 32 of the electrode pair 3 are electrically connected to one end and the other end of an AC power supply 33, respectively, and an AC voltage is applied between them. The AC voltage applied between electrodes 31 and 32 is, for example, a sine wave with a frequency of 5 MHz and an amplitude of 0.8 to 3 V. Alternatively, a DC voltage may be applied to the electrode pair 3 instead of an AC voltage. In this case, a DC power supply is provided instead of the AC power supply 33. Thus, while AC is preferred in this embodiment, particle capture by electrophoresis is also possible with DC.

[0015] As shown in Figure 2, electrode 31 of electrode pair 3 has a base portion 31a extending in the direction FD of fluid flow and a plurality of lines 31b extending from the base portion 31a in a direction perpendicular to the direction FD. Similarly, electrode 32 of electrode pair 3 has a base portion 32a extending in the direction FD of fluid flow and a plurality of lines 32b extending from the base portion 32a in a direction perpendicular to the direction FD. The width of lines 31b and line 32b is, for example, 40 μm. In the direction FD of fluid flow, lines 31b and line 32b are arranged alternately. That is, lines 31b and line 32b constitute interdigitated electrodes. When the AC power supply 33 is turned on, an AC voltage is applied between adjacent lines 31b and line 32b in the direction FD of fluid flow. Lines 31b and line 32b correspond to the first and second parts in this embodiment, respectively. As shown in Figure 2, adjacent lines 31b and 32b in the direction of fluid flow FD are spaced apart from each other.

[0016] The configuration of electrode pair 3 is not limited to those shown in Figures 1 and 2. For example, in electrode pair 3, at least one of bases 31a and 32a may be a metal foil such as copper foil connected in parallel to lines 31b and 32b, respectively. Alternatively, instead of at least one of bases 31a and 32a, wires or cables may be provided to connect lines 31b and 32b in parallel to one end and the other end of the AC power supply 33, respectively.

[0017] The capture film 4 is provided on the electrode pair 3 and covers at least a portion of the electrode pair 3. More specifically, the capture film 4 is provided on electrodes 31 and 32 and covers at least a portion of lines 31b and 32b. The capture film 4 is provided along at least one surface of the flow path. In this embodiment, as will be described later, the capture film 4 is provided along the bottom surface of the fluid channel. The capture film 4 is an insulating film such as a photoresist.

[0018] The capture membrane 4 is provided with various wells for capturing particles. Specifically, at least one connected well 41 is formed on the upstream side of the capture membrane 4 in the direction of fluid flow FD. As will be described in detail later, the connected well 41 according to this embodiment has a so-called skewer shape, in which multiple circular first wells 41a are connected. Furthermore, at least one second well 42 is formed on the downstream side of the capture membrane 4 in the direction of fluid flow FD, below the connected well 41. The connected well 41 and the second well 42 include through holes provided in the capture membrane 4 and lines 31b and 32b located on the lower surface of the through holes. Various wells such as the connected well 41, the first well 41a, and the second well 42 are also called microwells. In this embodiment, an example in which the connected well 41 is placed on the upstream side of the capture membrane 4 and the second well 42 is placed on the downstream side is described as a preferred example, but the configuration is not limited to this. For example, the arrangement of the second well may be omitted, and the capture membrane 4 may be constructed by arranging the connected wells 41 side by side. Alternatively, a connecting well 41 with a smaller diameter first well 41a may be placed on the upstream side, and a connecting well 41 with a larger diameter first well 41a may be placed on the downstream side.

[0019] The height, or thickness, of the capture film 4 is, for example, 3.6 to 4 μm. The height of the capture film 4 may differ between the portion where the connecting well 41 is formed and the portion where the second well 42 is formed. As another example, the capture film 4 may be divided into the portion where the connecting well 41 is formed and the portion where the second well 42 is formed. As yet another example, the capture film 4 may have different heights for each portion covering each line, or it may be divided into portions covering each line.

[0020] In this embodiment, the bottom of the connecting well 41 exposes the upper surface of either line 31b or line 32b. That is, the connecting well 41 is provided on either line 31b or line 32b and is not provided so as to straddle both lines 31b and line 32b. Similarly, the bottom of the second well 42 exposes the upper surface of either line 31b or line 32b. That is, the second well 42 is provided on either line 31b or line 32b and is not provided so as to straddle both lines 31b and line 32b. This makes it easy to form the connecting well 41 and the second well 42 and avoids a decrease in dielectrophoretic force when the size of the wells is reduced.

[0021] Furthermore, in this embodiment, the multiple connected wells 41 are offset in a direction perpendicular to the direction FD for each line. For example, in Figure 2, the connected well 41 on line 32b is offset in a direction perpendicular to the direction FD of fluid flow relative to the connected well 41 on the adjacent line 31b. In this way, the connected wells 41 located downstream are offset in a direction perpendicular to the direction FD of fluid flow relative to the connected wells 41 located upstream. This makes it easier for particles that could not be captured in the connected wells 41 on one line to be captured in the connected wells 41 on other lines, thereby improving the capture efficiency of the connected wells 41. Similarly, the second well 42 located downstream is offset in a direction perpendicular to the direction FD of fluid flow relative to the second well 42 located upstream. This improves the capture efficiency of the second well 42. In this embodiment, a preferred example is described in which the connected wells 41 are offset in a direction perpendicular to the direction FD of fluid flow and arranged in an inclined direction, but the configuration is not limited to this. For example, the arrangement direction of the connected wells 41 may be along the direction FD or perpendicular to FD.

[0022] As shown in Figure 1, the cover member 5 is provided to cover the capture membrane 4. The cover member 5 is made of silicone rubber such as polydimethylsiloxane (PDMS). The cover member 5 comprises, for example, a frame portion 51 and a lid portion 52. The frame portion 51 is provided with an opening 53 for forming a fluid channel through which fluid flows. The lid portion 52 is provided with an inlet opening 54 and an outlet opening 55 for this fluid channel. At least one of the openings 54 and 55 is connected to a pump or syringe (not shown), and fluid flows into the fluid channel through the opening 54 and is discharged from the fluid channel through the opening 55.

[0023] In the example shown in Figure 1, the corners of the frame portion 51 and the lid portion 52 of the cover member 5 are fixed to the corners of the base plate 2 by screws or the like. More specifically, the frame portion 51 of the cover member 5 is fixed on the base plate 2, and the lid portion 52 of the cover member 5 is fixed on top of the frame portion 51. The cover member 5 may also be fixed to the base plate 2 by adhesive or the like. Furthermore, the frame portion 51 and the lid portion 52 of the cover member 5 may be formed as a single unit.

[0024] <Details of Linkwell> Next, the connected well 41 according to this embodiment will be described in detail with reference to Figure 3. Figure 3 is a schematic plan view of the connected well 41 according to one embodiment.

[0025] As shown in Figure 3, the connected well 41 according to this embodiment has a plurality of first wells 41a that are connected to each other. In this embodiment, the plurality of first wells 41a are connected to each other by connecting wells 41b.

[0026] Furthermore, in this embodiment, in the connecting well 41, each of the multiple first wells 41a is circular, and the connecting well 41b is rectangular. Thus, the connecting well 41 according to this embodiment has a skewer-like shape, with the first wells 41a forming the dumpling portion and the connecting well 41b forming the skewer portion. At least one of the multiple first wells 41a may be elliptical, or a polygon such as a hexagon or octagon. Also, the width of the middle portion of the connecting well 41b may be wider or narrower than the width of the portion where the connecting well 41b and the first well 41a meet.

[0027] In the linked wells 41, the size of each of the first wells 41a, i.e., the diameter, is set to be less than or equal to the size of the first target particle, which is a particle intended to be captured in the linked wells 41. The first target particle is, for example, a small single cell such as a lymphocyte or red blood cell, and its diameter is set to match the size of such a single cell. While it is preferable for the first target particle to be a single cell such as a lymphocyte or red blood cell, it may also be applied to a cluster consisting of at least one of lymphocytes and red blood cells. The size of particles such as the first target particle can be expressed, for example, by the diameter, major axis, or minor axis of the particle, but in this embodiment, the diameter will be described as "size". If the size of the first target particle extends within a certain range, the size of the first well 41a is set to be, for example, below the lower limit of that range. More specifically, the size of the first well 41a may be set to be about 2 μm smaller than the size of the first target particle. This is because if the size of the first well 41a is larger than the size of the first target particle, there is a risk that larger diameter particles other than the target particle will be nonspecifically captured. By making the size of the first well 41a smaller by a certain percentage (for example, about 20%) than the size of the first target particle, larger diameter particles will not be captured, while the first target particle can be captured in a state where it rests on top of the first well 41a. On the other hand, the size of the first well 41a is set to be larger than the radius of the first target particle. This is because if the size of the first well 41a is very small, the effect of dielectrophoresis will not be sufficiently obtained, and a force sufficient to capture the first target particle will not be generated. Alternatively, the size of the first well 41a may be determined, for example, by the size of the first target particle and the height of the capture film 4 in the part where the first well 41a is formed. The height of the capture film 4 should be set so that a lower height and a closer distance between the electrode and the first target particle ensures a stronger capturing force, while also allowing a portion of the first target particle to be pulled into the inside of the first well 41a. In this embodiment, the size of the particles to be captured by the first well 41a is assumed to be about 8 μm, and the diameter of the first well 41a is 6 μm.

[0028] In this embodiment, the sizes of the multiple first wells 41a in the connected well 41 are equal to each other. However, the size of at least one of the multiple first wells 41a may differ from that of the other first wells 41a. For example, the size of the first wells 41a may gradually increase from one end of the connected well 41 to the other.

[0029] Furthermore, in this embodiment, in the connected well 41, the multiple first wells 41a are aligned in a direction AD that is oblique to the direction FD in which the fluid flows. That is, on the capture membrane 4, the direction AD in which the multiple first wells 41a are aligned intersects the direction FD in which the fluid flows at an angle. The angle between direction AD and direction FD is such that, for example, when a first target particle is captured in the forward, i.e., upstream first well 41a in one connected well 41, it does not prevent the capture of other first target particles in the backward, i.e., downstream first well 41a. Specifically, the angle between direction AD and direction FD is such that a certain first well 41a and the first well 41a connected immediately behind it are offset by, for example, a length of 0.8 to 1.0 times the diameter of the first well 41a in a direction perpendicular to direction FD.

[0030] The angle at which the axial direction AD of the connected wells 41 is inclined with respect to the direction FD, which is the direction of fluid flow, will be explained. According to the structure of the connected wells 41 in this embodiment, as will be described later, even if the leading first well 41a fails to capture the first target particle in the flow path and the first target particle flows downstream, the subsequent connected first wells 41a can capture the first target particle, and as a result, the capture capability of the first target particle is improved. Now, consider the case where the axial direction AD is inclined at an angle of 90 degrees with respect to the direction FD, which is the direction of fluid flow, that is, an angle perpendicular to direction FD. At this angle, if any of the first wells 41a of the connected wells 41 fail to capture the first target particle, the other connected first wells 41a cannot exert the ability to capture it either. On the other hand, consider the case where the angle of direction AD is parallel to direction FD. At this angle, the ability of the subsequent first wells 41a to capture the first target particle that the leading first well 41a has missed can be ensured. However, if a first target particle is already trapped in the leading first well 41a of the connected channels, even if a second first target particle flows into the same connected well 41, the first target particle already trapped in the leading first well 41a will obstruct its flow into the subsequent first well 41a. Therefore, subsequent first wells 41a in the connected channels cannot trap the second and subsequent first target particles, resulting in a decrease in the trapping capacity of the series of connected wells 41. Furthermore, when the direction AD is at an angle parallel to the direction FD, the area occupied by the connected wells 41 in the lateral width direction of the channel is narrow, thus narrowing the particle trapping range in the channel.

[0031] For the reasons stated above, tilting direction AD diagonally with respect to direction FD enhances the ability of the connected series of first wells 41a to capture the first target particle, and also ensures a wide capture range in the lateral direction of the flow path. It is preferable that the angle of inclination with respect to direction FD is not 45 degrees, but rather an angle that is offset by a length of 0.8 to 1.0 times the diameter of the first well 41a in the direction perpendicular to direction FD, so that direction AD and direction FD form an acute angle. This is because, if the leading first well 41a fails to capture the first target particle in the connected well 41, the electrophoretic force generated by the connecting well 41b and subsequent first wells 41a attached to the leading first well 41a can pull the first target particle towards the axis of the connected well 41, causing it to move in a clinging manner. In order to ensure a wide range over which the series of connected wells 41 can exert their trapping ability in the lateral direction of the flow path, it is geometrically desirable to make the angle of inclination of direction AD obtuse, at the expense of the ability to recapture any missed first target particles. However, due to the aforementioned force that attracts the first target particles, by setting the angle between direction AD and direction FD to an acute angle, it is possible to ensure a range over which the series of connected wells 41 can exert their trapping ability in the lateral direction of the flow path, while also ensuring the ability to recapture any missed first target particles. Note that the angle between direction AD and direction FD is not limited to the above range and can be set to various values.

[0032] Furthermore, in this embodiment, the width of the connecting well 41b is smaller than the size of each first well 41a. Here, the width of the connecting well 41b is the length of the connecting well 41b in the direction perpendicular to direction AD. In this embodiment, the width of the connecting well 41b is 3 μm.

[0033] The presence of the connecting well 41b makes the distance d1 between the centers of adjacent first wells 41a greater than the size of each first well 41a. The distance d1 is set to be equal to, for example, the size of the first target particle that is intended to be captured by the connecting well 41. If the size of the first target particle spans a certain range, the distance d1 is set to be equal to, for example, the upper limit of that range. In this embodiment, the distance d1 is 8 μm.

[0034] In the example described above, the linked well 41 had four first wells 41a. However, it is not limited to this, and the number of first wells 41a in the linked well 41 may be three or fewer, or five or more. By increasing the number of first wells 41a in the linked well 41, the number of first target particles captured by the particle capture device 1 can be increased. However, the number of first wells 41a in the linked well 41 should be such that the linked well 41 does not extend beyond the line 31b or line 32b of the electrode pair 3, or that the linked well 41 is located in the center of the line 31b or line 32b where the electric field is relatively uniform.

[0035] Furthermore, in the above example, the multiple first wells 41a in the connected well 41 were arranged in a straight line. However, the configuration is not limited to this; the multiple first wells 41a in the connected well 41 may be arranged in a curved line, or they may be arranged in a way that bends in the middle. For example, in the connected well 41 shown in Figure 3, the first half of the first wells 41a may be tilted to the left relative to the figure, and the second half of the first wells 41a may be tilted to the right relative to the figure, thereby forming a "V" shaped connected well 41. Alternatively, when providing first wells 41a in multiple rows, the first wells 41a in one row may be tilted to the left in Figure 3, and the first wells 41a in another row may be tilted to the right in Figure 3. However, it is preferable to configure the first wells 41a in all rows to be tilted to the same side so that particles that cannot be captured by the first wells 41a in one row can be captured by the first wells 41a in subsequent rows.

[0036] <Second Well Details> As shown in Figure 2, the second well 42 is a single circular well. The size of the second well 42, i.e., its diameter, is larger than the size of each of the multiple first wells 41a. The size of the second well 42 is set to be, for example, greater than or equal to the size of the second target particle that is intended to be trapped in the second well 42, and less than or equal to twice the size of the second target particle. This prevents more than one second target particle from being trapped in a single second well 42. The second target particle is, for example, a circulating tumor cell or a cluster containing circulating tumor cells. In this embodiment, the diameter of the second well 42 is 12 μm. The distance between the second wells 42 is also greater than, for example, the distance d1 between the centers of adjacent first wells 41a in a connected well 41. In this embodiment, the distance between the second wells 42 is 50 μm. The second well 42 may be elliptical, or it may be a polygon such as a hexagon or octagon. The diameter of the second well 42 may be any within the range of 10 to 22 μm.

[0037] <Operation of the particle capture device> Next, an example of the operation of the particle capture device 1 will be described with reference to Figures 4 and 5. Figure 4 is a cross-sectional view showing an example of the operation of the particle capture device 1 according to one embodiment. Figure 5 is an enlarged view showing an example of the operation of the particle capture device 1 according to one embodiment, and is an enlarged view of the area around the connecting well 41.

[0038] As shown in Figure 4, in the particle capture device 1 according to this embodiment, a fluid channel FC through which fluid flows is defined by the electrode pair 3, the capture membrane 4, and the cover member 5. More specifically, the electrode pair 3 and the capture membrane 4 constitute the bottom surface of the fluid channel FC, the frame portion 51 of the cover member 5 constitutes the side surface of the fluid channel FC, and the lid portion 52 of the cover member 5 constitutes the top surface of the fluid channel FC. In addition, the openings 54 and 55 of the cover member 5 constitute the inlet and outlet of the fluid channel FC, respectively.

[0039] The fluid contains, for example, particles P1 to P4. For instance, particle P1 is a single cell released into the fluid, particle P2 is a cluster of cells released into the fluid, particle P3 is a single cell trapped in linked well 41, and particle P4 is a cluster of cells trapped in second well 42.

[0040] As shown in Figure 5, when an AC voltage is applied between lines 31b and 32b, an electric field EF is formed between lines 31b and 32b. This electric field EF exerts a dielectrophoretic force F on particles P in the fluid. DEP Provides sufficient dielectrophoretic force F. DEP Given a force F acting on particle P, flow The particles resist the current and are absorbed and captured in various wells, such as the connected well 41. In this way, the particle capture device 1 captures particles contained in the fluid flowing in the flow direction FD into various wells by dielectrophoresis.

[0041] <Particles targeted for capture> Next, with reference to Figure 6, we will describe in more detail the first target particle, which is intended to be captured in the linked well 41, and the second target particle, which is intended to be captured in the second well 42.

[0042] Figure 6 shows an example of a target particle in a particle capture device 1 according to one embodiment. In Figure 6, (a) represents a lymphocyte. The size of this lymphocyte is 6-8 μm. (b) is a cluster consisting of two lymphocytes, with a distance of 6-8 μm between the centers of the lymphocytes. (c) is a cluster consisting of one lymphocyte and one circulating tumor cell (CTC), with a distance greater than 8 μm between the centers. (d) represents a single circulating tumor cell. The size of a circulating tumor cell is generally greater than 10 μm. (e) is a cluster consisting of a circulating tumor cell and a neutrophil, with a distance greater than 10 μm between the centers. Note that the clusters shown in (c) and (e) correlate with poor prognosis in patients.

[0043] In Figure 6, the "Trap" row indicates whether or not these particles are captured by the linked well 41. Particles marked with ◎ can be captured well, and a certain level of capture capability is also observed for particles marked with ▼. On the other hand, particles marked with × are not captured. That is, the lymphocyte in (a) is well captured by the linked well 41, and the cluster of two lymphocytes in (b) can also be captured by the linked well 41. On the other hand, as shown in (c) to (e), a single circulating tumor cell and a cluster containing circulating tumor cells are not easily captured by the linked well 41. In other words, in this embodiment, the particles in (a) and (b) are examples of first target particles that are intended to be captured by the linked well 41. At least some of the particles in (c) to (e) are examples of second target particles that are intended to be captured by the second well 42. In other words, by using the structure of the linked well 41, particles of the target particle size can be selectively and efficiently captured, while particles larger than the target size can be allowed to flow downstream without being captured.

[0044] The ability of the connected wells 41 to selectively capture particles of a desired size will be explained below with reference to Figures 7 to 18.

[0045] <Electric fields formed in various wells> First, to clarify the effect of various wells on particle trapping, we will describe the results of a comparative simulation analysis of the magnitude of the electric field strength gradient, performed using the COMSOL Multiphysics® software. The magnitude of the electric field strength gradient is proportional to the dielectrophoretic force that directly acts on particle trapping. More specifically, the dielectrophoretic force per unit area acting on a particle is proportional to the square of the gradient of the electric field strength (∇E 2 It is proportional to ). The results of this simulation are shown in Figure 7.

[0046] Figure 7 shows the simulation results of electric fields formed in various wells in a particle capture device according to one embodiment and a comparative example. In Figure 7, (a), (b), and (c) represent examples of electric fields formed in single, circular wells with diameters of 18 μm, 12 μm, and 6 μm, respectively. Of these, (b) corresponds to the second well 42 of this embodiment. (d) represents an example of an electric field formed in a connected well 41 according to this embodiment. (e) represents an example of an electric field formed in an unconnected well 410 according to a comparative example. The unconnected well 410 has a plurality of first wells 41a that are not connected to each other. That is, the unconnected well 410 is a well that has a cluster portion but no skewer portion. In the example of Figure 7(e), the diameter of each first well 41a in the unconnected well 410 is 6 μm. Also, the distance between the centers of adjacent first wells 41a in the unconnected well 410 is 8 μm, the same as in the connected well 41. (f) represents the relationship between intensity and electric field strength in (a) to (e).

[0047] As shown in Figures 7(a) to (c), in a single, circular well, the gradient of the electric field strength per unit area increases as the size of the well, i.e., the diameter, decreases. Therefore, when a small well is provided alone, as in (c), the dielectrophoretic force becomes excessive, and there is a risk that particles larger than the target particles will be nonspecifically trapped in that well.

[0048] On the other hand, as shown in Figures 7(d) and 7(e), by providing multiple single wells at a high density, the magnitude of the electric field strength in each single well is suppressed. Furthermore, as shown in Figure 7(d), by connecting the single wells with connecting wells, the electric field strength is further suppressed and equalized overall. The electric field strengths of the connected wells 41 and the unconnected wells 410 will be described in more detail below with reference to Figure 8.

[0049] Figure 8 is a graph showing the magnitude of the gradient of the electric field strength formed on each first well 41a in a connected well 41 according to one embodiment and an unconnected well 410 according to a comparative example. (a) represents the sum of the electric field strengths on each first well 41a in the connected well 41 and the unconnected well 410. More specifically, the sum is calculated by integrating the electric field strengths at each location over the area. (b) represents the sum of the electric field strengths within a circle with a diameter of 8 μm centered on each first well 41a, i.e., in the lymphocyte region. In both (a) and (b), the lightly hatched graph represents the case of the connected well 41, and the darkly hatched graph represents the case of the unconnected well 410. Also, in both (a) and (b), it represents the sum of the electric field strengths at a height of 4.1 μm from the top surface of the electrode pair 3. Furthermore, #1 to #4 represent the sum of the electric field strengths centered on the first wells 41a located 1 to 4th from the upstream side, respectively.

[0050] As shown in Figure 8(a), at all positions #1 to #4, the sum of the electric field strengths on the first well 41a in the connected well 41 is smaller than the sum of the electric field strengths on the first well 41a in the unconnected well 410. Furthermore, the sum of the electric field strengths differs more significantly at positions #1 and #4 at both ends than at positions #2 and #3 in the center. In other words, in the connected well 41, the difference in electric field strength between the ends and the center is smaller compared to the unconnected well 410, indicating that the electric field strengths are more evenly distributed.

[0051] Furthermore, as shown in Figure 8(b), at positions #1 and #4 at both ends, similar to the above, the sum of the electric field strengths in the lymphocyte region of the connected well 41 is smaller than the sum of the electric field strengths in the lymphocyte region of the unconnected well 410. On the other hand, at positions #2 and #3 in the central part, the sum of the electric field strengths is greater in the connected well 41 than in the unconnected well 410. This is because in the connected well 41, a portion of the connected well 41b is present in the lymphocyte region, and the electric field strengths in the connected well 41b are added. Therefore, it can be said that the electric field strengths in the lymphocyte region are more evenly distributed in the connected well 41.

[0052] Based on the above, the connected well 41 allows for the suppression of the overall gradient of the electric field strength compared to a single well and an unconnected well 410, while also enabling the formation of an equalized electric field at both ends and in the center compared to an unconnected well 410.

[0053] <Second target particle capture experiment> Next, it will be explained that the connected well 41 according to this embodiment can suppress nonspecific capture of the second target particle compared to the unconnected well 410 according to the comparative example.

[0054] Figure 9 shows the results of a capture experiment of a second target particle TP2 in a particle capture device 1 according to one embodiment. In Figure 9, the dotted lines indicate connected wells 41 and 2nd well 42, which represent wells in which the second target particle TP2 was not captured, while the solid line indicates a well in which the second target particle TP2 was captured. Figure 10 shows the results of a capture experiment of a second target particle TP2 in a particle capture device according to a comparative example. In Figure 10, the solid line indicates an unconnected well 410, which represents a well in which the second target particle TP2 was not captured. The inverted triangle below it indicates that the second target particle TP2 was captured, and the arrow indicates the direction of movement of the uncaptured second target particle TP2. Figure 10(a) shows the state at time 0 seconds, and Figure 10(b) shows the state at time 7 seconds.

[0055] In the capture experiments shown in Figures 9 and 10, DU145 cells (prostate cancer cell line) were used as the second target particle TP2. The diameter of DU145 cells was 10 μm or more, with an average diameter of 13 μm.

[0056] As shown in Figure 9, the DU145 cells, which are the second target particle TP2, are not captured in the linked well 41, but are captured in the second well 42, which is located downstream of the linked well 41.

[0057] On the other hand, as shown in Figure 10, it can be seen that some DU145 cells are nonspecifically captured in the unconnected well 410. This is because, compared to the connected well 41 which can form an equalized electric field, the electric field strength is large and the electric field is non-uniform in the first well 41a at both ends of the unconnected well 410. As a result, regions with high dielectrophoretic force are locally generated, and the first well 41a nonspecifically captures even the second target particle TP2, which is not originally intended to be captured.

[0058] Therefore, in the connected well 41, a uniform electric field can be formed by providing a connecting portion, which suppresses the nonspecific capture of the second target particle TP2 compared to the unconnected well 410.

[0059] <Target Particle Capture Experiment (First Target)> Next, it will be explained that the connected well 41 according to this embodiment can improve the capture efficiency of the first target particle compared to the unconnected well 410 according to the comparative example.

[0060] Figure 11 shows the results of a capture experiment of a first target particle in a connected well 41 according to one embodiment and in an unconnected well 410 according to a comparative example. Each circle in Figure 11 represents a captured first target particle.

[0061] In the capture experiment shown in Figure 11, 100,000 Ramos cells (lymphoid hematopoietic cells) were used as the first target particles. The diameter of the Ramos cells ranged from 5.4 μm to 12.5 μm, with an average diameter of 6.9 μm. This capture experiment used a particle capture apparatus with multiple linked wells 41 and unlinked wells 410 arranged in a 1:1 ratio. More specifically, linked wells 41 were provided in the upper region R1 of Figure 11, and unlinked wells 410 were provided in the lower region R2. As shown in Figure 11, it can be seen that more Ramos cells were captured in the linked wells 41 compared to the unlinked wells 410.

[0062] Figure 12 is a graph showing the results of a capture experiment of first target particles in a connected well 41 according to one embodiment and an unconnected well 410 according to a comparative example, and is an aggregation of the results in Figure 11. Figure 12 shows the results of comparing the number of captured Ramos cells when the particle capture rate in the connected well 41 exceeds 50%. Figure 12(a) shows the ratio of first target particles captured in the connected well 41 and the unconnected well 410, respectively. In Figure 12(a), the symbol A represents the ratio captured in the connected well 41, and the symbol B represents the ratio captured in the unconnected well 410. Figure 12(b) shows the ratio of first target particles captured in each first well 41a at positions #1 to #4 for both the connected well 41 and the unconnected well 410. In Figure 12(b), the darkly hatched graphs represent the case of the connected well 41, and the lightly hatched graphs represent the case of the unconnected well 410.

[0063] As shown in Figure 12(a), more Ramos cells are captured in the linked well 41 than in the unlinked well 410. More specifically, the number of Ramos cells captured in the linked well 41 is more than 1.5 times the number of Ramos cells captured in the unlinked well 410. In other words, the capture efficiency in the linked well 41 is more than 1.5 times higher than that in the unlinked well 410.

[0064] Furthermore, as shown in Figure 12(b), in the linked well 41, the difference in capture rate between the first wells 41a located at both ends (#2 and #3) and the first wells 41a located at the center (#2 and #3) is small, indicating that the capture efficiency of Ramos cells in the center is significantly higher compared to the unlinked well 410. This is thought to be because, in the linked well 41, the magnitude of the electric field intensity gradient in the lymphocyte region of the first well 41a in the center is increased.

[0065] Thus, the linked well 41 can improve the capture efficiency of the first target particle compared to the unlinked well 410.

[0066] Furthermore, in the connected well 41, the first target particles that are not completely captured in the upstream first well 41a and become free are recaptured in the downstream first well 41a via the connecting well 41b, thereby improving the capture efficiency. This will be explained with reference to Figures 13 and 14.

[0067] <Recapture of the first target particle in the linked well> Figure 13 shows the results of another capture experiment of the first target particle TP1 in a connected well 41 according to one embodiment. Figure 14 shows the results of another capture experiment of the first target particle TP1 in an unconnected well 410 according to a comparative example. Figures 13 and 14 are time-lapses taken in the order (a) to (h), respectively. In Figures 13 and 14, the inverted triangle indicates that the first target particle TP1 is captured below it, and the arrow indicates the direction of movement of the first target particle TP1 that is not captured.

[0068] In the capture experiments shown in Figures 13 and 14, Ramos cells were used as the primary target particle TP1, similar to the capture experiment in Figure 11. Similarly, a particle capture apparatus was used in which multiple linked wells 41 and unlinked wells 410 were arranged in a 1:1 ratio. However, in this experiment, the AC voltage applied to electrode pair 3 was lowered by 1V compared to the capture experiment in Figure 11, and the fluid flow rate was increased by 0.3 μl / min, creating conditions that made particle capture more difficult.

[0069] As shown in Figure 13(a), when a Ramos cell, which is the first target particle TP1, approaches the junctional well 41, as shown in (b) to (d), the particle moves in a sawtooth pattern along the junctional well 41. As the particle moves along the junctional well 41, its movement speed decreases. Subsequently, as shown in (e), the particle is captured in the downstream first well 41a of the junctional well 41. That is, although the particle was not fully captured in the upstream first well 41a, it moves along the junctional well 41 and is captured again in the downstream first well 41a. Subsequently, as shown in (e) to (h), when another Ramos cell approaches the junctional well 41, this particle also moves along the junctional well 41 and is captured in the downstream first well 41a.

[0070] On the other hand, as shown in Figures 14(a) to (h), when the Ramos cell, which is the first target particle TP1, approaches the unconnected well 410, it does not exhibit the behavior described above. In other words, the direction of movement of the first target particle TP1 does not change significantly from the direction FD in which the fluid flows, and it does not behave as if it is moving along the unconnected well 410. Therefore, it can be seen that in the unconnected well 410, the particle moves linearly between the unconnected wells 410, and the capture efficiency is lower compared to the connected well 41.

[0071] The behavior of particles approaching the connected well 41 under dielectrophoresis will be explained with reference to Figures 15 and 16. Figure 15 shows the state of the first target particle TP1 on the connecting well 41b in the connected well 41 according to one embodiment. Figure 16 shows an example of the electric field on the connecting well 41b in the connected well 41 according to one embodiment. In Figure 16, each arrow represents the electric field formed at their respective distances from the electrode pair 3.

[0072] As shown in Figure 15, the connection well 41b has a height h1 and a width w. The height h1 is the height of the trapping film 4 in the portion where the connection well 41b is formed. When the first target particle TP1 is located on the connection well 41b, the lower end of the first target particle TP1 will be located below the upper surface of the trapping film 4 by a height h2. That is, the lower end of the first target particle TP1 is closer to the electrode pair 3 by a height h2 than the upper surface of the trapping film 4. As a specific example, if we assume that the diameter of the first target particle TP1 is 5.5 to 8.5 μm, then the height h2 will be 0.3 to 0.4 μm.

[0073] As shown in Figure 16, the electric field strength on the connecting well 41b increases significantly as it approaches the upper surface of the electrode pair 3. By providing the connecting well 41b, the distance between the first target particle TP1 and the electrode is kept short, and the dielectrophoretic force acting on the first target particle TP1 is maintained. As a result, the first target particle TP1 is less likely to dissociate from the connecting well 41. In the connecting well 41, even if the first target particle TP1 to be captured escapes capture in the leading first well 41a and flows downstream, the particle can be guided to the next connected first well 41a along the electric field emitted by the connecting well 41b. Therefore, the capture capability of the first target particle TP1 is improved in the connecting well 41 compared to the unconnected well 410.

[0074] Thus, in the connected well 41, the particles move along the connected well 41, allowing them to receive continuous electrophoretic force in the connected well 41b, thereby further improving the capture efficiency of the first target particle TP1.

[0075] <Experiment on the separation and capture of the first and second target particles> Next, we will describe the results of a sample capture experiment using the particle capture device 1 according to this embodiment, which captures a first target particle TP1 and a second target particle TP2.

[0076] Figure 17 shows the results of a separate capture experiment of a first target particle TP1 and a second target particle TP2 in a particle capture device 1 according to one embodiment. Each circle in Figure 17 represents the captured first target particle TP1 and second target particle TP2.

[0077] In the capture experiment shown in Figure 17, Ramos cells were used, similar to the capture experiment in Figure 11. Of the Ramos cells, particles with a diameter of 8.4 μm or less correspond to the first target particle TP1, and the remaining particles correspond to the second target particle TP2. As shown in Figure 17, it can be seen that Ramos cells of different sizes were captured in linked well 41 and the second well 42, respectively.

[0078] Figure 18 is a graph showing the results of a sorting and capture experiment of the first target particle TP1 and the second target particle TP2 in the particle capture device 1 according to one embodiment, and is an aggregation of the results in Figure 17. Figures 18(a) and 18(b) are histograms showing the Ramos cells captured in the linked well 41 and the second well 42 aggregated by diameter, respectively. In both Figures 18(a) and 18(b), the count is in 0.5 μm increments, and the left side of the dashed line represents the number of particles with a diameter of 8.4 μm or less.

[0079] As shown in Figure 18(a), Ramos cells with a diameter of 8.4 μm or less were captured in linked well 41. The diameters of the Ramos cells captured in linked well 41 ranged from 5.4 to 8.4 μm, with an average diameter of 6.87 μm. Furthermore, the Ramos cells captured in linked well 41 accounted for more than 95% of the total cells.

[0080] On the other hand, as shown in Figure 18(b), Ramos cells with a diameter of approximately 9.0 μm or larger were captured in the second well 42. The diameters of the Ramos cells captured in the second well 42 ranged from 7.6 to 12.1 μm, with an average diameter of 10.0 μm.

[0081] Thus, according to the particle capture device 1 of this embodiment, small Ramos cells can be selectively captured in the connected well 41 and the remaining Ramos cells can be captured in the second well 42. In other words, according to the particle capture device 1 of this embodiment, the first target particle TP1 can be selectively captured in the connected well 41 and the second target particle TP2 can be selectively captured in the second well 42.

[0082] <Effects and Effects> As described above, in the particle capture device 1 according to this embodiment, the connected wells formed in the capture membrane 4 have a plurality of first wells 41a that are connected to each other. This makes it possible to reduce the size of each first well 41a while avoiding a decrease in the total area of ​​the connected wells 41. Therefore, it is possible to avoid an unnecessary increase in dielectrophoretic force and suppress nonspecific capture. Accordingly, the particle capture device 1 according to this embodiment can selectively capture small particles among the particles contained in the fluid.

[0083] Traditionally, when separating particles such as cells by size, the filtration method is used. In the filtration method, a solution containing particles is drawn under negative pressure and passed through a filter, trapping larger particles on the filter and allowing smaller particles to pass below. However, in the filtration method, the particles are deformed by physical compression, making highly accurate separation by particle size difficult. Furthermore, even if the target particles include smaller particles, those target particles will be lost.

[0084] According to this embodiment, by capturing particles contained in the fluid using dielectrophoresis, deformation of the particles during capture is avoided compared to the filter method, and highly accurate separation based on particle size can be facilitated. Furthermore, unlike the filter method, even if the target particles include smaller particles, they can be detected without being discarded.

[0085] Furthermore, by having multiple first wells 41a in the connected well 41, the number of first wells 41a in the particle capture device 1 can be increased, allowing for the capture of more first target particles TP1. This is effective, for example, when the majority of particles in the fluid are first target particles TP1.

[0086] Furthermore, in the connected wells 41, the electric field strength between the first wells 41a is equalized compared to the unconnected wells 410 because multiple first wells 41a are connected to each other by connecting wells 41b. More specifically, the dielectrophoretic force is suppressed in the first wells 41a at both ends, further suppressing the nonspecific capture of particles larger than the first target particle TP1. On the other hand, in the first well 41a in the center, the electric field strength gradient is increased by connecting wells 41b to both ends, thereby increasing the capture efficiency of the first target particle TP1. Moreover, particles that have been captured in one first well 41a but have dissociated can be captured again in the adjacent first well 41a. Therefore, according to the connected wells 41 of this embodiment, the capture efficiency of the first target particle TP1 can be improved in all first wells 41a compared to the unconnected wells 410.

[0087] In the connected wells 41, it is preferable that the distance d1 between the centers of adjacent first wells 41a is greater than the size of the first wells 41a and does not exceed the diameter of the second target particle TP2. First, in the connected wells 41, because the distance d1 between the centers of adjacent first wells 41a is greater than the size of the first wells 41a, the connected wells 41 can efficiently capture first target particles TP1 over a certain size range. That is, when a first target particle TP1 is captured in one first well 41a, another first target particle TP1 can be captured in the adjacent first well 41a. In this embodiment, since the diameter of the first wells 41a is 6 μm and the distance d1 is 8 μm, the connected wells 41 can capture particles with diameters of, for example, 6 μm to 8 μm.

[0088] On the other hand, in the linked well 41, the distance d1 between the centers of adjacent first wells 41a is set to be shorter than the diameter of the second target particle TP2. For example, it is preferable that the distance d1 between the centers of the first wells 41a be approximately the same as the size of the first target particle TP1. That is, the distance d1 is smaller than the size of the second target particle TP2. This suppresses the nonspecific capture of clusters consisting of, for example, the first target particle TP1 and the second target particle TP2 in the linked well 41. As a specific example, a heterocluster of lymphocytes and circulating tumor cells, as shown in Figure 6(c), is suppressed from being captured in the linked well 41 because the distance between its centers is greater than the distance d1.

[0089] Furthermore, in the connected wells 41, the multiple first wells 41a are arranged in a direction AD that is oblique to the direction FD in which the fluid flows. This makes it easier for multiple first target particles TP1 to be captured in a single connected well 41. For example, when a first target particle TP1 is captured in the upstream first well 41a, other first target particles TP1 will go around the captured first target particle TP1 and be captured in the rear first well 41a. This further improves the capture efficiency of the first target particles TP1. In addition, by making the angle between direction FD and direction AD smaller, the number of connected wells 41 that can be installed per unit area in the particle capture device 1 can be increased, and the number of first target particles TP1 that can be captured per unit area can be improved.

[0090] Furthermore, because the size of each of the multiple first wells 41a is less than or equal to the size of the first target particle TP1, the first target particle TP1 is not dropped into the connected wells 41, but is instead captured by resting on top of it. This allows one particle that is a certain size larger than the first well 41a to be captured in each first well 41a, thereby improving the particle capture efficiency. Note that the diameter of the first well 41a may be larger than the diameter of the first target particle TP1 but less than twice as large. In this case, it is possible to prevent two or more first target particles TP1 from being captured in a single first well 41a.

[0091] Furthermore, since the second well 42 is formed downstream of the connecting well 41 in the capture membrane 4, the second target particle TP2 can be selectively captured from the fluid after the first target particle TP1 has been captured. In other words, the particles contained in the fluid can be arrayed according to their size. Also, since smaller particles generally tend to flow faster in the fluid than larger particles, the capture efficiency of the particle capture device 1 can be improved by placing the connecting well 41, which captures small particles, on the upstream side and the second well 42, which captures large particles, on the downstream side.

[0092] (Variation 1) Note that each first well 41a of the connecting well 41 may be connected to one another without going through the connecting well 41b. Hereinafter, such a case will be described as Modification 1 with reference to Figure 19. Figure 19 is a schematic plan view of the connecting well 41A according to Modification 1 of one embodiment.

[0093] As shown in Figure 19, the connected well 41A in this modified example has a plurality of first wells 41a that are connected to each other. In this modified example, the plurality of first wells 41a are directly connected to each other without the need for a connecting well 41b. A narrow portion 41c is formed between the plurality of first wells 41a, and the width of the narrow portion 41c is narrower than the width of the first well 41a. The width of the narrow portion 41c is equal to, for example, the width of the connecting well 41b described above. Note that the distance d2 between the centers of the first wells 41a in this modified example is smaller than the distance d1 between the centers of the first wells 41a in the embodiment.

[0094] In this modified example, by connecting multiple first wells 41a to each other, it is possible to reduce the size of each first well 41a while avoiding a decrease in the total area of ​​the connected wells 41. In other words, according to this modified example, as in the embodiment, it is possible to selectively capture smaller particles among the particles contained in the fluid.

[0095] (Modification 2) Referring to Figure 20, a particle capture device 1A according to Modification 2 of one embodiment will be described. Figure 20 is a plan view of the particle capture device 1A according to Modification 2 of one embodiment. One of the differences between this modification and the embodiment described above is the presence or absence of the third well 43. Hereinafter, this embodiment will be described focusing on the differences from the embodiment described above.

[0096] As shown in Figure 20, in this modified example, at least one third well 43 is further formed in the trapping membrane 4 downstream of the connecting well 41 in the fluid flow direction FD. Each third well 43 is, for example, a single circular well. In the example in Figure 20, multiple third wells 43 are provided downstream of the second well 42. The third wells 43 may be provided downstream of the connecting well 41 and upstream of the second well 42. Alternatively, the third wells 43 may be provided on the same line as the second well 42, or they may be provided mixed with the second well 42.

[0097] The size of the third well 43, i.e., its diameter, is larger than the size of each of the multiple first wells 41a, and different from the size of the second well 42. In the example in Figure 20, the size of the third well 43 is larger than the size of the second well 42. However, the size of the third well 43 may also be smaller than the size of the second well 42.

[0098] According to this modified version, for example, relatively small particles from the second target particles TP2 can be selectively captured in the second well 42, while the remaining particles are captured in the third well 43. In other words, particles contained in the fluid can be separated and captured into a wider range of sizes.

[0099] (Variation 3) Referring to Figure 21, a particle capture device 1B according to Modification 3 of one embodiment will be described. Figure 21 is a schematic diagram of the particle capture device 1B according to Modification 3 of one embodiment. This modification has a larger single well than the particle capture device 1A according to Modification 2 described above. The following description of this embodiment will focus on the differences from Modification 2.

[0100] As shown in Figure 21, the particle capture device 1B comprises a connected well 41 in which four first wells 41a with a diameter of 6 μm are connected to each other, a second well 42 with a diameter of 12 μm, and third wells 43 with diameters of 15, 18, and 22 μm. Here, the connected wells 41 are for capturing lymphocytes, a type of white blood cell (WBC), and the total number of cells in the first wells 41a is 3 to 4 × 10⁶. 5 The wells are designed to capture individual circulating tumor cells (CTCs) and relatively small clusters containing circulating tumor cells. The third well, 43, is designed to capture relatively large clusters containing circulating tumor cells.

[0101] According to this modified example, particles contained in the fluid can be separated and captured into even more sizes. The wells provided in the particle capture device 1B are arranged such that small particles are captured on the upstream side and large particles are captured on the downstream side, with a connected well 41 and a single well, the second well 42. This arrangement is not limited to the combination of connected wells and a single well as shown in Figure 21. For example, the diameter of the particles captured on the upstream and downstream sides can be switched by placing a connected well 41 with a small diameter of the first well 41a upstream and a connected well 41 with a large diameter of the first well 41a downstream.

[0102] (Modification 4) Referring to Figure 22, a particle capture device 1C according to Modification 4 of one embodiment will be described. Figure 22 is a plan view of the particle capture device 1C according to Modification 4 of one embodiment. One of the differences between this modification and the embodiment described above is the configuration of the electrode pair. The following description of this embodiment will focus on the differences from the embodiment described above.

[0103] As shown in Figure 22, the particle capture device 1C according to this modified example comprises two electrode pairs, namely electrode pair 3A and electrode pair 3B. Electrode pair 3A is provided on the upstream side in the direction of fluid flow FD, and electrode pair 3B is provided on the downstream side. Electrode pair 3A and electrode pair 3B are electrically connected to different AC power sources, AC power source 33A and AC power source 33B, respectively. A connecting well 41 is provided above electrode pair 3A, while a second well 42 is not provided. Similarly, a second well 42 is provided above electrode pair 3B, while a connecting well 41 is not provided. Electrode pair 3A and electrode pair 3B correspond to the first electrode pair and second electrode pair in this modified example, respectively.

[0104] In this modified example, particles trapped in the second well 42 can be released separately from particles trapped in the connecting well 41. For example, after trapping particles in the connecting well 41 and the second well 42, the AC power supply 33B is turned off, and the AC voltage applied to the electrode pair 3B is turned off. This eliminates the dielectrophoretic force acting on the particles trapped in the second well 42, and by flowing fluid, the particles trapped in the second well 42 can be dissociated and discharged from the outlet. On the other hand, since the AC power supply 33A remains on, the trapped state of the particles trapped in the connecting well 41 is maintained while the fluid is flowing. Subsequently, the AC power supply 33A is turned off, and the AC voltage applied to the electrode pair 3A is turned off. This eliminates the dielectrophoretic force acting on the particles trapped in the connecting well 41, and by flowing fluid, the particles trapped in the connecting well 41 can be dissociated and discharged from the outlet.

[0105] According to this modified version, the particles trapped in the connected well 41 and the particles trapped in the second well 42 can be recovered separately and easily used for subsequent analysis.

[0106] Furthermore, in the particle capture device 1 according to the embodiment shown in Figure 2, particles captured in the second well 42 can be released separately from particles captured in the connected well 41. In this case, for example, while an AC voltage is applied, the electrical connection between the line 31b and line 32b where the second well 42 is located and the AC power supply 33 is severed by cutting the base 31a and base 32a near the center into two sections, upstream and downstream, using a laser or the like.

[0107] (Variation 5) Referring to Figure 23, a particle capture device 1D according to Modification 5 of one embodiment will be described. Figure 23 is a plan view of the particle capture device 1D according to Modification 5 of one embodiment. This modification corresponds to Modification 4 in which the electrode pair and AC power supply are further divided. The following description of this embodiment will focus on the differences from the embodiments described above.

[0108] As shown in Figure 23, the particle capture device 1D according to this modified example comprises four electrode pairs 3C. Each electrode pair 3C comprises an electrode 31C and an electrode 32C. Electrodes 31C and 32C are connected to one end and the other end of an AC power supply 33C, respectively. The electrode pair 3C where the connecting well 41 is located corresponds to the first electrode pair in this modified example, and the electrode pair 3C where the second well 42 is located corresponds to the second electrode pair in this modified example. In the example of Figure 23, electrodes 31C and 32C each constitute a line. The number of electrode pairs 3C in the particle capture device 1D may be three or fewer, or five or more.

[0109] In this modified version, particles trapped in the second well 42 can be released separately from particles trapped in the connected well 41. In this modified version, trapped particles can be collected line by line.

[0110] In this modified example, an AC power supply 33C is connected to each pair of lines. However, this is not limited to this, and an AC power supply may be connected to each line. This allows captured particles to be recovered line by line. In this case, for example, the same number of AC power supplies as the number of lines are prepared, one end of each AC power supply is connected to each line, and the other end of each AC power supply is connected to a common ground. In this case, each combination of line and ground corresponds to one electrode pair. Another modified example is to change the voltage applied to each line. This makes it possible to change the electrophoretic force and capture capacity even with linked wells 41 of the same shape or a single second well 42. For example, even in a series of linked wells 41, by lowering the voltage on the upstream side and raising the voltage on the downstream side, it is possible to prevent particles captured on the upstream side from becoming too dense, causing them to overlap and obstruct the flow path of the particles.

[0111] On the other hand, keeping the applied voltage constant for each line simplifies control and is expected to lead to more stable operation. In the embodiment shown in Figure 2, a connecting well 41 is provided on the upstream side of the flow path, and a large-diameter second well is provided on the downstream side, with the same voltage applied to each well. Compared to a configuration that creates a difference in capture capability between the upstream and downstream sides by providing a second well 42 to which a weak voltage is applied on the upstream side and a second well 42 to which a strong voltage is applied on the downstream side, the configuration of the embodiment shown in Figure 2 allows for a difference in capture capability between the upstream connecting well 41 and the downstream second well 42, even while applying the same voltage.

[0112] In the above explanation, the particles contained in the fluid were described in terms of living cells. However, the particles contained in the fluid are not limited to this; they may also be living organisms such as bacteria or viruses, biomacromolecules such as DNA, RNA or proteins, various dielectrics including resins or colloids, or conductors such as metal particles.

[0113] According to at least one embodiment described above, it is possible to selectively capture smaller particles among the particles contained in the fluid.

[0114] Although several embodiments have been described above, these embodiments are presented only as examples and are not intended to limit the scope of the invention. The novel apparatus and methods described herein can be implemented in a variety of other forms. Furthermore, various omissions, substitutions, and modifications can be made to the embodiments of the apparatus and methods described herein, without departing from the spirit of the invention. The appended claims and equivalents are intended to include such embodiments and modifications that are included in the scope and spirit of the invention. [Explanation of symbols]

[0115] 1 Particle capture device 2 Bottom plate 3 electrode pairs 31,32 electrode 31b, 32b lines 33 AC power supply 4. Capture membrane 41 Linked Wells 41a First Well 41b Connection well 42. Second Well 5 Cover component FC fluid channel TP1 1st target particle TP2 2nd target particle

Claims

1. A particle capture device that captures particles contained in a fluid flowing through a channel by dielectrophoresis, A pair of electrodes to which voltage is applied, A trapping membrane provided on the electrode pair and along at least one surface of the flow path, wherein the trapping membrane has connected wells for capturing particles, and the connected wells comprise a trapping membrane having a plurality of first wells connected to each other. A particle capture device equipped with the following features.

2. The particle capture device according to claim 1, wherein in the connected well, the plurality of first wells are connected to each other by connecting wells that are narrower than the size of each first well.

3. The particle capture device according to claim 2, wherein each of the plurality of first wells is circular and the connecting well is rectangular.

4. The particle capture device according to claim 2, wherein in the connected wells, the distance between the centers of adjacent first wells is greater than the size of each first well.

5. The particle capture device according to claim 2, wherein in the connected wells, the distance between the centers of adjacent first wells is equal to the size of a first target particle that is intended to be captured in the connected wells.

6. The particle capture device according to claim 1, wherein in the connected wells, the plurality of first wells are arranged in a direction oblique to the direction in which the fluid flows.

7. The particle capture device according to claim 1, wherein the size of each of the plurality of first wells is less than or equal to the size of a first target particle that is intended to be captured in the connected wells.

8. The particle capture device according to claim 1, wherein the size of each of the plurality of first wells is less than twice the size of the first target particle that is intended to be captured in the connected wells.

9. The particle capture device according to claim 1, wherein the sizes of the plurality of first wells are equal to each other.

10. The particle capture device according to claim 1, wherein the first target particle, which is intended to be captured in the linked well, is a lymphocyte, an erythrocyte, or a cluster consisting of at least one of a lymphocyte and an erythrocyte.

11. In the aforementioned trapping membrane, a second well for particle trapping is further formed downstream of the connecting well in the direction of fluid flow. The particle capture device according to any one of claims 1 to 10, wherein the size of the second well is larger than the size of each of the plurality of first wells.

12. The particle capture device according to claim 11, wherein the second target particle, which is intended to be captured in the second well, is a circulating tumor cell or a cluster containing circulating tumor cells.

13. The particle capture device according to claim 11, wherein the particles captured in the second well can be released separately from the particles captured in the connecting well.

14. The electrode pair comprises a first electrode pair and a second electrode pair connected to different power sources. The particle capture device according to claim 13, wherein the connecting well is located above the first electrode pair and the second well is located above the second electrode pair.

15. In the aforementioned trapping membrane, a third well for particle trapping is further formed downstream of the connecting well in the direction of fluid flow. The particle capture device according to claim 11, wherein the size of the third well is larger than the size of each of the plurality of first wells and different from the size of the second well.

16. The electrode pair comprises a first portion and a second portion adjacent to each other in the direction of fluid flow, The voltage is applied between the first part and the second part. The particle capture device according to claim 1, wherein the upper surface of either the first portion or the second portion is exposed at the bottom of the connecting well.

17. Multiple connected wells are provided, The particle capture device according to claim 1, wherein the connecting well located downstream in the direction of fluid flow is offset in a direction perpendicular to the direction of fluid flow with respect to the connecting well located upstream in the direction of fluid flow.

18. The particle capture device according to claim 1, wherein the capture film is an insulating film.

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