Microchip, microparticle sorting system, and microparticle sorting method

Abstract

The present technology relates to a microchip, a microparticle sorting system, and a microparticle sorting method that make it possible to more efficiently generate droplets containing desired microparticles. The microchip includes: a main channel through which a liquid containing microparticles flows; a first connection channel that communicates with the main channel; a second connection channel that communicates with the first connection channel; and a pressure chamber that communicates with the second connection channel. A plurality of liquid supply channels different from the first connection channel and the pressure chamber is connected to the second connection channel. The present technology can be applied to microparticle sorting systems.

Description

Microchip, microparticle separation system, and microparticle separation method

[0001] The present technology relates to a microchip, a microparticle separation system, and a microparticle separation method, and particularly to a microchip, a microparticle separation system, and a microparticle separation method that can more efficiently generate droplets containing desired microparticles.

[0002] In recent years, for example, encapsulating two different microparticles such as cells and functionalized beads, two different cells, etc. into one oil-in-droplet, and performing single-cell analysis (cells and barcode beads), analysis based on cell-cell interaction, cell screening, drug screening, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) screening, cell function analysis, etc. in the droplet has been increasingly popular.

[0003] When using droplets for such analysis, etc., there is an advantage that the throughput is higher than when using microwells and it can cope with high-scale operation.

[0004] Also, as a technology for generating droplets containing, for example, one microparticle, a technology has been proposed in which it is determined whether the microparticles flowing in the main channel are the microparticles to be collected, and the microparticles are collected into the collection channel according to the determination result (see, for example, Patent Document 1). In this technology, by collecting into the collection channel when it is determined that the microparticles are the microparticles to be collected, droplets containing only the microparticles to be collected can be efficiently generated.

[0005] Japanese Patent Application Laid-Open No. 2021-71397

[0006] By the way, even when generating droplets containing two or more microparticles such as two microparticles of different types from each other, it is desired to generate droplets more efficiently.

[0007] The present technology has been made in view of such a situation, and is intended to be able to generate droplets containing desired microparticles more efficiently.

[0008] The first aspect of this technology, the microchip, has a main channel through which a liquid containing fine particles flows, a first connecting channel communicating with the main channel, a second connecting channel communicating with the first connecting channel, and a pressure chamber communicating with the second connecting channel, wherein a plurality of liquid supply channels different from the first connecting channel and the pressure chamber are connected to the second connecting channel.

[0009] In the first aspect of this technology, a main channel through which a liquid containing fine particles flows, a first connecting channel communicating with the main channel, a second connecting channel communicating with the first connecting channel, and a pressure chamber communicating with the second connecting channel are provided, and a plurality of liquid supply channels different from the first connecting channel and the pressure chamber are connected to the second connecting channel.

[0010] The second aspect of this technology is a microparticle separation method comprising a main channel through which a first liquid containing microparticles flows, a first connecting channel communicating with the main channel, a second connecting channel communicating with the first connecting channel, and a pressure chamber communicating with the second connecting channel, wherein a plurality of liquid supply channels different from the first connecting channel and the pressure chamber are connected to the second connecting channel, and a second liquid that is immiscible with respect to the first liquid is supplied from the liquid supply channels to the second connecting channel, wherein the microparticle separation method for a microchip is achieved by varying the pressure in the pressure chamber, The method includes a first separation step of separating first target particles, which are particles, into a second liquid while they are contained in a first liquid, thereby generating a first droplet containing the first target particles, and a second separation step of varying the pressure in the pressure chamber to separate second target particles, which are minute particles, into a second liquid while they are contained in the first liquid, thereby generating a second droplet containing the second target particles, and colliding and combining the second droplet with the first droplet to generate a third droplet containing the first and second target particles.

[0011] In a second aspect of this technology, a microchip has a main channel through which a first liquid containing fine particles flows, a first connecting channel communicating with the main channel, a second connecting channel communicating with the first connecting channel, and a pressure chamber communicating with the second connecting channel, wherein a plurality of liquid supply channels different from the first connecting channel and the pressure chamber are connected to the second connecting channel, and a second liquid that is immiscible with respect to the first liquid is supplied from the liquid supply channels to the second connecting channel, and by varying the pressure in the pressure chamber, the first target, which is the fine particles, is controlled. The process includes a first separation step in which, with the particles contained in the first liquid, the first target particles are separated into the second liquid to generate a first droplet containing the first target particles, and a second separation step in which, by varying the pressure in the pressure chamber, the second target particles, which are minute particles, are separated into the second liquid with the first liquid to generate a second droplet containing the second target particles, and the second droplet is made to collide and merge with the first droplet to generate a third droplet containing the first and second target particles.

[0012] The third aspect of this technology, the microparticle sorting system, includes a main channel through which a liquid containing microparticles flows, a first connecting channel communicating with the main channel, a second connecting channel communicating with the first connecting channel, a pressure chamber communicating with the second connecting channel, and a control unit that controls a drive unit for varying the pressure in the pressure chamber. The second connecting channel is connected to a plurality of liquid supply channels different from the first connecting channel and the pressure chamber.

[0013] In a third aspect of this technology, a main channel through which a liquid containing fine particles flows, a first connecting channel communicating with the main channel, a second connecting channel communicating with the first connecting channel, a pressure chamber communicating with the second connecting channel, and a control unit for controlling a drive unit that varies the pressure in the pressure chamber are provided, and a plurality of liquid supply channels different from the first connecting channel and the pressure chamber are connected to the second connecting channel.

[0014] This figure shows an example configuration of a microparticle sorting system. This figure explains the configuration of the sheath flow formation section and the sorting section. This figure shows an example configuration of the sorting section when no stationary section is provided. This figure explains the sorting operation when no stationary section is provided. This figure shows an example configuration of the sorting section when no stationary section is provided. This figure shows an example configuration of the sorting section. This figure explains the generation of droplets containing two microparticles. This figure explains the generation of droplets containing two microparticles. This figure explains the generation of droplets containing two microparticles. This figure explains the generation of droplets containing two microparticles. This figure explains the generation of droplets containing two microparticles. This figure explains the generation of droplets containing two microparticles. This figure explains the generation of droplets containing two microparticles. This figure explains the drive waveform during sorting operation. This figure shows the result of droplet generation. This figure shows the collision and coalescence of droplets. This figure explains the behavior when droplets containing two microparticles are generated. This figure explains the behavior when droplets containing two microparticles are generated. This figure explains the behavior when droplets containing two microparticles are generated. This figure shows the droplet generation process. This figure shows the droplet generation process using a step-shaped drive waveform. This figure shows an example of a drive waveform when droplets containing two microparticles are generated. This is a diagram explaining delay time. This is a flowchart explaining the fine particle recovery process. This is a diagram explaining fractionation specification. This is a flowchart explaining droplet generation process. This is a diagram showing an example of the drive waveform when generating droplets containing 3 fine particles. This is a diagram explaining the generation of droplets containing 3 fine particles. This is a diagram explaining the generation of droplets containing 3 fine particles. This is a diagram explaining the generation of droplets containing 3 fine particles. This is a diagram showing an example of the computer configuration.

[0015] The following describes embodiments to which this technology is applied, with reference to the drawings.

[0016] <First Embodiment> <Example of Configuration of Microparticle Separation System> This technology enables the more efficient generation of droplets containing a desired number of microparticles by providing a stationary section in the connecting channel where droplets containing microparticles are generated, in which the droplets can remain stationary or nearly stationary.

[0017] Figure 1 shows an example configuration of one embodiment of a microparticle sorting system to which this technology is applied.

[0018] The microparticle sorting system 11 shown in Figure 1 includes a microfluidic chip 21, a light irradiation unit 22, an optical detection unit 23, a control unit 24, and a collection counter unit 25.

[0019] For example, the microparticle sorting system 11 consists of one or more devices having a control unit 24, etc., and a microfluidic chip 21, etc., which is a microparticle sorting mechanism, and functions as a microparticle sorting device that generates droplets (emulsion particles) containing two or more microparticles to be recovered (sorted).

[0020] The microfluidic chip 21 has a sample liquid channel 31, a sheath liquid channel 32-1, a sheath liquid channel 32-2, a sheath flow forming section 33, a main channel 34, a sorting section 35, a recovery channel 36, a waste liquid channel 37-1, a waste liquid channel 37-2, and an oil channel 38.

[0021] In the following, when there is no need to distinguish between the sheath fluid passage 32-1 and the sheath fluid passage 32-2, they will simply be referred to as sheath fluid passage 32. Similarly, when there is no need to distinguish between the waste liquid passage 37-1 and the waste liquid passage 37-2, they will simply be referred to as waste liquid passage 37.

[0022] The sample liquid channel 31 is a channel through which multiple microparticles, including at least the microparticles to be recovered (to be separated), flow, and more specifically, through which a sample liquid containing microparticles flows. The sample liquid is introduced into the sample liquid channel 31 from outside the microfluidic chip 21 via the sample inlet 41.

[0023] For example, the sample solution is a hydrophilic liquid, such as a culture medium primarily composed of water. Specifically, if the microparticles to be recovered are cells, the sample solution would be a cell suspension.

[0024] The sheath fluid channel 32 is a channel through which sheath fluid, mainly composed of physiological saline, flows. Sheath fluid is introduced into the sheath fluid channel 32 from outside the microfluidic tip 21 via the sheath inlet 42. The sheath fluid does not contain the fine particles to be recovered.

[0025] The sheath flow forming section 33 is a sheath forming channel where the sample liquid channel 31 and the two sheath liquid channels 32 merge. That is, the downstream end of the sample liquid channel 31 and the downstream ends of each of the two sheath liquid channels 32 are connected to the sheath flow forming section 33, and the sample liquid and the sheath liquid merge in the sheath flow forming section 33.

[0026] The sheath flow forming section 33 surrounds the sample flow, which is the sample liquid flowing from the sample liquid channel 31, with the sheath flow, which is the sheath liquid flowing from each sheath liquid channel 32, and converges the sample flow towards the center of the sheath flow forming section 33 (channel), thereby forming a laminar flow in which the sample liquid is surrounded by sheath liquid.

[0027] Hereinafter, the laminar flow formed in the sheath flow formation section 33, consisting of the sample liquid (sample flow) and the sheath liquid (sheath flow), will also be simply referred to as the sheath flow. This sheath flow is a fine particle-containing liquid that includes, for example, fine particles to be recovered, such as cells.

[0028] For example, in the sheath flow formation section 33, a laminar flow is formed in the central part of the sheath flow, that is, the laminar flow consisting of the sample liquid and the sheath liquid, such that the fine particles are arranged in approximately a single line. In this way, each of the fine particles to be recovered is advected through the flow path at approximately the same speed while being arranged in approximately a single line. This makes it possible to suppress variations in the detection of the fine particles to be recovered in the downstream optical detection section 23.

[0029] The main channel 34 is connected to the downstream side of the sheath flow forming section 33, that is, on the side of the sheath flow forming section 33 opposite to the side to which the sample liquid channel 31 and the sheath liquid channel 32 are connected.

[0030] The main channel 34 is a channel through which a laminar flow (sheath flow) containing the fine particles to be recovered (hereinafter also referred to as the recovered particles), that is, a liquid consisting of the sample liquid and the sheath liquid, flows. The upstream end of the main channel 34 is connected to the sheath flow formation section 33, and the downstream end of the main channel 34 is connected to the sorting section 35. Therefore, the laminar flow (sheath flow) formed in the sheath flow formation section 33 flows through the main channel 34 and reaches the sorting section 35.

[0031] The sorting unit 35 functions as a fine particle sorting unit that sorts the particles to be recovered that have flowed through the main channel 34.

[0032] The sorting section 35 has a connecting passage 43 and a pressure chamber 44. The sorting section 35 also has a vibrating plate (not shown) provided adjacent to the upper surface of the pressure chamber 44, and a piezo actuator (not shown) connected to the vibrating plate. Furthermore, an oil passage 38, more specifically the portion of the oil passage 38 connected to the connecting passage 43, and a waste liquid passage 37, more specifically the portion of the waste liquid passage 37 connected to the connecting passage 43, are also components of the sorting section 35.

[0033] The connecting channel 43 is a channel called an orifice, and the particles to be recovered are separated (drawn in) from the main channel 34 to the connecting channel 43.

[0034] Upstream of the connecting channel 43, the main channel 34 and two waste liquid channels 37 are connected, and downstream of the connecting channel 43, the pressure chamber 44 is connected. In particular, the upstream end of the connecting channel 43 has a three-way branching channel structure, with the connection point between the connecting channel 43 and the main channel 34 sandwiched on both sides by the two waste liquid channels 37. Furthermore, between the connection points of the connecting channel 43 to the main channel 34 and the waste liquid channels 37 and the connection point to the pressure chamber 44, there are two connection points to the oil channel 38, and the section between these connection points is designated as a stationary section. Within the stationary section, the generated droplets can be kept stationary or nearly stationary.

[0035] The oil channel 38 is a liquid supply channel connected perpendicularly to the connecting channel 43. The oil channel 38 introduces a liquid (oil) that is immiscible with respect to the sheath flow (sample liquid and sheath liquid), such as a hydrophobic oil, into the connecting channel 43. That is, oil is introduced into the oil channel 38 from outside the microfluidic chip 21 via the oil inlet 45, and this oil is introduced (flows) from the oil channel 38 into the connecting channel 43. For example, if the oil channel 38 were connected at an angle to the connecting channel 43, secondary flow (vortices) may easily occur on the acute angle side of the connection between the oil channel 38 and the connecting channel 43. In contrast, in the microfluidic chip 21, since the oil channel 38 is connected perpendicularly (or nearly perpendicularly) to the connecting channel 43, the generation of secondary flow is suppressed, and a stable flow can be formed in the stationary section and before and after the stationary section.

[0036] The connecting passage 43, the pressure chamber 44, and the recovery passage 36 are kept constantly filled with oil introduced from the oil passage 38.

[0037] The pressure chamber 44 is located between the connecting channel 43 and the recovery channel 36 and is in communication with the connecting channel 43 and the main channel 34. When the piezo actuator constituting the sorting section 35 is driven and the diaphragm is pushed inward or outward into the pressure chamber 44, the pressure inside the pressure chamber 44 fluctuates. That is, the pressure inside the pressure chamber 44 and the connecting channel 43 that communicates with the pressure chamber 44 fluctuates.

[0038] In the sorting section 35, these pressure fluctuations are utilized to collect (separate) the particles to be recovered from the main channel 34 into the connecting channel 43. That is, the particles to be recovered are separated (drawn) into the oil in the connecting channel 43 while contained in a liquid consisting of the sample liquid and the sheath liquid, thereby forming droplets (emulsion particles) containing the particles to be recovered and consisting of the sample liquid and the sheath liquid. In other words, an emulsion is formed with hydrophobic oil as the dispersion medium and hydrophilic liquid consisting of the sample liquid and the sheath liquid as the dispersed phase. The liquid that constitutes the dispersed phase of this emulsion forms droplets containing the desired particles to be recovered.

[0039] As will be explained in more detail later, for example, in the sorting section 35, two sorting operations are performed to generate (form) a single droplet containing two different types of target particles. Specifically, the first sorting operation generates a first droplet (a droplet containing one microparticle) containing a predetermined target particle. Then, the second sorting operation generates a second droplet containing other target particles of a different type from the predetermined target particle, and this second droplet collides and merges with the first droplet to generate a droplet containing two target particles (a droplet containing two microparticles). Collision and merging here means that the two droplets collide, their interfaces fuse, and the two droplets merge to form a single droplet. The droplet thus generated is advected from the connecting channel 43 through the pressure chamber 44 to the recovery channel 36.

[0040] In the following description, we will explain an example of generating two microparticle-containing droplets containing two particles of different types, but the two particles to be recovered contained in the two microparticle-containing droplets may be of the same type. Furthermore, the pressure chamber 44 only needs to be in communication with the connecting channel 43, and the connecting channel 43 and the pressure chamber 44 may be connected via a predetermined channel.

[0041] If the sorting operation (sorting operation) of the particles to be recovered by the sorting unit 35 is not performed, the sheath flow containing fine particles that have been advected in the main flow channel 34 will not be separated (recovered) into the connecting flow channel 43, but will be discharged from the main flow channel 34 to the waste liquid flow channel 37.

[0042] In other words, the sheath flow that flows into the waste liquid channel 37-1 is discharged to the outside of the microfluidic chip 21 via the waste liquid outlet 46-1, and the sheath flow that flows into the waste liquid channel 37-2 is discharged to the outside of the microfluidic chip 21 via the waste liquid outlet 46-2. Hereafter, when there is no need to distinguish between waste liquid outlet 46-1 and waste liquid outlet 46-2, they will simply be referred to as waste liquid outlet 46.

[0043] The recovery channel 36 is a channel connected to the downstream side of the pressure chamber 44 for recovering droplets containing microparticles, that is, particles to be recovered. The recovery channel 36 discharges (derives) the droplets that have flowed (inflowed) from the pressure chamber 44 to the outside of the microchannel chip 21 through the collection outlet 47.

[0044] In the microparticle sorting system 11, a light irradiation unit 22, an optical detection unit 23, and a collection counter unit 25 are arranged near the microchannel chip 21 as described above.

[0045] Specifically, the light irradiation unit 22 and the optical detection unit 23 are arranged in the portion of the main channel 34 of the microchannel chip 21.

[0046] The light irradiation unit 22 is composed of a light source such as a laser, and irradiates the main channel 34 with light such as laser light as irradiation light. For example, the operation of the light irradiation unit 22 is controlled by the control unit 24.

[0047] When the irradiation light from the light irradiation unit 22 irradiates the microparticles (particles to be recovered) flowing in the main channel 34, scattered light and fluorescence caused by the irradiation light are emitted from the microparticles. For example, when the irradiation light functions as excitation light, fluorescence is emitted from the microparticles in response to the irradiation of the irradiation light.

[0048] The optical detection unit 23 receives the scattered light and fluorescence from the main channel 34 (microparticles), and supplies a light reception intensity signal indicating the light reception intensity to the control unit 24. That is, in the optical detection unit 23, the scattered light and fluorescence incident from the main channel 34 in response to the irradiation of the irradiation light are detected. Note that the light to be detected by the optical detection unit 23 may be either scattered light or fluorescence, or both scattered light and fluorescence. Also, the scattered light may be either forward scattered light or side scattered light, or both, and lights with different wavelengths may be detected as fluorescence.

[0049] The control unit 24 detects the particles to be collected flowing through the main channel 34 based on the light intensity signal supplied from the optical detection unit 23, and controls the operation of the sorting unit 35 according to the detection result. In particular, the control unit 24 controls the drive of the piezo actuator (drive unit) that constitutes the sorting unit 35. In addition, the control unit 24 also appropriately utilizes the droplet count supplied from the collection counter unit 25 when controlling the operation of the sorting unit 35.

[0050] The collection counter unit 25 has a light irradiation unit and a light receiving unit located near the recovery channel 36. The collection counter unit 25 irradiates the recovery channel 36 with light using the light irradiation unit and detects droplets (emulsion particles) flowing through the recovery channel 36 by receiving scattered light and fluorescence that enters the recovery channel 36 from the light receiving unit in response to the light. The collection counter unit 25 counts the number of detected droplets and supplies the resulting droplet count to the control unit 24.

[0051] For example, in the collection counter unit 25, droplets (emulsion particles) are detected by forward scattered light, and minute particles, i.e., particles to be recovered within the droplets, are detected by fluorescence. Alternatively, the collection counter unit 25 may only receive scattered light and fluorescence, and the control unit 24 may perform droplet detection and detection of minute particles (particles to be recovered) contained within the droplets based on the light reception results. For example, as a method for droplet detection etc. performed in the collection counter unit 25, the method described in International Publication No. 2023 / 153297 can be used.

[0052] Figure 2 is a magnified view of the sheath flow formation section 33 and sorting section 35 in the microfluidic chip 21.

[0053] As shown in Figure 2, the sheath flow forming section 33 has a flow channel structure in which the sample liquid flow channel 31 and two sheath liquid flow channels 32 merge to form a single main flow channel 34.

[0054] Furthermore, the sorting section 35 has a three-way branching channel structure in which one main channel 34 branches into one connecting channel 43 and two waste liquid channels 37.

[0055] <Regarding the generation of droplets containing one microparticle> The generation (formation) of droplets in the microfluidic chip 21 will be explained. Here, we will explain the generation of droplets containing one target particle (one microparticle-containing droplet), and the generation of droplets containing two target particles (two microparticle-containing droplet) will be described later.

[0056] During droplet generation, i.e., during the collection (separation) of target particles, the sample liquid is first introduced into the sample liquid channel 31, and the sheath liquid is introduced into the sheath liquid channel 32. Then, the sample liquid and the sheath liquid merge in the sheath flow forming section 33 and flow through the main channel 34 as a sheath flow (laminar flow).

[0057] In addition, the light irradiation unit 22 irradiates the area with light, the optical detection unit 23 detects scattered light and fluorescence, and the resulting received light intensity signal is supplied to the control unit 24.

[0058] The control unit 24 detects minute particles, such as particles to be recovered, flowing through the main channel 34 based on the light intensity signal supplied from the optical detection unit 23. Hereinafter, the detection of minute particles will also be referred to as event detection, and the detection of minute particles will also be referred to as an event being detected. In event detection, the type of detected minute particle is also determined, that is, whether the detected minute particle is a particle to be recovered (detection of particles to be recovered). For example, in event detection, pre-specified minute particles such as cells are detected as particles to be recovered. Such event detection can be described as a detection process that detects minute particles flowing (passing) through the main channel 34.

[0059] When the control unit 24 detects a particle to be recovered, that is, when it detects that the particle to be recovered has reached the position in the main flow path 34 where the optical detection unit 23 is provided, it determines whether that particle is the target particle to be recovered.

[0060] For example, if the goal is to ultimately generate droplets containing two different types of target particles, then if one of the two types of target particles is detected, the detected target particle is determined to be the target particle.

[0061] If the control unit 24 determines that the particle is the target particle to be recovered, it performs a proximity determination based on the received light intensity signal.

[0062] In proximity determination, when a sorting operation (separation operation) is performed to collect (separate) the detected target particles, it is determined whether there are other minute particles in the vicinity of the target particles that could be collected (separated) together with the target particles into the connecting channel 43. In other words, it is determined whether there are no other minute particles in the vicinity of the target particles that are at a distance that would cause them to be collected simultaneously, and whether it is possible to collect only the target particles and generate droplets containing only those target particles.

[0063] Therefore, the microparticle sorting system 11 includes a sorting step for sorting microparticles before the generation of droplets (droplets containing one microparticle).

[0064] Therefore, the microparticle sorting system 11 makes it possible to accurately form only one droplet of the target (specified) type of microparticle to be recovered, even when using a sample solution containing various types of microparticles, i.e., microparticles of different species suspended in it.

[0065] If the proximity detection determines that only the target particles can be collected, the sorting unit 35 performs a sorting operation (separation operation) when the target particles detected by the control unit 24 (optical detection unit 23) reach the connecting channel 43. In other words, the piezo actuator that generates droplets is driven.

[0066] When the sorting operation is performed, the particles to be recovered are guided from the main channel 34 to the connecting channel 43, where droplets containing the particles to be recovered are generated. More specifically, a sheath flow containing the particles to be recovered is drawn from the main channel 34 to the connecting channel 43, thereby generating droplets containing the particles to be recovered. These droplets then flow from the connecting channel 43 through the pressure chamber 44 to the recovery channel 36 (where they are recovered).

[0067] Conversely, if the detected minute particles are not the target particles to be recovered, or if proximity detection determines that it is not possible to recover only the target particles, the sorting operation will not be performed.

[0068] In this case, the oil introduced (supplied) from the oil channel 38 and branched off to the upstream side of the connecting channel 43 is discharged from the connecting channel 43 to the main channel 34, and then splits into two separate wastewater channels 37, flowing through the wastewater channels 37. Therefore, fine particles that flow through the main channel 34 and reach the upstream end of the connecting channel 43 are not drawn into the connecting channel 43 (not recovered), but flow into the wastewater channels 37 together with the oil discharged from the connecting channel 43, and are discharged from the wastewater outlet 46.

[0069] In this way, when the sorting operation is not being performed, the oil discharged from the connecting channel 43 prevents fine particles from flowing into the connecting channel 43 and prevents fine particles from accumulating at the entrance of the connecting channel 43.

[0070] For example, if the connection channel is configured without a stationary section, the sorting section can be configured as shown in Figure 3.

[0071] In the example shown in Figure 3, the sorting section ST11 is provided with a main channel FL11 through which a sheath flow consisting of sample liquid and sheath liquid flows. The sorting section ST11 also has a three-branch channel consisting of a connecting channel FL12 located at the downstream end of the main channel FL11, i.e., where the sheath flow reaches, and two waste liquid channels FL13 flanking the connecting channel FL12.

[0072] The sorting section ST11 is connected to the connecting channel FL12 and includes an oil channel FL14 for introducing oil, a pressure chamber FL15 connected to the downstream side of the connecting channel FL12, a vibrating plate (not shown) provided on the upper surface of the pressure chamber FL15, and a piezo actuator (not shown) for moving the vibrating plate.

[0073] In the example shown in Figure 3, the connecting channel FL12 is a two-stage channel consisting of an upstream connecting channel FL21 connected to the upstream side, i.e., the main channel FL11 and the wastewater channel FL13, and a downstream connecting channel FL22 connected to the downstream side, i.e., the pressure chamber FL15.

[0074] In particular, the cross-section (cross-sectional area) of the first-stage connecting channel, the upstream connecting channel FL21, is smaller than the cross-section of the second-stage connecting channel, the downstream connecting channel FL22, which is connected downstream of the upstream connecting channel FL21.

[0075] Furthermore, oil is introduced from a point along the connecting channel FL12. Specifically, in the diagram of the part of the downstream connecting channel FL22 that connects to the upstream connecting channel FL21, oil channels FL14 are connected to the upper and lower sides, and oil is introduced from these oil channels FL14 to the downstream connecting channel FL22.

[0076] In this example, the downstream connecting channel FL22 and the oil channel FL14 are connected vertically or nearly vertically, and oil is introduced from a total of two locations: the upper side and the lower side of the downstream connecting channel FL22 in the diagram.

[0077] A pressure chamber FL15 is connected to the downstream side of the downstream connecting channel FL22, and the cross-section (cross-sectional area) of the pressure chamber FL15 is larger than that of the downstream connecting channel FL22. Droplets generated by drawing sheath flow from the main channel FL11 to the connecting channel FL12 are advected from the connecting channel FL12 to the pressure chamber FL15.

[0078] Oil is always supplied (introduced) from the oil passage FL14 to the connecting passage FL12 at a constant flow rate. The oil that flows from the oil passage FL14 to the connecting passage FL12 branches off from the connection point between the oil passage FL14 and the connecting passage FL12 to the main passage FL11 side (upstream) and the pressure chamber FL15 side (downstream). The resistance of each passage is designed so that the downstream side from the connecting passage FL12 is always filled with oil.

[0079] Referring to Figure 4, the sorting operation of the sorting unit ST11 will be explained.

[0080] The arrows Q11 to Q13 in Figure 4 show the vicinity of the sorting unit ST11 at each timing point.

[0081] In the example shown in Figure 4, the sorting unit ST11 includes a diaphragm DG11, a spacer SP11, and a piezo actuator PZ11. In the sorting unit ST11, the upper surface portion that forms the pressure chamber FL15 is the diaphragm DG11, and the piezo actuator PZ11 is connected to the diaphragm DG11 via the spacer SP11.

[0082] Furthermore, the line graph L11 shows the waveform (drive waveform) of the drive signal for the piezo actuator PZ11. In the graph of line graph L11, the horizontal axis represents time, and the vertical axis represents the magnitude of the drive signal, that is, the magnitude of the voltage as a drive signal (drive voltage).

[0083] In the normal state when sorting is not being performed, the voltage applied to the piezo actuator PZ11, i.e., the drive signal voltage, is set to a predetermined voltage (hereinafter also referred to as the normal voltage).

[0084] When a normal voltage is applied to the piezo actuator PZ11, the piezo actuator PZ11 is extended, and the diaphragm DG11 is pushed inward into the pressure chamber FL15 by the piezo actuator PZ11. In other words, when sorting is not being performed (normal state), a predetermined pressure is applied to the pressure chamber FL15 by the diaphragm DG11.

[0085] In this normal state, suppose the target particle P11 is detected as shown by arrow Q11, and the proximity check determines that only the target particle P11 can be recovered.

[0086] In such cases, as shown by the broken line L11, the voltage of the drive signal is lowered at the moment the target particle P11 reaches the vicinity of the connecting channel FL12. More specifically, the voltage of the drive signal is gradually lowered until it reaches a predetermined voltage, at which point it is maintained.

[0087] In the example shown in Figure 4, during period T11, which has a length of Tf, the drive voltage decreases linearly from the normal voltage, and in the subsequent period T12, the drive voltage remains constant. Furthermore, the length Th of period T12 is longer than the length Tf of period T11. Note that while this example describes a linear change in drive voltage, the drive voltage could also change sinusoidally.

[0088] When the voltage applied to the piezo actuator PZ11 (drive voltage) is reduced by this drive, the piezo actuator PZ11 contracts as shown by arrow Q12, causing the diaphragm DG11 to be pulled upwards in the figure, i.e., outwards from the pressure chamber FL15.

[0089] As a result, the pressure inside the pressure chamber FL15 decreases, and a certain amount of the sample solution and sheath solution, which are mainly composed of water and contain the target particles P11 such as cells, are drawn into the main channel FL11, i.e., the inlet of the connecting channel FL12, into the connecting channel FL12. Then, as shown below arrow Q12, the drawn-in liquid (sample solution and sheath solution) gradually changes from a columnar shape to a spherical shape.

[0090] The connecting passage FL12 consists of an upstream connecting passage FL21, which is a smaller cross-sectional area section (first stage), and a downstream connecting passage FL22, which is a larger cross-sectional area section (second stage) to which the oil passage FL14 is connected. Furthermore, the cross-sectional area of ​​the pressure chamber FL15, which is connected to the downstream connecting passage FL22, is even larger than the cross-sectional area of ​​the downstream connecting passage FL22.

[0091] Therefore, the flow containing the target particles P11 drawn into the connecting channel FL12 (sheath flow), that is, the liquid consisting of the sample liquid and sheath liquid containing the target particles P11, flows from the upstream connecting channel FL21, which has a smaller cross-sectional area, to the downstream connecting channel FL22, which has a larger cross-sectional area, and further to the pressure chamber FL15. At this time, the liquid consisting of the sample liquid and sheath liquid containing the target particles P11 separates from the walls of the channels through which the liquid passes, becoming a flow (jet).

[0092] The downstream side of the connecting channel FL12 is filled with water, i.e., oil that is immiscible with the sample liquid and sheath liquid. Therefore, the liquid containing the target particles P11 that flows into the second stage, the downstream connecting channel FL22 and beyond, is initially in a columnar shape, but due to interfacial tension, it gradually changes into a spherical shape. In other words, droplets DP11 containing the target particles P11 are formed.

[0093] As shown by the broken line L11, after the period T12, the voltage of the drive signal is gradually increased to the normal voltage, and the drive state of the piezo actuator PZ11, i.e., the operating state of the sorting unit ST11, is returned to the normal state.

[0094] In the example shown in Figure 4, during period T13, which follows period T12 and has a length of Tr, the drive voltage increases linearly until it reaches the normal voltage. As a result, the piezo actuator PZ11 gradually extends, as shown by arrow Q13, and consequently, the diaphragm DG11 is pushed inward into the pressure chamber FL15 by the piezo actuator PZ11. Although this example describes a linear change in the drive voltage, the drive voltage may also change sinusoidally.

[0095] Even during sorting, oil flows (is introduced) at a constant rate from the oil passage FL14 to the connecting passage FL12. In addition, when the drive voltage is returned to the normal voltage and the piezo actuator PZ11 is driven back to its normal extended state, a flow in the opposite direction to the normal flow occurs near the connecting passage FL12, as shown below arrow Q13. This reverse flow is in the opposite direction to the direction of liquid discharge from the connecting passage FL12, that is, the direction toward the downstream side (the direction toward the pressure chamber FL15 from the connecting passage FL12).

[0096] The sorting operation that generates such droplets DP11 can also be described as an operation that forms an emulsion using a liquid consisting of a sample liquid and a sheath liquid as the dispersion phase and oil as the dispersion medium.

[0097] Because the droplet DP11 is advected to the back of the pressure chamber FL15 by the jet effect, and because oil is constantly supplied from the oil passage FL14, the oil near the outlet of the connecting passage FL12 is discharged (flows) downstream, the generated droplet DP11 does not flow back into the connecting passage FL12. In other words, the generated droplet DP11 is not released from the connecting passage FL12 into the main passage FL11.

[0098] Hereinafter, the length of the period T11 during which the drive voltage falls will also be referred to as the fall time, the length of the period T12 during which the drive voltage remains constant after the fall will also be referred to as the hold time, and the length of the period T13 during which the drive voltage rises will also be referred to as the rise time. Furthermore, the difference between the normal voltage, i.e., the drive voltage before the fall, and the constant voltage held after the fall (hereinafter also referred to as the hold voltage) will be referred to as the drive voltage difference or applied voltage.

[0099] In the above explanation, we described the case where no stationary section is provided within the connecting channel as an example. However, in the case of the microfluidic chip 21, the operation is basically the same as in the example described with reference to Figures 3 and 4, and droplets containing the particles to be collected are generated.

[0100] <Regarding the configuration of the sorting unit> The configuration of the sorting unit 35 will be explained below.

[0101] For example, when implementing a sorting operation, the sorting unit can be configured as described with reference to Figures 3 and 4.

[0102] In this case, as shown in Figure 5, the connecting channel FL12 is composed of a first-stage upstream connecting channel FL21 with a smaller cross-sectional area and a second-stage downstream connecting channel FL22 with a larger cross-sectional area, and the oil channel FL14 is connected to the downstream connecting channel FL22.

[0103] In Figure 5, the same reference numerals are used for parts corresponding to those in Figure 4, and their explanations are omitted as appropriate. Also, in Figure 5, the upper part of the figure shows the sorting section ST11 viewed from the direction of oil introduction, that is, the direction in which oil flows from the oil passage FL14 to the connecting passage FL12, and the lower part of the figure shows the sorting section ST11 viewed from a direction perpendicular to the direction of oil introduction. The arrows in the figure indicate the direction in which the oil flows.

[0104] As described above, when oil is introduced from the oil passage FL14 to the downstream connecting passage FL22, the introduced oil branches and flows to the main passage FL11 and the pressure chamber FL15.

[0105] Furthermore, when the sorting operation is performed, the sheath flow is drawn from the main channel FL11 to the upstream connecting channel FL21, generating droplets.

[0106] When generating droplets containing two fine particles, the sorting operation to generate the droplets is performed twice in succession. At this time, the movement speed (advection speed) of the droplets containing other particles to be recovered, generated by the second sorting operation, is made faster than the movement speed (advection speed) of the droplets containing the particles to be recovered, which have already been generated by the first sorting operation.

[0107] In this way, the droplets generated by the second sorting operation catch up to and collide with the droplets generated by the first sorting operation, resulting in the creation of a two-microparticle-containing droplet containing two target particles.

[0108] However, since the droplets generated by the first sorting operation are advected downstream after their creation, if a certain amount of time elapses between the first sorting operation and the second sorting operation, the collision and merging will fail.

[0109] Therefore, in this technology, as shown in Figure 6, a stationary section is provided in the connecting channel 43 that allows the droplet to remain stationary or nearly stationary, thereby improving the success rate of generating droplets containing two or more microparticles. In other words, it is possible to generate droplets containing two or more desired microparticles more efficiently.

[0110] In Figure 6, the upper part of the figure shows the sorting section 35 viewed from the direction of oil introduction, that is, the direction in which oil flows from the oil passage 38 to the connecting passage 43, while the lower part of the figure shows the sorting section 35 viewed from a direction perpendicular to the direction of oil introduction. The arrows in the figure indicate the direction in which the oil flows.

[0111] In this example, the connecting channel 43 is a two-stage channel consisting of an upstream connecting channel 101 provided on the upstream side and communicating with the main channel 34, and a downstream connecting channel 102 provided on the downstream side and connected to the pressure chamber 44. Furthermore, a part of the downstream connecting channel 102, that is, the central region (part) of the downstream connecting channel 102, is a stationary section 103 for stopping or nearly stopping the droplets.

[0112] The upstream connecting channel 101 is connected to the main channel 34 and the waste liquid channel 37, the downstream connecting channel 102 is in communication with the upstream connecting channel 101, and the pressure chamber 44 is in communication with the downstream connecting channel 102.

[0113] In the connecting channel 43, the cross-sectional area of ​​the first-stage connecting channel, the upstream connecting channel 101, is different from the cross-sectional area of ​​the second-stage connecting channel, the downstream connecting channel 102, which is connected downstream of the upstream connecting channel 101. Specifically, the cross-sectional area of ​​the downstream connecting channel 102 is larger (wider) than the cross-sectional area of ​​the upstream connecting channel 101. Also, the cross-section of the downstream connecting channel 102 is smaller than the cross-section of the pressure chamber 44.

[0114] In this example, the downstream connecting channel 102 (connecting channel 43) is directly connected to the pressure chamber 44, but the downstream connecting channel 102 may also be connected to the pressure chamber 44 via a channel. That is, a channel that functions as a connecting channel may be provided between the downstream connecting channel 102 (connecting channel 43) and the pressure chamber 44. In such a case, the channel should have a cross-section larger than the cross-section (cross-sectional area) of the downstream connecting channel 102. The channel provided between the downstream connecting channel 102 and the pressure chamber 44 may be a throat channel.

[0115] Furthermore, multiple oil passages 38 (oil passages 104 and 105), which are different from the upstream connecting passage 101 and the pressure chamber 44, are connected to the downstream connecting passage 102 as liquid supply passages. That is, the oil passages 38 have a first-stage oil passage 104 and a second-stage oil passage 105.

[0116] Here, we will describe an example in which two oil passages 38 are connected to the downstream connecting passage 102, but it is also possible to connect three or more oil passages 38 to the downstream connecting passage 102. In other words, it is sufficient for at least one oil passage 104 and at least one oil passage 105 to be connected to the downstream connecting passage 102.

[0117] The first stage oil passage 104 is connected to the upper and lower ends of the upstream (upstream connection passage 101) end portion of the downstream connection passage 102, that is, the portion that connects to the upstream connection passage 101. The second stage oil passage 105 is connected to the upper and lower ends of the downstream (pressure chamber 44) end portion of the downstream connection passage 102, that is, the portion that connects to the pressure chamber 44.

[0118] In particular, in this example, the downstream connecting channel 102 and the oil channel 104 are connected vertically or nearly vertically, and oil is introduced into the downstream connecting channel 102 from a total of two locations, the upper and lower sides, via the oil channel 104.

[0119] Similarly, the downstream connecting passage 102 and the oil passage 105 are connected vertically or nearly vertically, and oil is introduced into the downstream connecting passage 102 from a total of two locations, the upper and lower, via the oil passage 105.

[0120] In the downstream connecting channel 102, oil channels 104 and 105 are connected with a gap between them. In the downstream connecting channel 102, the section between the part to which oil channel 104 is connected and the part to which oil channel 105 is connected is designated as a stationary section 103.

[0121] When oil is introduced, oil is supplied at equal pressure from each of the multiple oil passages 38 to the downstream connecting passage 102. That is, oil is introduced from the oil passages 104 and 105 to the downstream connecting passage 102 so that the pressure at the connection point between the downstream connecting passage 102 and the oil passage 104, and the pressure at the connection point between the downstream connecting passage 102 and the oil passage 105 are equal (i.e., the pressures are equal).

[0122] In other words, the oil flow rates from each oil passage in the oil passage 104 and oil passage 105 are adjusted so that the same pressure is applied to the upstream and downstream ends of the stationary section 103. This adjustment of the oil flow rate may be achieved by the control unit 24 controlling the opening of a predetermined valve, or it may be done manually by the user.

[0123] When no sorting operation is being performed, the oil introduced (flowing in) from the oil passage 104 to the downstream connecting passage 102 flows towards the upstream connecting passage 101 and is discharged from the upstream connecting passage 101 to the main passage 34. Also, the oil introduced from the oil passage 105 to the downstream connecting passage 102 flows towards the pressure chamber 44. Therefore, the downstream side of the connecting passage 43, that is, the connecting passage 43, the pressure chamber 44, and the recovery passage 36, are always filled with oil.

[0124] When oil is introduced from oil passages 104 and 105 to the downstream connecting passage 102 in such a way that the pressure on the upstream and downstream sides of the stationary section 103 is equal (or approximately equal), the oil flow velocity in the stationary section 103 becomes zero (0) or approximately zero (very slow). Therefore, when droplets are generated, the generated droplets can be made to remain stationary or approximately stationary within the stationary section 103 by performing appropriate driving.

[0125] In the sorting unit 35, a vibrating plate 106, a spacer 107, and a piezo actuator 108 are provided as a configuration to realize sorting operation. In the sorting unit 35, similar to the example shown in Figure 4, the upper surface portion that forms the pressure chamber 44 is the vibrating plate 106, and the piezo actuator 108 is connected to the vibrating plate 106 via the spacer 107. The piezo actuator 108 is driven according to the control of the control unit 24 and functions as a drive unit that changes the pressure in the pressure chamber 44.

[0126] The control unit 24 drives the piezo actuator 108 and performs sorting by supplying a drive signal with a drive waveform determined from the fall time, hold time, voltage difference (difference in drive voltage before and after fall time), and rise time described above to the piezo actuator 108.

[0127] In the normal state when sorting is not being performed, the voltage applied to the piezo actuator 108 is set to a predetermined normal voltage, as in the case shown in Figure 4. In this case, the piezo actuator 108 is extended, and the diaphragm 106 is pushed inward into the pressure chamber 44 by the piezo actuator 108.

[0128] On the other hand, during the sorting operation, the same drive as in Figure 4 is performed. As a result, the piezo actuator 108 contracts, and the diaphragm 106 is pulled outward from the pressure chamber 44, causing the pressure inside the pressure chamber 44 to decrease. This draws a certain amount of the sample liquid and sheath liquid, which are mainly composed of water and contain the target particles such as cells, from the main channel 34 into the connecting channel 43, generating (forming) droplets containing the target particles.

[0129] <Regarding the generation of droplets containing two microparticles> Referring to Figures 7 to 13, the generation of droplets containing two target particles (droplets containing two microparticles) will be explained.

[0130] In Figures 7 to 13, the upper section shows the sorting unit 35 viewed from the direction of oil introduction, the middle section shows the sorting unit 35 viewed from a direction perpendicular to the direction of oil introduction, and the lower section shows the waveform of the drive signal (drive waveform) that drives the piezo actuator 108.

[0131] In particular, in the section showing the drive waveform, the vertical axis represents the drive voltage of the drive signal, and the horizontal axis represents time. Furthermore, the drive voltage at the position where "output voltage" is indicated on the horizontal axis corresponds to the drive voltage at the timings shown in the upper and middle sections of the figure for that section of the drive waveform. In other words, at the timings shown in the upper and middle sections of the figure, the drive voltage at the position where "output voltage" is indicated in the lower section of the figure is supplied to the piezo actuator 108 as a drive signal.

[0132] As shown in Figure 7, assume that the optical detection unit 23 (control unit 24) detects the target particle P21 when the sorting operation is not being performed.

[0133] Subsequently, as shown in Figure 8, when the detected recovered particles P21 have been advected to the vicinity of the connecting channel 43 (upstream connecting channel 101), the control unit 24 starts driving the piezo actuator 108 with the drive waveform shown in the broken line L21.

[0134] In Figure 8, the broken line L21 shows the waveform of the drive signal (drive waveform) during the first sorting operation. In this example, the drive voltage supplied to the piezo actuator 108 gradually decreases from the normal voltage, is then kept at a constant voltage for a predetermined holding time, and then gradually rises back up to the normal voltage.

[0135] When this type of drive (sorting operation) is performed, the particles P21 to be recovered are drawn into the connecting channel 43 (recovered). As a result, as shown in Figure 9, in the stationary section 103, droplets DP21, which are droplets containing one fine particle including the particles P21 to be recovered, are generated.

[0136] When the droplet DP21 is generated, the control unit 24 controls the drive of the piezo actuator 108 so that the generated droplet DP21 does not advect into the pressure chamber 44, but remains stationary or nearly stationary within the stationary section 103. In other words, the drive is performed with a drive waveform that keeps the generated droplet DP21 within the stationary section 103.

[0137] Once droplet DP21 is generated, the system waits until another target particle of a different type from the target particle P21 is detected.

[0138] After the formation of droplet DP21, assume that other particles P22 to be recovered are detected by the optical detection unit 23 (control unit 24), as shown in Figure 10.

[0139] Here, we assume that the recovered particles P21 and P22 are of different types.

[0140] For example, target particles and partner particles can be considered as particles of different types to be recovered. Here, target particles are the minute particles that are to be used for analysis or other processing using the droplets after droplet generation, i.e., minute particles such as cells that are to be observed. Partner particles are minute particles such as reagents or cells that are to be acted upon by the target particles.

[0141] Therefore, when target particles and partner particles are to be recovered, a droplet containing two microparticles, one of each, will be generated. Note that a droplet containing two or more target particles is not permitted, but a droplet containing two or more partner particles may be permitted. Hereafter, a droplet successfully generated containing one target particle and one or more partner particles, without any other microparticles, will be specifically referred to as a paired droplet.

[0142] Furthermore, in the following, the particle that is first recovered during the formation of a droplet containing two microparticles, i.e., the particle that is first encapsulated within the droplet, will be referred to as the first recovered particle or the first type of recovered particle. Also, the particle that is second to be recovered during the formation of a droplet containing two microparticles, i.e., the particle recovered after the first recovered particle, will be referred to as the second recovered particle or the second type of recovered particle.

[0143] Therefore, for example, when generating droplets containing two fine particles, if a droplet containing a target particle is generated first, and then a droplet containing a partner particle is generated, the first particle to be recovered is the target particle, and the second particle to be recovered is the partner particle. Also, in the example explained with reference to Figures 7 to 13, the particle to be recovered P21 is the first particle to be recovered, and the particle to be recovered P22 is the second particle to be recovered.

[0144] Furthermore, in the following, a droplet containing only the first target particles to be recovered will be referred to as the first droplet, and a droplet containing only the second target particles to be recovered will be referred to as the second droplet.

[0145] As shown in Figure 10, when the target particle P22 is detected, and then, as shown in Figure 11, when the target particle P22 has been advected to the vicinity of the connecting channel 43 (upstream connecting channel 101), the control unit 24 starts driving the piezo actuator 108 with the drive waveform shown in the broken line L22.

[0146] In this example, the drive waveform shown in line L21 for the first sorting operation and the drive waveform shown in line L22 for the second sorting operation are different waveforms. That is, the control unit 24 performs different drive control on the piezo actuator 108 for the first sorting operation and the second sorting operation. In particular, comparing line L21 and line L22, line L22 has a larger voltage difference in the drive signal (difference between normal voltage and holding voltage) and a longer holding time than line L21.

[0147] When the second sorting operation is performed, as shown in Figure 12, the target particle P22 is drawn into the connecting channel 43, and a droplet DP22 containing one minute particle, which includes the target particle P22, is generated. At the same time, droplet DP22 collides with droplet DP21 and merges with it. More specifically, Figure 12 shows the state in which the sample flow containing the target particle P22 is drawn into the connecting channel 43 and droplet DP22 is formed.

[0148] When droplet DP21 (the first droplet) is generated and at rest within the stationary section 103, droplet DP22 (the second droplet) is generated, and droplet DP22 collides with droplet DP21 and merges with it. That is, droplet DP22 collides with droplet DP21, and the two droplets DP21 and DP22 merge, generating a single droplet DP23 containing the target particles P21 and P22, as shown in Figure 13.

[0149] The droplet DP23, which contains two fine particles and is thus generated, is transferred from the connecting channel 43 to the pressure chamber 44, and then transferred from the pressure chamber 44 to the recovery channel 36. In other words, the droplet DP23 is recovered into the recovery channel 36 via the pressure chamber 44.

[0150] The sorting section 35 is designed to generate a velocity difference between the velocity (movement speed) of droplet DP21 and the velocity (movement speed) of droplet DP22 that is sufficient to cause interfacial fusion between droplet DP21 and droplet DP22 upon collision.

[0151] Therefore, without providing electrodes near the connecting channel 43 to fuse the interfaces using an electric field, or a mechanism to introduce an interface fusion liquid, droplets DP21 and DP22 can be fused at the interface with a simple configuration to generate a single droplet DP23.

[0152] The velocity difference between droplet DP21 and droplet DP22 can be created by the difference in cross-sectional area between the upstream connecting channel 101 and the downstream connecting channel 102.

[0153] Referring to Figures 14 and 15, the generation results when droplet generation was actually performed using the microfluidic chip 21 will be explained.

[0154] As shown in Figure 14, the waveform of the drive signal that drives the piezo actuator 108 (drive waveform) is determined by the fall time, hold time, voltage difference, and rise time. In Figure 14, the horizontal axis represents time, and the vertical axis represents the magnitude of the drive signal, i.e., the voltage.

[0155] In Figure 14, the fall time T21 is the time from when the drive voltage begins to fall from the normal voltage until it reaches a constant voltage. The hold time T22 is the time from when the drive voltage falls until it reaches a constant voltage, and the rise time T23 is the time from when the drive voltage begins to rise until it returns to the normal voltage. Furthermore, the difference between the normal voltage and the voltage after the drive voltage falls is the voltage difference (voltage) D11.

[0156] When the piezo actuator 108 was driven in the microfluidic chip 21 with a drive waveform having a fall time of 10 μsec, a hold time of 10 μsec, a rise time of 10 μsec, and a voltage difference of 45 V, the results shown in Figure 15 were obtained.

[0157] In this example, the operation of the piezo actuator 108 for droplet generation (sorting operation) is started at a timing of "0 μsec".

[0158] Furthermore, it can be confirmed that droplet DP41 is generated 1000 μsec after the start of operation, and that droplet DP41 remains stationary in the stationary section 103 even 4800 μsec after the start of operation.

[0159] Figure 16 shows the droplet generation result when two droplets collide and merge.

[0160] In this example, during the generation of the first droplet, the piezo actuator 108 is driven with a drive waveform that has a fall time of 60 μsec, a hold time of 30 μsec, a rise time of 60 μsec, and a voltage difference of 36 V. Furthermore, 1000 μsec after the generation of the first droplet, the second droplet is generated, and during the generation of the second droplet, the piezo actuator 108 is also driven with a drive waveform that has a fall time of 60 μsec, a hold time of 30 μsec, a rise time of 60 μsec, and a voltage difference of 36 V.

[0161] In this example, the driving (sorting operation) of the piezo actuator 108 for the generation of the first droplet is started at a timing of "0 μsec," and it can be seen that 143 μsec after the start of driving, the sheath flow is drawn into the connecting channel 43 and forms a liquid column.

[0162] Furthermore, the first droplet is already formed 1000 μsec after the start of operation, and the formation of the second droplet begins at this time.

[0163] It can be seen that 1100 μsec after the start of the drive for the generation of the first droplet, the sheath flow that will become the second droplet is drawn in, 1148 μsec after the start of the drive, the first and second droplets collide and merge, and 1410 μsec after the start of the drive, they have become a single droplet through collision and merger.

[0164] Referring to Figures 17 to 19, the behavior when the second droplet collides and merges with the first droplet will be explained.

[0165] For example, as shown in Figure 17, suppose a first droplet DP51 containing the target particle P41 is generated, and while the first droplet DP51 is in the stationary section 103, the target particle P42 has been advected to the vicinity of the connecting channel 43 (upstream connecting channel 101).

[0166] Subsequently, when the sorting operation is performed, as shown in Figure 18, the liquid consisting of the sample liquid and the sheath liquid, which contains the particles to be recovered P42, is drawn into the connecting channel 43, and a second droplet DP52 containing the particles to be recovered P42 is generated.

[0167] When the sorting operation is performed, the first droplet DP51 that has already been generated and is in the stationary section 103 is advected towards the pressure chamber 44 due to fluctuations in the pressure of the pressure chamber 44.

[0168] However, because the cross-sectional area of ​​the first stage upstream connecting channel 101 and the second stage downstream connecting channel 102 are different, a velocity difference occurs between the first droplet DP51 and the second droplet DP52. In other words, the second droplet DP52 is advected faster than the first droplet DP51.

[0169] Therefore, as shown in Figure 19, the second droplet DP52 catches up with the first droplet DP51 and collides and merges, generating a droplet containing the target particles P41 and P42.

[0170] For example, suppose the cross-sectional area of ​​the downstream connecting channel 102 is four times the cross-sectional area of ​​the upstream connecting channel 101. In this case, the liquid flow rates during sorting in the upstream connecting channel 101 and the downstream connecting channel 102 will be equal, so the initial velocity of the second droplet DP52 after passing through the upstream connecting channel 101 will be four times the velocity of the first droplet DP51.

[0171] Therefore, the kinetic energy of the second droplet DP52 is sufficient to allow it to catch up with and collide with the first droplet DP51, causing the interface between the first droplet DP51 and the second droplet DP52 to fuse.

[0172] <Regarding the driving of the piezo actuator> When the piezo actuator 108 is driven with a drive waveform that has long fall time, hold time, and rise time in order to generate a large first droplet, there is unnecessary behavior during droplet generation.

[0173] Figure 20 shows the droplet formation process when the device is driven with a drive waveform that has a fall time of 60 μsec, a hold time of 30 μsec, a rise time of 60 μsec, and a voltage difference of 36 V.

[0174] In Figure 20, the upper section shows the vicinity of the connection channel 43 during droplet generation, and the lower section shows the drive waveform. In particular, in the section showing the drive waveform, the vertical axis represents the drive voltage of the drive signal, and the horizontal axis represents time. The voltage at the position where "Output Voltage" is indicated corresponds to the drive voltage at the timing shown in the upper section of the figure for that drive waveform.

[0175] In this example, the piezo actuator 108 is driven (sorting operation) at a timing of "0 μsec". Due to the falling edge and voltage holding portion of the drive waveform, the droplets enter deep into the downstream connection channel 102. Here, the droplet size is at its maximum 143 μsec after the start of the drive.

[0176] Subsequently, due to the action of the rising portion in the drive waveform, a reverse flow (flow towards the main flow channel 34) is generated in the connecting channel 43, so that most of the droplets that were initially drawn in are released from the connecting channel 43 (upstream connecting channel 101) into the main flow channel 34.

[0177] Therefore, the volume of the droplet that ultimately remains stationary in the downstream connecting channel 102 becomes smaller. Here, it can be seen that at a timing of 1000 μsec after the start of operation, the final droplet size is smaller compared to the droplet size at 143 μsec after the start of operation.

[0178] The reason why not all droplets are discharged from the connecting channel 43 to the main channel 34 is that when droplets are drawn from the upstream connecting channel 101 to the downstream connecting channel 102, which has a larger cross-sectional area, the flow separates from the wall surface and becomes a jet, causing the flow behavior to become nonlinear.

[0179] Furthermore, the reason the trapped droplet reaches its largest size (maximum volume) at a timing 143 μsec after the start of driving, when the output of the drive signal to the piezo actuator 108 is near completion, is that there is a delay in the fluid response to the temporal change in the drive waveform (drive voltage) due to the elastic deformation of the constituent members.

[0180] Thus, in the example shown in Figure 20, the final droplet size is small relative to the amount of liquid drawn in (sheath flow), so the droplet generation efficiency cannot be said to be good.

[0181] To generate droplets (first droplets) more efficiently, it is preferable to drive the piezo actuator 108 with a step-shaped drive waveform, as shown in Figure 21.

[0182] In Figure 21, the upper panel shows the drive waveform of the piezo actuator 108, and the lower panel shows the vicinity of the connection channel 43 when driven by the drive signal of that drive waveform. Specifically, in the upper panel of the figure, the vertical axis represents the drive voltage of the drive signal, and the horizontal axis represents time.

[0183] In this example, the drive waveform shown in the upper part of the figure is a step waveform in which the voltage falls from the normal voltage for a predetermined fall time, and then the voltage is held constant. The lower part of the figure shows the droplet formation process when driven with a drive waveform with a fall time of 30 μsec and a voltage difference of 22.5 V.

[0184] In this example, the piezo actuator 108 is started to drive (sort operation) at a timing of "0 μsec," and the sheath flow is drawn into the connecting channel 43. At a timing of 143 μsec after the start of driving, the size of the droplets has increased, and at a timing of 239 μsec after the start of driving, droplet generation is complete.

[0185] In the example shown in Figure 21, there is no significant difference between the final droplet size and the maximum droplet size, indicating efficient droplet generation. In particular, in this example, since no reverse flow occurs at low voltages (small voltage differences), larger droplets can be obtained compared to the example of the pulse-shaped drive waveform shown in Figure 20.

[0186] Furthermore, by adding a small rising wave after the step-shaped waveform to the drive waveform, the first droplet can be brought to rest closer to the upstream connecting channel 101, thereby more reliably causing the second droplet and the first droplet to collide and merge.

[0187] In other words, by performing a drive such as the sorting operation shown in Figure 22, for example, two-microparticle-containing droplets can be generated more efficiently and reliably.

[0188] In Figure 22, the broken line L61 in the upper section shows the timing of the sorting operation, the curve L62 in the middle section shows the drive waveform of the piezo actuator 108, and the lower section shows the state of the vicinity of the connection flow path 43 at each timing. In particular, in the middle section of the figure, the horizontal axis represents time, and the vertical axis represents the voltage of the drive signal (drive voltage). Also, in the lower section of the figure, some symbols have been omitted to make the figure easier to read.

[0189] In this example, the downward convex portion of the polyline L61 indicates the timing of the sorting operation. As shown in curve L62, when the timing of the first sorting operation arrives, the drive voltage falls from the normal voltage and is then kept constant for the duration of the holding time. When this drive is performed, the first droplet DP61 is generated, as shown in the first position from the left in the lower row of the figure.

[0190] After the holding time has elapsed, the drive voltage rises slightly to a predetermined voltage, as shown in curve L62, and then settles to a constant voltage. In this case, the voltage after the rise is lower than the normal voltage. Hereafter, the difference between the voltage at the end of the first sorting operation, i.e., the voltage after the rise, and the normal voltage will also be referred to as the offset or offset voltage.

[0191] By incorporating such a rising edge into the drive waveform during the first sorting operation, a reverse flow occurs within the connecting channel 43, as shown in the second image from the left in the lower row of the figure, causing the first droplet DP61 to move toward the main channel 34. In this case, by appropriately setting the rise time and offset voltage, the first droplet DP61 can be made to rest at any desired position within the connecting channel 43.

[0192] As the timing of the second sorting operation shown by curve L62 approaches, the drive voltage falls, then remains constant for the duration of the holding time before rising again, finally returning to the normal voltage. When this type of drive occurs, as shown in the third image from the left in the bottom row of the figure, a second droplet DP62 is generated, and this second droplet DP62 collides and merges with the first droplet DP61, generating a single droplet DP63 (a droplet containing two microparticles), as shown in the fourth image from the left in the bottom row.

[0193] During the second sorting operation, the drive voltage supplied to the piezo actuator 108 is set to be lower than the voltage after the falling edge during the first sorting operation, and the rise of the drive voltage is also set to include not only the amount of the falling edge but also the offset.

[0194] As described above, during the first sorting operation, the control unit 24 controls the drive of the piezo actuator 108 with a drive signal whose drive waveform falls from the normal voltage to a predetermined voltage, is held at a constant voltage for a holding time, and then rises to a voltage lower than the normal voltage (hereinafter also referred to as the post-rising voltage).

[0195] Furthermore, during the second sorting operation, the control unit 24 controls the drive of the piezo actuator 108 with a drive signal that has a drive waveform that, after the voltage falls from the rising edge voltage to a predetermined voltage even lower, is held at a constant voltage for a holding time, and then rises to the normal voltage.

[0196] This type of control allows for the more efficient and reliable generation of droplets containing two microparticles.

[0197] <Regarding proximity determination> The delay time is defined as the time from when the optical detection unit 23 (control unit 24) detects the particles to be collected until the sorting operation actually begins, that is, the time from when the piezo actuator 108 is driven until the operation to generate droplets begins.

[0198] Furthermore, the recovery rate is defined as the percentage of droplets generated during the sorting operation that contain the target particles for recovery and are successfully recovered, i.e., the percentage of droplets that successfully contain only the target particles for recovery (success rate).

[0199] When the recovery for each delay time is plotted, the characteristics shown in Figure 23 are obtained. In Figure 23, the horizontal axis represents the delay time, and the vertical axis represents the recovery.

[0200] In the characteristics shown in Figure 23, as the delay time is gradually increased, the recovery gradually rises and increases from a certain time (length), and then the recovery reaches 100%. After the time when the recovery reaches 100%, there is a period of time during which the recovery is approximately 100%, and after that period, the recovery falls and decreases, and then becomes approximately 0%.

[0201] Therefore, when actually performing the sorting operation, it is sufficient to set the delay time to a fixed value within the interval where recovery is approximately 100%, or to a time to which velocity compensation is applied. Setting with velocity compensation applied means, for example, detecting events at different positions on the main flow path 34, measuring (estimating) the velocity of the particles to be recovered from the results of event detection at each of those positions, and setting the delay time according to the measurement results. As a method of velocity compensation, for example, the method described in Japanese Patent Application Publication No. 2014-202573 can be adopted.

[0202] Furthermore, if the time at which the particle to be recovered is detected by the optical detection unit 23 (control unit 24) is denoted as time Td, and the delay time as DT, then the time at which the sorting operation actually begins (or the time at which the particle to be recovered reaches the sorting unit 35 (connection channel 43)) Ts can be expressed as time Ts = Td + DT.

[0203] In this case, if other particles flowing nearby arrive within ΔT1 of time Ts, there is a possibility that these other particles will be collected together with the target particle. In other words, if other particles flowing behind the target particle arrive between time Ts and time (Ts + ΔT1), there is a possibility that the target particle and the other particles will be collected in the same droplet, resulting in a failure to form the droplet.

[0204] Similarly, if other particles flowing in close proximity to the target particle arrive within a time interval of ΔT2 from time Ts, there is a possibility that these other particles will also be collected along with the target particle. In other words, if other particles flowing ahead of the target particle arrive between time (Ts-ΔT2) and time Ts, there is a possibility that the target particle and the other particles may be collected in the same droplet, resulting in a failure of droplet formation.

[0205] Therefore, in the proximity determination described above, if other minute particles arrive between time (Ts-ΔT2) and time (Ts+ΔT1), which is around the arrival time Ts calculated based on the detection time, for a particle to be recovered at time Td, it may be determined that the particle to be recovered (sorting operation) will not be performed. By doing so, it is possible to generate droplets containing only one particle to be recovered with higher accuracy, i.e., more efficiently.

[0206] Furthermore, if droplets containing only one target particle each can be generated using, for example, a first type of target particle such as a target particle and a second type of target particle such as a partner particle, then these droplets can be collided and merged to generate two microparticle-containing droplets containing one of each of the two different types of target particles with high accuracy. In other words, droplets containing only the desired target particles can be generated with a higher success rate (more efficiently).

[0207] Note that the time ΔT1 and time ΔT2 described above differ between the generation of the first droplet and the generation of the second droplet. Therefore, in proximity determination, the determination is made based on appropriate time ΔT1 and time ΔT2 depending on whether it is the generation of the first droplet or the second droplet.

[0208] <Regarding the collection order of fine particles> This section explains the collection order of particles of different types that are to be collected.

[0209] When generating a two-particle-containing droplet that includes a predetermined type of target particle and another type of target particle, such as a partner particle, it is sufficient that the droplet ultimately contains both types of target particles. Therefore, for example, when generating a droplet containing a target particle and a partner particle, the order in which the target particle and the partner particle are recovered does not matter.

[0210] As described above, when two fine particle-containing droplets are generated, the first particle to be recovered (separated) will be referred to as the first recovered particle, and the next particle to be recovered (separated) will be referred to as the second recovered particle.

[0211] When generating two fine particle-containing droplets containing two types of target particles, two methods can be considered: one in which the recovery order of the target particles is predetermined and fixed (hereinafter also referred to as the fixed recovery order method), and another in which the recovery order is variable (hereinafter also referred to as the variable recovery order method).

[0212] For example, a fixed recovery order method is one in which a predetermined type of fine particle is designated as the first target particle for recovery, and a different, predetermined type of fine particle, distinct from the first target particle, is designated as the second target particle for recovery. In other words, a fixed recovery order method is one in which the recovery order is predetermined, for example, by designating the target particle as the first target particle for recovery and the partner particle as the second target particle for recovery.

[0213] In the fixed collection order method, the first type of particle to be collected is always collected first, followed by the second type of particle to be collected. Therefore, even if the second type of particle to be collected is detected before the first type of particle to be collected, it will not be collected (separated).

[0214] In contrast, the variable recovery order method is a technique in which, of two different types of particles to be recovered, the particle of the type detected first by event detection is designated as the first particle to be recovered, and of the other two different types of particles to be recovered, the particle of a different type from the first particle to be recovered is designated as the second particle to be recovered. That is, for example, the variable recovery order method is a method in which the particle detected first among the target particle and partner particle is recovered (separated) as the first particle to be recovered, and then the particle that was not designated as the first particle among the target particle and partner particle is recovered as the second particle to be recovered.

[0215] Whether droplet generation is performed using a fixed collection order method or a variable collection order method may be specified by the user or determined by the control unit 24.

[0216] For example, when the control unit 24 decides whether to use a fixed collection order method or a variable collection order method to generate droplets, the decision may be made based on the pre-obtained component concentration ratio of each target particle in the sample solution, or the ratio of the number of each target particle (count ratio) obtained from the detection results of the optical detection unit 23 (control unit 24). In such a case, for example, the count ratio can be obtained by a measurement performed in advance.

[0217] <Explanation of the microparticle recovery process> Next, the operation of the microparticle sorting system 11 will be explained.

[0218] Here, we will explain the process assuming that droplets containing two different types of target particles are generated. In such a case, for example, the fine particle recovery process shown in Figure 24 is performed. The fine particle recovery process will be explained below with reference to the flowchart in Figure 24.

[0219] In step S11, a suspension of fine particles containing the fine particles to be recovered is prepared as a sample solution.

[0220] In this process, a predetermined target particle (target cell) and a partner particle (partner cell) are designated as the particles to be recovered, and a fine particle suspension containing a large number of these recovered particles is prepared.

[0221] In step S12, a preliminary measurement is performed and the fractionation of particles to be recovered is specified.

[0222] In other words, in the pre-measurement, the intensity of scattered light and fluorescence (received light intensity) obtained by irradiating target particles and partner particles with light from the light irradiation unit 22 is measured. Then, a histogram of the obtained scattered light and fluorescence is generated, or the received light intensity of the scattered light and fluorescence is plotted in two dimensions, and the fractions of target particles and partner particles are specified. In this case, only scattered light may be used, only fluorescence may be used, or a combination of scattered light and fluorescence may be used.

[0223] Specifically, in the preliminary measurement, by irradiating with light, the received intensity of fluorescence 1 at a predetermined wavelength and the received intensity of fluorescence 2 at a different wavelength from fluorescence 1 were obtained for a large number of minute particles, including target particles and partner particles.

[0224] In such cases, as shown in Figure 25, for example, the received light intensities of fluorescence 1 and fluorescence 2 obtained for each minute particle are plotted in a two-dimensional space with the received light intensity of fluorescence 1 on the horizontal axis and the received light intensity of fluorescence 2 on the vertical axis. That is, the points (positions) in the two-dimensional space correspond to the combination of received light intensities of fluorescence 1 and fluorescence 2 obtained for each minute particle.

[0225] When plotting is performed in this manner, region R11 in the two-dimensional space is designated as gate A for the target particle (target cell), and region R12 is designated as gate B for the partner particle (partner cell).

[0226] For example, region R11, designated as gate A, is a region that includes points corresponding to the received light intensity of fluorescence 1 and fluorescence 2 obtained for the target particle. In other words, region R11 is a region in the plot results that includes points corresponding to the received light intensity of fluorescence 1 and fluorescence 2 obtained for the minute particle that is likely the target particle.

[0227] Therefore, for example, when the optical detection unit 23 obtains light intensity signals for fluorescence 1 and fluorescence 2 for a minute particle flowing through the main channel 34, if the point corresponding to the light intensity indicated by those light intensity signals is located within region R11 in the two-dimensional space (two-dimensional coordinate space) shown in Figure 25, then that minute particle is considered a target particle.

[0228] Furthermore, for example, region R12, designated as gate B, is a region that includes points corresponding to the light detection intensities of fluorescence 1 and fluorescence 2 obtained for the partner particle.

[0229] For example, the control unit 24 stores information indicating gates A and B as gate information used for event detection. The gate information may be input by a user or the like and supplied to the control unit 24 from an input unit (not shown), or it may be generated by the control unit 24 based on the light intensity signal supplied from the optical detection unit 23 during a pre-measurement.

[0230] When gate information is generated by the control unit 24, in the pre-measurement, a portion of the fine particle suspension obtained in step S11 is introduced as a sample liquid from the sample inlet 41 into the sample liquid channel 31. In addition, sheath liquid is introduced from the sheath inlet 42 into the sheath liquid channel 32, and oil is introduced from the oil inlet 45 into the oil channel 38.

[0231] Furthermore, the light irradiation unit 22 irradiates the minute particles flowing through the main channel 34 with irradiation light, and the scattered light, fluorescence, or both scattered light and fluorescence incident from the minute particles in response to the irradiation light are received by the optical detection unit 23, and the received light intensity signal obtained as a result of the light reception is supplied to the control unit 24. Based on the supplied received light intensity signal, the control unit 24 plots it in the two-dimensional space shown in Figure 25, and generates gate information from the plot result and information indicating the fluorescence characteristics of the target particle and partner particle. Alternatively, the control unit 24 may display the plot result on a display unit (not shown), and the user may specify gate A or gate B by referring to the plot result.

[0232] In addition, the control unit 24 may determine whether each minute particle is a target particle or a partner particle based on the light intensity signal of each minute particle obtained in the pre-measurement, and calculate and store the count ratio of each particle to be recovered from the determination result.

[0233] Returning to the flowchart in Figure 24, in step S13, the control unit 24 selects a method for collecting the fine particles.

[0234] For example, suppose a user operates an input unit (not shown) to specify either the fixed-order collection method or the variable-order collection method as described above. In such a case, the control unit 24, based on the signal supplied from the input unit in response to the user's operation, uses the method specified by the user as the collection method during actual measurement, i.e., during droplet generation.

[0235] Furthermore, if the user has previously entered the component concentration ratios of each particle to be recovered in the fine particle suspension (sample solution) prepared in step S11, the control unit 24 will select a recovery method based on the entered component concentration ratios.

[0236] Furthermore, if the count ratio of each target particle has been obtained through prior measurement, the control unit 24 selects a recovery method based on the count ratio.

[0237] Specifically, for example, the control unit 24 sets a predetermined threshold X and selects either a variable recovery order method or a fixed recovery order method as the recovery method, depending on whether the component concentration ratio or count ratio of the target particles is less than X%.

[0238] Furthermore, it may be predetermined whether the recovery method is a fixed recovery order method or a variable recovery order method.

[0239] Once the retrieval method is selected, the actual measurement begins. Alternatively, the actual measurement may begin immediately after the preliminary measurement.

[0240] In step S14, a fine particle suspension is introduced. That is, part or all of the fine particle suspension obtained in step S11 is introduced as a sample liquid from the sample inlet 41 into the sample liquid channel 31. This sample liquid flows from the sample liquid channel 31 to the sheath flow forming section 33.

[0241] Furthermore, when sheath liquid is introduced from the sheath inlet 42 into the sheath liquid channel 32, the sheath liquid flows through the sheath liquid channel 32 to the sheath flow forming section 33. In the sheath flow forming section 33, the sample liquid from the sample liquid channel 31 and the sheath liquid from the sheath liquid channel 32 merge, and a laminar flow (sheath flow) consisting of the sample liquid and sheath liquid flows through the main channel 34. When the laminar flow reaches the connection point between the main channel 34 and the connecting channel 43, if no sorting operation is being performed, it flows from the main channel 34 to the waste liquid channel 37.

[0242] Furthermore, when oil is introduced from the oil inlet 45 into the oil passage 38, the oil flows from the oil passage 38 into the connecting passage 43. At this time, oil is introduced at equal pressure from the oil passages 104 and 105, which make up the oil passage 38, into the connecting passage 43. A portion of the oil that flows into the connecting passage 43 passes through the pressure chamber 44 and then through the recovery passage 36. Another portion of the oil that flows into the connecting passage 43 is discharged from the connecting passage 43 to the connection point with the main passage 34 and flows into the waste liquid passage 37.

[0243] When the actual measurement begins, the irradiation light from the light irradiation unit 22 also begins. The optical detection unit 23 receives scattered light and fluorescence incident from the main channel 34, or more specifically from the minute particles passing through the main channel 34, in response to the irradiation light from the light irradiation unit 22, and sequentially supplies the resulting received light intensity signal to the control unit 24.

[0244] Furthermore, once measurement begins, the collection counter unit 25 also starts detecting droplets as appropriate, and the detection results are sequentially supplied to the control unit 24. The collection counter unit 25 detects droplets (emulsion particles) flowing through the recovery channel 36 and counts the number of detected droplets, and the count results are supplied to the control unit 24. The collection counter unit 25 may also be configured to determine the type of fine particles contained in the droplets.

[0245] In step S15, the microparticle sorting system 11 generates droplets containing two microparticles. That is, in step S15, the microparticle sorting system 11 performs event detection and, according to the result of the event detection, performs a sorting operation at an appropriate timing to generate droplets containing the target particles to be recovered.

[0246] In step S16, the droplets containing two fine particles generated in step S15 are collected. That is, when droplets (droplets containing two fine particles) are generated in step S15, they are transferred from the pressure chamber 44 to the collection channel 36, and are collected in an external container or the like through the collection channel 36 and the collection outlet 47.

[0247] The above measurement procedures are carried out for an appropriate period of time, and the fine particle recovery process ends when the measurement is complete.

[0248] <Explanation of droplet generation process> For example, in step S15 of Figure 24, the droplet generation process shown in Figure 26 is performed as a process to generate droplets containing two microparticles.

[0249] The droplet generation process by the microparticle sorting system 11 will be explained below with reference to the flowchart in Figure 26.

[0250] In step S51, the control unit 24 determines whether or not an event has been detected.

[0251] In other words, the control unit 24 performs event detection based on the light intensity signals supplied sequentially from the optical detection unit 23. During event detection, minute particles flowing through the main channel 34 are detected based on the light intensity signals.

[0252] Event (microparticle) detection may be performed by any method, such as detection based on the received intensity signal of scattered light or detection based on the received intensity signal of fluorescence. Alternatively, event detection may be performed based on bright-field or dark-field images. In event detection, in addition to determining whether microparticles are present, it is also appropriate to determine whether the microparticles are of a first type of target particle for collection, a second type of target particle for collection, or other types of microparticles (microparticles that are not target for collection).

[0253] As a specific example, if the control unit 24 holds gate information as explained with reference to Figure 25, the control unit 24 detects an event based on the light reception intensity signals of fluorescence 1 and fluorescence 2 supplied from the optical detection unit 23 and the gate information. That is, the control unit 24 determines whether or not there are minute particles and identifies (discriminates) the type of minute particles by determining whether the points in the two-dimensional space corresponding to the light reception intensity of fluorescence 1 and fluorescence 2, indicated by the light reception intensity signals, are within the region R11 corresponding to gate A or the region R12 corresponding to gate B.

[0254] If it is determined in step S51 that no event was detected, that is, if no fine particles flowing through the main channel 34 were detected, the subsequent processing returns to step S51, and the above-described process is repeated.

[0255] If, in step S51, it is determined that an event has been detected, the process then proceeds to step S52.

[0256] In step S52, the control unit 24 determines whether the minute particle detected as an event in step S51 is a first target particle for recovery, that is, whether it is a first type of target particle for recovery designated as a first target particle.

[0257] Specifically, for example, suppose the fine particles to be recovered are a target particle and a partner particle, and in step S13 of Figure 24, the recovery order fixing method is selected, and it is predetermined that the target particle is the first particle to be recovered. In such a case, when the target particle is detected by event detection, it is determined in step S52 that it is the first particle to be recovered.

[0258] Furthermore, suppose, for example, that the microparticles to be recovered are target particles and partner particles, and that the variable recovery order method is selected in step S13 of Figure 24. In such a case, if either the target particle or the partner particle is detected by event detection, it is determined in step S52 that it is the first particle to be recovered.

[0259] If it is determined in step S52 that the particle is not the first particle to be recovered, the subsequent processing returns to step S51, and the above-described process is repeated.

[0260] In contrast, if it is determined in step S52 that the particle is the first particle to be recovered, in step S53 the control unit 24 performs the proximity determination described above based on the result of the most recent event detection and determines whether it is possible to form a droplet of only one particle.

[0261] Specifically, for example, the control unit 24 calculates the arrival time Ts (= Td + DT) from the detection time Td of the first particle to be recovered detected as an event in step S51, and determines whether or not a fine particle corresponding to another event arrives within a predetermined period of length that includes that arrival time Ts. In other words, it determines whether or not the arrival time of a fine particle corresponding to another event is included within a predetermined period of length. For example, the predetermined period of length is the period from the above-mentioned time (Ts - ΔT2) to time (Ts + ΔT1).

[0262] The control unit 24 calculates the arrival times of other events (other microparticles) and determines that if the arrival times of other events (other microparticles) are not included within a predetermined period of time, that is, if there are no other microparticles in the vicinity of the first target particle to be recovered, then it is possible to dropletize only one particle (only the first target particle to be recovered).

[0263] However, if the first particle to be recovered is a partner particle, and it is permissible for a pair droplet to contain multiple partner particles, then it is determined that droplet formation is possible even if the arrival time of other partner particles is included within a predetermined period of time.

[0264] If it is determined in step S53 that droplet formation is not possible, the sorting operation is not performed to prevent the generation of unnecessary droplets, and the process returns to step S51, where the above-described process is repeated.

[0265] In response to this, if it is determined in step S53 that droplet formation is possible, in step S54 the control unit 24 outputs a sort time according to the setting conditions for the first particles to be recovered. The control unit 24 generates the first droplet by driving the piezo actuator 108 according to the output sort time.

[0266] For example, for the first target particle to be recovered, the driving conditions of the piezo actuator 108, namely the drive waveform determined from the fall time, voltage difference, holding time, and rise time, and the delay time DT until the driving of the piezo actuator 108 is started are predetermined as setting conditions. In addition, the post-rise voltage, as explained with reference to Figure 22, can also be set as a setting condition for the first target particle to be recovered, so that the driving is performed with a drive waveform consisting of a step waveform (a waveform with a step shape) and a rise waveform.

[0267] The control unit 24 supplies a drive signal to the piezo actuator 108 according to the setting conditions predetermined for the first target particle to be recovered, that is, with a predetermined drive waveform and drive start timing, thereby driving the piezo actuator 108. In other words, the sorting operation is performed by the sorting unit 35.

[0268] The piezo actuator 108 performs the drive described, for example, with reference to Figure 22, according to the control of the control unit 24. That is, the piezo actuator 108 pulls the diaphragm 106 outward from the pressure chamber 44, and after the diaphragm 106 is held in a certain position, it is pushed inward to an intermediate position.

[0269] This causes the pressure in the pressure chamber 44 to fluctuate, generating a first droplet containing the first target particles for recovery. In other words, an emulsion is formed with the sample liquid and sheath liquid as the dispersion phase and the oil as the dispersion medium, and the first droplet is generated (formed) in the emulsion during emulsion formation. The first droplet thus generated remains stationary or nearly stationary at a desired position within the stationary section 103 of the connecting channel 43.

[0270] As described above, in response to the detection result of an event (microparticle), the control unit 24 controls the drive of the piezo actuator 108 to fluctuate the pressure in the pressure chamber 44, thereby separating the first particles to be recovered that are flowing through the main channel 34 into the oil and generating a first droplet. In other words, the control unit 24 controls the execution of the first separation process. In this first separation process, the first particles to be recovered are separated into the oil while they are contained in a liquid consisting of the sample liquid and the sheath liquid, thereby generating a first droplet containing the first particles to be recovered and consisting of the sample liquid and the sheath liquid.

[0271] After the process in step S54 is performed and the sorting time of the first droplet is determined, the process proceeds to step S55.

[0272] In step S55, the control unit 24 determines whether or not an event has been detected. In step S55, the same processing as in step S51 is performed.

[0273] If it is determined in step S55 that no event was detected, the process in step S55 is repeated until it is determined that an event was detected.

[0274] On the other hand, if it is determined in step S55 that an event has been detected, in step S56 the control unit 24 determines whether the minute particle detected as an event in step S55 is a second target particle for recovery, that is, whether it is a second type of target particle for recovery designated as a second target particle.

[0275] Specifically, for example, suppose the microparticles to be recovered are a target particle and a partner particle, and in step S13 of Figure 24, the recovery order fixing method is selected, and it is predetermined that the partner particle will be the second particle to be recovered. In such a case, when a partner particle is detected by event detection, it is determined in step S56 to be the second particle to be recovered.

[0276] Furthermore, suppose, for example, that the microparticles to be recovered are target particles and partner particles, and that the variable recovery order method is selected in step S13 of Figure 24. Also, suppose, for example, that the partner particle is the first particle to be recovered, and that the first droplet is generated. In such a case, when the microparticle detected in step S55 is the target particle, it is determined in step S56 to be the second particle to be recovered.

[0277] If it is determined in step S56 that the particle is not the second target particle for recovery, the subsequent processing returns to step S55, and the above-described process is repeated.

[0278] In contrast, if it is determined in step S56 that the particle is a second particle to be recovered, in step S57 the control unit 24 performs a proximity determination based on the result of the most recent event detection and determines whether it is possible to form a droplet of only one particle.

[0279] For example, in step S57, a proximity determination is made in the same way as in step S53. That is, if the arrival time of another event (microparticle) is not included in the period from time (Ts-ΔT2) to time (Ts+ΔT1), it is determined that droplet formation is possible. Note that the times ΔT1 and ΔT2 described above may be different for the first target particle to be recovered and the second target particle to be recovered.

[0280] If it is determined in step S57 that droplet formation is not possible, the process returns to step S55, and the above-described process is repeated.

[0281] In response to this, if it is determined in step S57 that droplet formation is possible, in step S58 the control unit 24 outputs a sort time according to the setting conditions for the second particle to be recovered. The control unit 24 drives the piezo actuator 108 according to the output sort time to generate a second droplet, and then causes the second droplet to collide and merge with the first droplet to generate a droplet containing two fine particles. Similar to the first droplet, the second droplet and the droplet containing two fine particles are formed by the liquid (sample liquid and sheath liquid) that constitutes the dispersed phase of the emulsion.

[0282] For example, for the second set of particles to be recovered, different setting conditions are predetermined for driving the piezo actuator 108 than those for the first set of particles to be recovered, such as the drive waveform and delay time DT determined from the fall time, voltage difference, holding time, and rise time.

[0283] The control unit 24 supplies a drive signal to the piezo actuator 108 according to the setting conditions defined for the second target particle to be recovered, that is, with a predetermined drive waveform and drive start timing, thereby driving the piezo actuator 108.

[0284] The piezo actuator 108 performs the drive described, for example, with reference to Figure 22, according to the control of the control unit 24. In this case, although the drive waveform is different, the drive is performed in the same way as in step S54.

[0285] As a result, the pressure in the pressure chamber 44 fluctuates, and a second droplet containing the second target particle for recovery is generated. At this time, the second droplet catches up with the already generated first droplet and collides and merges, generating a droplet containing both the first and second target particles for recovery (a droplet containing two fine particles). The droplet thus generated is then transferred from the pressure chamber 44 to the recovery channel 36.

[0286] As described above, in response to the detection result of the event (fine particle), the control unit 24 controls the drive of the piezo actuator 108 to fluctuate the pressure in the pressure chamber 44, thereby separating the second target particles flowing through the main channel 34 into the oil and generating a second droplet in a second separation process. In other words, the control unit 24 controls the execution of the second separation process. In particular, in the second separation process, the control unit 24 controls the drive of the piezo actuator 108 so that a different pressure fluctuation occurs in the pressure chamber 44 than in step S54 (first separation process) described above.

[0287] In the second separation step, the second target particles to be recovered are separated into oil while they are contained in a liquid consisting of the sample liquid and the sheath liquid, and a second droplet consisting of the sample liquid and the sheath liquid, containing the second target particles to be recovered, is generated.

[0288] In step S59, the control unit 24 determines whether or not to terminate the process of generating droplets containing two fine particles.

[0289] If it is determined in step S59 that the process is complete, the droplet generation process is terminated.

[0290] In contrast, if it is determined in step S59 that the process of generating droplets containing two fine particles has not yet been completed, the process then proceeds to step S60.

[0291] In step S60, the control unit 24 determines whether or not an event has been detected. In step S60, the same processing as in step S51 is performed.

[0292] If it is determined in step S60 that no event was detected, the process in step S60 is repeated until it is determined that an event was detected.

[0293] On the other hand, if it is determined in step S60 that an event has been detected, in step S61 the control unit 24 determines whether the time from the generation of the second droplet, in other words, the droplet containing two fine particles, to the arrival time calculated based on the event detection time is equal to or greater than a predetermined wait time.

[0294] For example, if the next first droplet is generated immediately after the second droplet is generated, that is, if two-microparticle-containing droplets are generated at a high rate, multiple droplets may accumulate near the connecting channel 43, specifically in the downstream connecting channel 102 and the pressure chamber 44 (a congested state). This could lead to unstable droplet generation, meaning that it may become impossible to accurately generate two-microparticle-containing droplets.

[0295] Therefore, the microparticle sorting system 11 is designed so that the generation of the next first droplet does not begin until a predetermined weight time has elapsed after the generation of the second droplet, thereby enabling the generation of two microparticle-containing droplets more efficiently, and more reliably.

[0296] If it is determined in step S61 that the wait time has not elapsed, that is, if the wait time has not yet elapsed, the process returns to step S60, and the process described above is repeated.

[0297] If, in step S61, it is determined that the time is longer than the wait time, then the processing in steps S62 to S64 is performed, and a new sort time for the first droplet is output.

[0298] Note that the processing in steps S62 to S64 is the same as the processing in steps S52 to S54, so the explanation is omitted. However, if it is determined in step S62 that the particle is not the first particle to be recovered, and if it is determined in step S63 that droplet formation is not possible, the process then returns to step S60. Also, once the processing in step S64 is performed and the first droplet is generated, the process then returns to step S55.

[0299] As described above, the microparticle sorting system 11 generates a first droplet and keeps it stationary or nearly stationary within the stationary section 103, then generates a second droplet and causes the first droplet and the second droplet to collide and merge, thereby generating a two-microparticle-containing droplet.

[0300] In this way, droplets containing the desired fine particles, namely the first and second target particles, can be generated more efficiently.

[0301] <Modification> The above describes an example of generating a droplet containing a first target particle and a second target particle. However, this technology is not limited to this and can also be applied to generating a single droplet containing three or more target particles, such as a single droplet containing three or more target particles of different types (a droplet containing fine particles).

[0302] In this case, droplets containing one microparticle, which is the particle to be recovered, are generated sequentially, and these newly generated droplets containing one microparticle are then made to collide and merge with a single droplet containing multiple particles to be recovered, which was generated by collision and merging immediately before the generation of the first droplet containing one microparticle.

[0303] In other words, after N microparticle-containing droplets containing N (N≧2) target particles are generated, a 1-microparticle-containing droplet containing one target particle is generated, and this 1-microparticle-containing droplet is then collided and merged with the N-microparticle-containing droplet to generate (N+1) microparticle-containing droplets.

[0304] Referring to Figures 27 to 30, an example of generating a single droplet containing three target particles (a droplet containing three microparticles) will be explained.

[0305] Figure 27 shows the timing of the sorting operation and the drive waveform during each sorting operation. Figures 28 to 30 show the state of the vicinity of the connection channel 43 at each timing. Note that in Figures 28 to 30, some reference numerals have been omitted for clarity.

[0306] In Figure 27, the line L81 shows the timing of the sorting operation, and the curve L82 shows the drive waveform of the piezo actuator 108 during the sorting operation. In particular, the downward convex portion of the line L81 indicates the timing of the sorting operation. In the portion shown in the curve L82, the horizontal axis represents time, and the vertical axis represents the voltage of the drive signal (drive voltage).

[0307] As shown in curve L82, when it is time for the first sorting operation, the drive voltage falls from the normal voltage and then remains constant for the duration of the hold time. At time t1, when the hold time has elapsed, the drive voltage rises slightly, and at time t2, when it reaches a predetermined voltage (hereinafter also referred to as the voltage after the first rise), the first sorting operation is completed. In other words, the first sorting process described above has been performed.

[0308] At time t1, after the holding time has elapsed, as shown on the left side of Figure 28, the sheath flow containing the particles to be recovered is drawn into the connecting channel 43, and droplets DP81 containing those particles are generated.

[0309] Furthermore, after the drive voltage rises slightly, at time t2 when the first sorting operation is completed, as shown on the right side of Figure 28, a reverse flow occurs in the connecting channel 43, and the generated droplets DP81 are slightly advected towards the main channel 34 side (upstream side) and come to rest within the stationary section 103 of the connecting channel 43.

[0310] Once the first sorting operation is complete, the drive voltage remains at the same level as the voltage after the first rise, as shown by curve L82 in Figure 27, until the start of the second sorting operation.

[0311] When it is time for the second sorting operation, the drive voltage falls from the voltage after the first rise, and then remains constant from time t3 to time t4, that is, for a predetermined holding time. At time t4, after the holding time has elapsed, the drive voltage rises slightly, and at time t5, when it reaches a predetermined voltage lower than the voltage after the first rise (hereinafter also referred to as the voltage after the second rise), the second sorting operation is completed. In other words, the second sorting process described above has been performed.

[0312] Note that, although the first and second sorting operations here are different operations (different drive waveforms), these operations may be made to be the same. For example, the drive waveforms for the first and second sorting operations may be the same pulse-type waveform as shown in Figure 14, etc.

[0313] At time t3, when the drive voltage falls during the second sorting operation, as shown on the left side of Figure 29, the sheath flow containing the particles to be recovered is drawn into the connecting channel 43, and droplets DP82 containing those particles are generated. At this time, droplets DP81 that have already been generated in the connecting channel 43 (static section 103) are advected toward the pressure chamber 44 side, i.e., the downstream side.

[0314] Because the velocity of the generated droplet DP82 is faster than the velocity of the droplet DP81 advecting downstream, droplet DP82 catches up to droplet DP81 and collides and merges, creating a single droplet DP83 containing two target particles, as shown in the center of Figure 29.

[0315] At time t5, when the drive voltage rises and the second sorting operation is completed, as shown on the right side of Figure 29, a reverse flow occurs in the connecting channel 43, and the droplets DP83 generated by collision and coalescence are slightly advected towards the main channel 34 and come to rest within the stationary section 103 of the connecting channel 43.

[0316] Once the second sorting operation is complete, the drive voltage remains at the same level as the voltage after the second rise, as shown by curve L82 in Figure 27, until the start of the third sorting operation.

[0317] When it is time for the third sorting operation, the drive voltage falls from the voltage after the second rise, and then remains constant from time t6 to time t7, that is, for a predetermined holding time. At time t7, when the holding time has elapsed, the drive voltage rises, and when it returns to the normal voltage at time t8, the third sorting operation is completed, and the operating state of the piezo actuator 108 returns to the normal state. The drive waveform during the third sorting operation is different from the drive waveforms during the first and second sorting operations.

[0318] At time t6, when the drive voltage falls during the third sorting operation, as shown on the left side of Figure 30, the sheath flow containing the particles to be recovered is drawn into the connecting channel 43, and droplets DP84 containing those particles are generated. At this time, the droplets DP83 already generated in the connecting channel 43 (static section 103) are advected toward the pressure chamber 44 side, i.e., the downstream side.

[0319] Because the velocity of the generated droplet DP84 is faster than the velocity of the droplet DP83 advecting downstream, droplet DP84 catches up with droplet DP83 and collides and merges, creating a single droplet DP85 containing three target particles, as shown in the center of Figure 30. This droplet DP85 contains the target particles that were contained in droplet DP81, droplet DP82, and droplet DP84.

[0320] Once the drive voltage rises and the third sorting operation is completed, the generated droplets DP85 are transferred from the connecting channel 43 to the pressure chamber 44, as shown on the right side of Figure 30, and then transferred from the pressure chamber 44 to the recovery channel 36.

[0321] The process of generating droplets DP84 can be described as a third separation step in which, in response to the detection result of an event (fine particle), the control unit 24 controls the drive of the piezo actuator 108 to fluctuate the pressure in the pressure chamber 44, thereby separating the particles to be recovered flowing through the main channel 34 into the oil and generating droplets DP84.

[0322] For example, in the third preparative step, the control unit 24 controls the drive of the piezo actuator 108 so that different pressure fluctuations occur in the pressure chamber 44 compared to those in at least one of the first and second preparative steps. In other words, in the third preparative step, the drive is performed with a different drive waveform than that in at least one of the first and second preparative steps.

[0323] In the third separation step, the particles to be recovered are separated into oil while they are contained in a liquid consisting of the sample liquid and the sheath liquid, and droplets DP84 consisting of the sample liquid and the sheath liquid, containing the particles to be recovered, are generated.

[0324] As described above, the microparticle sorting system 11 can generate droplets containing three or more target particles more efficiently, in the same manner as when generating droplets containing two microparticles.

[0325] According to this technology, it is possible to generate droplets containing multiple desired fine particles more efficiently.

[0326] For example, one proposed method for generating droplets containing two microparticles involves passing the target particle and another target particle through different channels to merge them, and then introducing oil after the merger to generate droplets containing those two target particles (see, for example, International Publication No. 2017 / 070056).

[0327] In this method, the events of each target particle flowing through the introduction channel reaching the confluence point with other target particles are known to be independent and random events, resulting in a Poisson process. In this case, the number of droplets containing one target particle and one other target particle is small, and the efficiency is not considered good. It is also possible to isolate each minute particle from the sample liquid beforehand, but this would be time-consuming and costly.

[0328] Furthermore, a method has been proposed in which, for example, two droplets containing the target particles, each with a different diameter, are generated separately and then merged. After this, an electric field is applied using electrodes to fuse the droplet interfaces, thereby combining the two droplets and generating a droplet containing both target particles (see, for example, Japanese Patent Publication No. 2009-524825).

[0329] However, even with this method, the process of generating droplets containing the two target particles is a Poisson process, making it impossible to efficiently generate droplets containing two microparticles. Furthermore, even in this case, it is necessary to isolate each microparticle beforehand, which is time-consuming and costly. In addition, this method requires the application of an electric field using electrodes, which complicates the structure of the microchannel used to generate the droplets.

[0330] Unlike the currently proposed methods described above, which are heavily influenced by chance (Poisson process), this technology can more efficiently generate a single droplet containing two or more target particles.

[0331] Specifically, in this technology, for example, by providing a stationary section 103 in the connecting channel 43, the influence of chance is suppressed, and the target droplets can be generated with high accuracy (efficiency). In particular, by providing a stationary section 103, the waiting time between generating a droplet and generating the next droplet to collide and merge with the previous droplet can be increased. Therefore, for example, a sample liquid with a low concentration of fine particles can be used, and droplet generation with high utilization efficiency of fine particles can be achieved. Moreover, in this technology, the droplet generation efficiency can be further improved by detecting fine particles with the optical detection unit 23 (control unit 24) and generating droplets according to the detection result. In addition, proximity determination can be performed to further improve the droplet generation efficiency.

[0332] Furthermore, this technology allows for the direct generation of droplets containing one or more target types of particles without prior purification (isolation) of a sample solution containing two or more different types of target particles or other fine particles. Therefore, it reduces the time and cost of droplet generation and enables efficient droplet generation.

[0333] Furthermore, in this technology, for example, droplets are collided and merged with a sufficient velocity difference to generate droplets containing two or more target particles through interfacial fusion. Therefore, electrodes for generating an electric field for interfacial fusion and a configuration for introducing an interfacial dissolving solution are not required. Consequently, this technology enables highly accurate (efficient) droplet generation using a simple microfluidic chip 21.

[0334] In the above, we have described an example of generating emulsions (emulsion particles) as liquid droplets, but this technology can also be applied to generating multiple emulsions, which are liquid droplets with multiple layers (two or more layers).

[0335] For example, if an emulsion (emulsion particles) is passed through the main channel 34 and then separated into the connecting channel 43, a double emulsion (two-layer emulsion) is formed during the emulsion separation process. In this case, if the emulsion is further separated to form a new double emulsion, and the two double emulsions are brought into a collision, they will combine to form a single double emulsion. For example, it is conceivable to pass a liquid mainly composed of water and emulsion particles covered with oil through the sample liquid channel 31, pass oil through the sheath liquid channel 32, and introduce a liquid mainly composed of water from the oil channel 38. In this case, the liquid mainly composed of water is covered with oil, and an emulsion is formed in which the oil is contained within the liquid mainly composed of water introduced from the oil channel 38. That is, a double emulsion (droplet) consisting of oil and the liquid mainly composed of water is formed within the liquid mainly composed of water.

[0336] <Description of a computer to which this technology is applied> The series of processes described above can be executed by hardware or by software. When the series of processes are executed by software, the programs that make up the software are installed on the computer. Here, the term "computer" includes computers built into dedicated hardware, as well as general-purpose personal computers, for example, that can perform various functions by installing various programs.

[0337] Figure 31 is a block diagram showing an example of the hardware configuration of a computer that executes the series of processes described above using a program.

[0338] In a computer, the processing circuit 901, ROM (Read Only Memory) 902, and RAM (Random Access Memory) 903 are interconnected by a bus 904.

[0339] An input / output interface 905 is further connected to the bus 904. An input unit 906, an output unit 907, a recording unit 908, a communication unit 909, and a drive 910 are connected to the input / output interface 905.

[0340] The input unit 906 may include physical or virtual means of operation that the user operates to input information, such as a keyboard, mouse, or touch panel, as well as means of inputting information by the user through voice, eye gaze, etc. Furthermore, the input unit 906 may include sensors for inputting various physical quantities to the computer.

[0341] For example, the input unit 906 may include sensors that acquire physical quantities such as light (including infrared light other than visible light) and sound, such as cameras and microphones. Alternatively, the input unit 906 may include sensors that acquire other physical quantities such as temperature, moisture content, acceleration, and distance.

[0342] The output unit 907 may include means for presenting information to the user by stimulating the user's senses, such as a display, speaker, or haptic device. The recording unit 908 consists of a hard disk, non-volatile or volatile memory, etc., and records various types of information (including programs).

[0343] The communication unit 909 is a network interface, etc., and performs wired or wireless communication with the outside. The drive 910 drives removable media 911 such as a magnetic disk, optical disk, magneto-optical disk, or semiconductor memory.

[0344] The processing circuit 901 includes a processor that executes programs such as a CPU (Central Processing Unit) and a DSP (Digital Signal Processor). The processing circuit 901 (its processor) loads the program recorded in the recording unit 908 into the RAM 903 via the input / output interface 905 and the bus 904, and executes it, thereby performing the series of processes described above.

[0345] The processing circuit 901 can output the processing results of a series of processes from the output unit 907, for example, via the bus 904 and the input / output interface 905, as needed. The processing circuit 901 can also record the processing results in the recording unit 908 or transmit them from the communication unit 909.

[0346] The program executed by the computer (processing circuit 901) can be provided by recording it on a removable medium 911, such as a package medium. The program can also be provided via wired or wireless transmission media, such as a local area network, the internet, or digital satellite broadcasting.

[0347] In a computer, a program can be installed in the recording unit 908 via the input / output interface 905 by inserting the removable media 911 into the drive 910. Alternatively, a program can be received by the communication unit 909 from another device, such as a server, via a wired or wireless transmission medium, and installed in the recording unit 908. Furthermore, programs can be pre-installed in the ROM 902 or the recording unit 908.

[0348] The programs executed by the computer may be programs that are processed chronologically in the order described herein, or they may be programs that are processed in parallel or at necessary times, such as when a call is made.

[0349] The processes that a computer performs according to a program do not necessarily have to follow the order described in the flowchart. In other words, the processes that a computer performs according to a program include processes that are executed in parallel or individually (e.g., parallel processing and object-based processing).

[0350] The program may be processed by a single computer (processor), or it may be processed in a distributed manner by multiple computers. Furthermore, the program may be transferred to a remote computer and executed there.

[0351] When the computer executes a program to perform the series of processes described above, the processing circuit 901 functions as the control unit 24, and for example, the input unit 906 functions as the optical detection unit 23 or the collection counter unit 25.

[0352] In this specification, a system means one component or a collection of multiple components (devices, modules (parts), etc.). Therefore, one or more components of a computer, for example, only the processor, or a combination of the processor and memory (for example, only the processing circuit 901, or a combination of the processing circuit 901 to the bus 904, etc.), constitute a system. Regarding a collection of multiple components, it is not necessary whether all components reside in the same enclosure. Therefore, multiple devices housed in separate enclosures and connected via a network, or a single device containing multiple modules within a single enclosure, are all systems. Furthermore, for example, the entire computer, or a combination of a computer and other devices such as a server (not shown), also constitute a system.

[0353] The components (blocks) of the apparatus illustrated in this specification are functional conceptual blocks, and the actual apparatus does not need to have the illustrated configuration. That is, the apparatus can have any configuration in which the functions of the illustrated components are divided into any units and / or integrated, for example, a configuration having one block in which the functions of all components are integrated.

[0354] Furthermore, the embodiments of this technology are not limited to those described above, and various modifications are possible without departing from the spirit of this technology.

[0355] For example, this technology can be configured as cloud computing, where a single function is shared and processed collaboratively by multiple devices via a network.

[0356] Furthermore, each step described in the flowchart above can be performed by a single device, or it can be divided and performed by multiple devices.

[0357] Furthermore, if a single step includes multiple processes, those processes can be executed by a single device or shared among multiple devices.

[0358] Furthermore, this technology can also be configured as follows:

[0359] (1) A microchip having a main channel through which a liquid containing fine particles flows, a first connecting channel communicating with the main channel, a second connecting channel communicating with the first connecting channel, and a pressure chamber communicating with the second connecting channel, wherein a plurality of liquid supply channels different from the first connecting channel and the pressure chamber are connected to the second connecting channel. (2) The microchip according to (1), wherein liquid can be supplied from each of the plurality of liquid supply channels to the second connecting channel such that the pressure at the connection portion between each of the plurality of liquid supply channels and the second connecting channel is equal. (3) The microchip according to (1) or (2), wherein the cross-sectional area of ​​the first connecting channel and the cross-sectional area of ​​the second connecting channel are different. (4) The microchip according to (3), wherein the cross-sectional area of ​​the second connecting channel is larger than the cross-sectional area of ​​the first connecting channel. (5) The microchip according to any one of (1) to (4), further having a connecting channel provided between the second connecting channel and the pressure chamber. (6) The second connecting channel is connected to the liquid supply channel by at least one first liquid supply channel and at least one second liquid supply channel, the first liquid supply channel is connected to the first connecting channel side of the second connecting channel, and the second liquid supply channel is connected to the pressure chamber side of the second connecting channel, the microchip according to any one of (1) to (5). (7) The first liquid supply channel and the second liquid supply channel are connected perpendicularly or substantially perpendicularly to the second connecting channel, the microchip according to (6). (8) The first liquid supply channel and the second liquid supply channel are connected to the second connecting channel with a gap between them, and the second connecting channel has a stationary section between the first liquid supply channel and the second liquid supply channel where the liquid flow velocity is zero or substantially zero, the microchip according to (6) or (7).(9) A microchip microparticle separation method comprising: a main channel through which a first liquid containing microparticles flows; a first connecting channel communicating with the main channel; a second connecting channel communicating with the first connecting channel; and a pressure chamber communicating with the second connecting channel, wherein a plurality of liquid supply channels different from the first connecting channel and the pressure chamber are connected to the second connecting channel, and a second liquid that is immiscible with respect to the first liquid is supplied from the liquid supply channels to the second connecting channel, comprising: a first separation step of varying the pressure in the pressure chamber to separate the first target particles, which are microparticles, into the second liquid while the first target particles are contained in the first liquid, thereby generating a first droplet containing the first target particles; A method for separating fine particles, comprising: a second separation step of separating the second target particles, which are fine particles, into the second liquid while they are contained in the first liquid by varying the pressure in the pressure chamber, generating a second droplet containing the second target particles, and combining the second droplet with the first droplet to generate a third droplet containing the first target particles and the second target particles. (10) The method for separating fine particles according to (9), wherein in the first separation step, a drive unit for varying the pressure in the pressure chamber is driven with a first drive waveform, and in the second separation step, the drive unit is driven with a second drive waveform different from the first drive waveform. (11) The fine particle sorting method according to (10), wherein the drive waveform of the drive unit is the waveform of the drive voltage supplied to the drive unit, and is determined by the fall time of the drive voltage, the difference between the drive voltage before and after the fall, the holding time during which the drive voltage is held at a constant level, and the rise time of the drive voltage. (12) The first drive waveform is a waveform in which the drive voltage supplied to the drive unit falls from a predetermined voltage, becomes a constant voltage, and then rises to a voltage lower than the predetermined voltage.(13) A method for separating fine particles according to any one of (9) to (12), further comprising a detection step of detecting the fine particles flowing in the main channel, determining whether there are other fine particles that can be separated together with the first target particle or the second target particle near the first target particle or the second target particle based on the detection result of the fine particles, and performing the first separation step or the second separation step according to the result of the determination. (14) A method for separating fine particles according to any one of (9) to (13), further comprising a third separation step of varying the pressure in the pressure chamber so that the third target particle, which is a fine particle, is separated into the second liquid while the third target particle is contained in the first liquid, generating a fourth droplet containing the third target particle, and combining the fourth droplet with the third droplet to generate a fifth droplet containing the first target particle, the second target particle, and the third target particle. (15) The fine particle separation method according to (14), wherein the pressure fluctuation in the pressure chamber is different in at least one of the first separation step and the second separation step and in the third separation step. (16) The fine particle separation method according to any one of (9) to (15), wherein the first droplet and the second droplet are formed by the first liquid, which is the dispersed phase of an emulsion with the second liquid as the dispersion medium. (17) The fine particle separation method according to any one of (9) to (16), wherein the second liquid is supplied from each of the plurality of liquid supply channels to the second connecting channel such that the pressure at the connection portion between each of the plurality of liquid supply channels and the second connecting channel is equal. (18) The fine particle separation method according to any one of (9) to (17), wherein the cross-sectional area of ​​the second connecting channel is larger than the cross-sectional area of ​​the first connecting channel. (19) A fine particle separation system comprising: a main channel through which a liquid containing fine particles flows; a first connecting channel communicating with the main channel; a second connecting channel communicating with the first connecting channel; a pressure chamber communicating with the second connecting channel; and a control unit for controlling a drive unit that varies the pressure in the pressure chamber, wherein the second connecting channel is connected to a plurality of liquid supply channels different from the first connecting channel and the pressure chamber.

[0360] 11 Microparticle sorting system, 21 Microfluidic chip, 22 Light irradiation unit, 23 Optical detection unit, 24 Control unit, 25 Collection counter unit, 34 Main channel, 35 Sorting unit, 36 Recovery channel, 37-1, 37-2, 37 Waste liquid channels, 38 Oil channel, 43 Connecting channel, 44 Pressure chamber, 101 Upstream connecting channel, 102 Downstream connecting channel, 103 Stationary section, 104 Oil channel, 105 Oil channel, 106 Vibrating plate, 108 Piezo actuator

Claims

1. A microchip having a main channel through which a liquid containing fine particles flows, a first connecting channel communicating with the main channel, a second connecting channel communicating with the first connecting channel, and a pressure chamber communicating with the second connecting channel, wherein the second connecting channel is connected to a plurality of liquid supply channels different from the first connecting channel and the pressure chamber.

2. The microchip according to claim 1, wherein liquid can be supplied from each of the multiple liquid supply channels to the second connecting channel such that the pressure at the connection portion between each of the multiple liquid supply channels and the second connecting channel is equal.

3. The microchip according to claim 1, wherein the cross-sectional area of ​​the first connecting channel and the cross-sectional area of ​​the second connecting channel are different.

4. The microchip according to claim 3, wherein the cross-sectional area of ​​the second connecting channel is larger than the cross-sectional area of ​​the first connecting channel.

5. The microchip according to claim 1, further comprising a connecting channel provided between the second connecting channel and the pressure chamber.

6. The microchip according to claim 1, wherein the second connecting channel is connected as a liquid supply channel to at least one first liquid supply channel and at least one second liquid supply channel, the first liquid supply channel is connected to the first connecting channel side of the second connecting channel, and the second liquid supply channel is connected to the pressure chamber side of the second connecting channel.

7. The microchip according to claim 6, wherein the first liquid supply channel and the second liquid supply channel are connected perpendicularly or substantially perpendicularly to the second connecting channel.

8. The microchip according to claim 6, wherein the first liquid supply channel and the second liquid supply channel are connected to the second connecting channel with a gap between them, and the second connecting channel has a stationary section between the first liquid supply channel and the second liquid supply channel where the liquid flow velocity is zero or nearly zero.

9. A microchip microparticle separation method comprising: a main channel through which a first liquid containing microparticles flows; a first connecting channel communicating with the main channel; a second connecting channel communicating with the first connecting channel; and a pressure chamber communicating with the second connecting channel, wherein a plurality of liquid supply channels different from the first connecting channel and the pressure chamber are connected to the second connecting channel, and a second liquid that is immiscible with respect to the first liquid is supplied from the liquid supply channels to the second connecting channel, comprising: a first separation step of varying the pressure in the pressure chamber to separate the first target particles, which are microparticles, into the second liquid while the first target particles are contained in the first liquid, thereby generating a first droplet containing the first target particles; A method for separating fine particles, comprising a second separation step of varying the pressure in the pressure chamber so that the second target particles, which are fine particles, are separated into the first liquid while they are contained in the first liquid, generating a second droplet containing the second target particles, and combining the second droplet with the first droplet to generate a third droplet containing the first and second target particles.

10. The fine particle sorting method according to claim 9, wherein in the first sorting step, a drive unit that varies the pressure in the pressure chamber is driven with a first drive waveform, and in the second sorting step, the drive unit is driven with a second drive waveform different from the first drive waveform.

11. The method for separating fine particles according to claim 10, wherein the drive waveform of the drive unit is the waveform of the drive voltage supplied to the drive unit, and is determined by the fall time of the drive voltage, the difference between the drive voltage before and after the fall, the holding time during which the drive voltage is maintained at a constant level, and the rise time of the drive voltage.

12. The method for separating fine particles according to claim 10, wherein the first drive waveform is a waveform in which the drive voltage supplied to the drive unit falls from a predetermined voltage, becomes a constant voltage, and then rises to a voltage lower than the predetermined voltage.

13. A method for separating fine particles according to claim 9, further comprising a detection step of detecting the fine particles flowing in the main channel, wherein, based on the detection result of the fine particles, a determination is made as to whether there are other fine particles that can be separated together with the first target particle or the second target particle in the vicinity of the first target particle or the second target particle, and the first separation step or the second separation step is performed according to the result of the determination.

14. The method for separating fine particles according to claim 9, further comprising a third separation step of varying the pressure in the pressure chamber to separate the third target particles, which are fine particles, into the second liquid while the third target particles are contained in the first liquid, to generate a fourth droplet containing the third target particles, and to combine the fourth droplet with the third droplet to generate a fifth droplet containing the first target particles, the second target particles, and the third target particles.

15. The method for separating fine particles according to claim 14, wherein the pressure fluctuation in the pressure chamber is different in at least one of the first separation step and the second separation step and in the third separation step.

16. The method for separating fine particles according to claim 9, wherein the first droplet and the second droplet are formed by the first liquid, which is the dispersed phase of an emulsion with the second liquid as the dispersion medium.

17. The method for separating fine particles according to claim 9, wherein the second liquid is supplied from each of the multiple liquid supply channels to the second connecting channel such that the pressure at the connection portion between each of the multiple liquid supply channels and the second connecting channel is equal.

18. The method for separating fine particles according to claim 9, wherein the cross-sectional area of ​​the second connecting channel is greater than the cross-sectional area of ​​the first connecting channel.

19. A fine particle separation system comprising: a main channel through which a liquid containing fine particles flows; a first connecting channel communicating with the main channel; a second connecting channel communicating with the first connecting channel; a pressure chamber communicating with the second connecting channel; and a control unit for controlling a drive unit that varies the pressure in the pressure chamber, wherein the second connecting channel is connected to a plurality of liquid supply channels different from the first connecting channel and the pressure chamber.

Images