Microparticle sorting system and microparticle sorting method

Abstract

The present technology relates to a microparticle sorting system and a microparticle sorting method that make it possible to more efficiently generate droplets containing desired microparticles. This microparticle sorting method for a microparticle sorting mechanism having a main flow path through which microparticles flow and a pressure chamber communicating with the main flow path comprises: a detection step for detecting microparticles flowing through the main flow path; a first sorting step for sorting the microparticles flowing through the main flow path by changing the pressure in the pressure chamber according to the results of detecting the microparticles, and generating droplets containing the microparticles; and a second sorting step for sorting the microparticles flowing through the main flow path by changing the pressure in the pressure chamber, so that a pressure change different from that in the first sorting step occurs, according to the results of detecting the microparticles, and thereby generating droplets containing the microparticles. The present technology can be applied to a microparticle sorting system.

Description

Micro Particle Sorting System and Micro Particle Sorting Method

[0001] The present technology relates to a micro particle sorting system and a micro particle sorting method, and more particularly to a micro particle sorting system and a micro particle sorting method that can more efficiently generate droplets containing desired micro particles.

[0002] In recent years, for example, encapsulating two different micro particles 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 micro particle, a technology has been proposed in which it is determined whether the micro particles flowing in the main channel are the micro particles to be recovered, and the micro particles are recovered to the recovery channel according to the determination result (see, for example, Patent Document 1). In this technology, by performing recovery to the recovery channel when it is determined that the micro particles are the micro particles to be recovered, droplets containing only the micro particles to be recovered can be efficiently generated.

[0005] International Publication No. 2021 / 084814

[0006] By the way, even when generating droplets containing two or more micro particles such as two micro particles 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 enables more efficient generation of droplets containing desired micro particles.

[0008] The first aspect of this technology is a method for separating fine particles, comprising a fine particle separating mechanism having a main channel through which fine particles flow and a pressure chamber communicating with the main channel, the method comprising: a detection step of detecting the fine particles flowing through the main channel; a first separating step of separating the fine particles flowing through the main channel by varying the pressure in the pressure chamber according to the detection result of the fine particles and generating droplets containing the fine particles; and a second separating step of separating the fine particles flowing through the main channel by varying the pressure in the pressure chamber such that a different pressure fluctuation occurs than in the first separating step, according to the detection result of the fine particles and generating droplets containing the fine particles.

[0009] In the first aspect of the present technology, a fine particle separation method is provided in a fine particle separation mechanism having a main channel through which fine particles flow and a pressure chamber communicating with the main channel. The process includes: a detection step of detecting the fine particles flowing through the main channel; a first separation step of separating the fine particles flowing through the main channel by varying the pressure in the pressure chamber according to the detection result of the fine particles and generating droplets containing the fine particles; and a second separation step of separating the fine particles flowing through the main channel by varying the pressure in the pressure chamber such that a different pressure fluctuation occurs than in the first separation step, according to the detection result of the fine particles and generating droplets containing the fine particles.

[0010] The second aspect of this technology is a microparticle sorting system comprising a main channel through which microparticles flow, a pressure chamber communicating with the main channel, a detection unit for detecting the microparticles flowing through the main channel, and a control unit for controlling a drive unit that fluctuates the pressure in the pressure chamber. The control unit controls the drive unit according to the detection result of the microparticles to fluctuate the pressure in the pressure chamber, thereby sorting the microparticles flowing through the main channel and controlling the execution of a first sorting step in which the microparticles are sorted by fluctuating the pressure in the pressure chamber in accordance with the detection result of the microparticles, thereby sorting the microparticles flowing through the main channel and controlling the execution of a second controlling the microparticles.

[0011] In a second aspect of this technology, a microparticle sorting system has a main channel through which microparticles flow, a pressure chamber communicating with the main channel, a detection unit for detecting the microparticles flowing through the main channel, and a control unit for controlling a drive unit for varying the pressure in the pressure chamber. The system controls the drive unit according to the detection result of the microparticles to control the execution of a first sorting step in which the pressure in the pressure chamber is varied to sort the microparticles flowing through the main channel and generate droplets containing the microparticles. The system controls the drive unit according to the detection result of the microparticles to control the execution of a second sorting step in which the pressure in the pressure chamber is varied to sort the microparticles flowing through the main channel and generate droplets containing the microparticles, causing a different pressure fluctuation than in the first sorting step.

[0012] This is a diagram showing an example of the configuration of a microparticle sorting system. This diagram explains the configuration of the sheath flow formation section and the sorting section. This is a diagram showing the structure of the sorting section. This diagram explains the sorting operation. This is a diagram explaining the generation of droplets containing two microparticles. This is a diagram explaining the generation of droplets containing two microparticles. This is a diagram explaining the delay time. This is a diagram showing an example of failure to generate droplets containing two microparticles. This is a diagram showing an example of failure to generate droplets containing two microparticles. This is a diagram showing the simulation results of the target utilization rate. This is a diagram showing the simulation results of the pair generation rate. This is a diagram showing the simulation results of Purity. This is a diagram showing the process of generating droplets containing two microparticles. This is a diagram showing the result of generating droplets containing two microparticles. This is a flowchart explaining the microparticle recovery process. This is a diagram explaining the specification of fractionation. This is a flowchart explaining the droplet generation process. This is a diagram explaining the generation of droplets containing three microparticles. This is a diagram explaining the generation of droplets containing three microparticles. This is a diagram explaining the generation of droplets containing three microparticles. This is a diagram explaining the generation of droplets containing three microparticles. This is a diagram explaining the generation of droplets containing cells and reagents. This is a diagram explaining sheath flow generation with an added reagent flow layer. This figure illustrates an example of generating droplets containing cells and reagents. This figure illustrates the configuration of a microfluidic chip. This figure shows the results of a fluid simulation. This figure shows an example of a computer configuration.

[0013] The following describes embodiments to which this technology is applied, with reference to the drawings.

[0014] <First Embodiment> <Example of Microparticle Separation System Configuration> This technology enables the more efficient generation of droplets containing a desired number of microparticles by causing a faster-moving droplet containing other microparticles to collide and merge with a droplet containing microparticles within a connecting channel where droplets containing microparticles are generated.

[0015] Figure 1 shows an example configuration of one embodiment of a microparticle sorting system to which this technology is applied.

[0016] 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.

[0017] 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).

[0018] 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.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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, an oil channel 38 is connected between the connection point 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.

[0033] 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 then introduced (flows) from the oil channel 38 into the connecting channel 43.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] Incidentally, hereinafter, an example of generating a two-micro-particle-containing droplet including two types of particles to be recovered that are different from each other will be described. However, the two particles to be recovered included in the two-micro-particle-containing droplet may be of the same type. Further, the pressure chamber 44 only needs to communicate with the connection channel 43, and the connection channel 43 and the pressure chamber 44 may be connected via a predetermined channel.

[0039] When the sorting operation (sorting operation) of the particles to be recovered by the sorting unit 35 is not performed, the sheath flow containing the microparticles that has advected in the main channel 34 is discharged from the main channel 34 to the waste liquid channel 37 without being sorted (recovered) to the connection channel 43.

[0040] That is, the sheath flow that has flowed into the waste liquid channel 37-1 is discharged to the outside of the microchannel chip 21 via the waste liquid outlet 46-1, and the sheath flow that has flowed into the waste liquid channel 37-2 is discharged to the outside of the microchannel chip 21 via the waste liquid outlet 46-2. Hereinafter, when there is no particular need to distinguish between the waste liquid outlet 46-1 and the waste liquid outlet 46-2, they are simply also referred to as the waste liquid outlet 46.

[0041] 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 advected (flowed in) from the pressure chamber 44 to the outside of the microchannel chip 21 via the collection outlet 47.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] When the irradiation light from the light irradiation unit 22 irradiates the microparticles (particles to be recovered) flowing in the main flow path 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.

[0046] The optical detection unit 23 receives the scattered light and fluorescence from the main flow path 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 flow path 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.

[0047] Based on the light reception intensity signal supplied from the optical detection unit 23, the control unit 24 detects the particles to be recovered flowing (passing through) in the main flow path 34, and controls the operation of the sorting unit 35 according to the detection result. In particular, the control unit 24 controls the driving of the piezo actuator (driving unit) constituting the sorting unit 35. Also, when controlling the operation of the sorting unit 35, the control unit 24 appropriately uses the count number of droplets supplied from the collection counter unit 25.

[0048] The collection counter unit 25 has a light irradiation unit and a light reception unit arranged in the vicinity of the recovery flow path 36. The collection counter unit 25 irradiates the recovery flow path 36 with light by the light irradiation unit, and detects the droplets (emulsion particles) flowing in the recovery flow path 36 by receiving the scattered light and fluorescence incident from the recovery flow path 36 to the light reception unit in response to the light. The collection counter unit 25 counts the number of detected droplets and supplies the resulting droplet count number to the control unit 24.

[0049] 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.

[0050] Figure 2 is a magnified view of the sheath flow formation section 33 and sorting section 35 in the microfluidic chip 21.

[0051] 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.

[0052] 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.

[0053] <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 droplets) will be described later.

[0054] 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).

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] Therefore, the microparticle sorting system 11 includes a sorting step for sorting microparticles before the generation of droplets (droplets containing one microparticle).

[0062] 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.

[0063] As shown in Figure 3, the sorting section 35 has a three-branched flow path consisting of a connecting flow path 43 located at the position where the sheath flow, which consists of the sample liquid and the sheath liquid, reaches, and two waste liquid flow paths 37 flanking the connecting flow path 43.

[0064] Furthermore, the sorting unit 35 is connected to the connecting passage 43 and includes an oil passage 38 for introducing oil, a pressure chamber 44 connected to the downstream (backstream) side of the connecting passage 43, a vibrating plate (not shown) provided on the upper surface of the pressure chamber 44, and a piezo actuator (not shown) for moving the vibrating plate.

[0065] In the example shown in Figure 3, the connecting channel 43 is a two-stage channel consisting of an upstream connecting channel 71 connected to the upstream side, i.e., the main channel 34 and the wastewater channel 37, and a downstream connecting channel 72 connected to the downstream side, i.e., the pressure chamber 44.

[0066] In particular, the cross-section (cross-sectional area) of the first-stage connecting channel, the upstream connecting channel 71, is smaller than the cross-section of the second-stage connecting channel, the downstream connecting channel 72, which is connected downstream of the upstream connecting channel 71.

[0067] Furthermore, oil is introduced from a point along the connecting channel 43. Specifically, in the diagram of the portion of the downstream connecting channel 72 that connects to the upstream connecting channel 71, oil channels 38 are connected to the upper and lower sides, and oil is introduced from these oil channels 38 into the downstream connecting channel 72.

[0068] In this example, the downstream connecting channel 72 and the oil channel 38 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 72 in the diagram.

[0069] A pressure chamber 44 is connected to the downstream side of the downstream connecting channel 72, and the cross-section (cross-sectional area) of the pressure chamber 44 is larger than that of the downstream connecting channel 72. Droplets generated by drawing sheath flow from the main channel 34 to the connecting channel 43 are advected from the connecting channel 43 to the pressure chamber 44. In other words, droplets containing the particles to be recovered are recovered through the pressure chamber 44 into the recovery channel 36.

[0070] In this example, the downstream connecting channel 72 (connecting channel 43) is directly connected to the pressure chamber 44, but the downstream connecting channel 72 may also be connected to the pressure chamber 44 via a channel. That is, a channel may be provided between the downstream connecting channel 72 (connecting channel 43) and the pressure chamber 44. In such a case, the channel should have a cross-sectional area larger than the cross-sectional area of ​​the downstream connecting channel 72.

[0071] Oil is always supplied (introduced) from the oil passage 38 to the connecting passage 43 at a constant flow rate. The oil that flows from the oil passage 38 to the connecting passage 43 branches off from the connection point between the oil passage 38 and the connecting passage 43 to the main passage 34 side (upstream side) and the pressure chamber 44 side (downstream side), and the resistance of each passage is designed so that the area from the connecting passage 43 to the recovery passage 36 is always filled with oil.

[0072] 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. That is, the piezo actuator that generates droplets is driven. Once the sorting operation is performed, the target particles are guided from the main channel 34 to the connecting channel 43.

[0073] 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.

[0074] In this case, the oil introduced (supplied) from the oil passage 38 and branched off to the upstream connecting passage 71 of the connecting passage 43 is discharged from the upstream connecting passage 71 and then separates into the two wastewater passages 37, flowing through the wastewater passages 37. Therefore, fine particles that flow through the main passage 34 and reach the upstream end of the connecting passage 43 (upstream connecting passage 71) are not drawn into (recovered by) the upstream connecting passage 71, but instead flow into the wastewater passage 37 along with the oil discharged from the upstream connecting passage 71, and are discharged from the wastewater outlet 46.

[0075] In this way, when the sorting operation is not being performed, the oil discharged from the upstream connecting channel 71 prevents fine particles from flowing into the connecting channel 43, and also prevents fine particles from accumulating at the entrance of the connecting channel 43 (upstream connecting channel 71).

[0076] Refer to Figure 4 for a further explanation of the sorting operation.

[0077] The arrows Q11 to Q13 in Figure 4 show the state of the vicinity of the sorting unit 35 at each timing.

[0078] In the example shown in Figure 4, the sorting unit 35 includes a diaphragm 101, a spacer 102, and a piezo actuator 103. In the sorting unit 35, the upper portion that forms the pressure chamber 44 is the diaphragm 101, and the piezo actuator 103 is connected to the diaphragm 101 via the spacer 102. The piezo actuator 103 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.

[0079] Furthermore, the broken line L11 shows the waveform (drive waveform) of the drive signal for the piezo actuator 103 supplied from the control unit 24 to the piezo actuator 103. In the graph of the broken line 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).

[0080] In the normal state when sorting is not being performed, the voltage applied to the piezo actuator 103, i.e., the drive signal voltage, is set to a predetermined voltage (hereinafter also referred to as the normal voltage).

[0081] When a normal voltage is applied to the piezo actuator 103, the piezo actuator 103 is extended, and the diaphragm 101 is pushed inward into the pressure chamber 44 by the piezo actuator 103. In other words, when sorting is not being performed (normal state), a predetermined pressure is applied to the pressure chamber 44 by the diaphragm 101.

[0082] 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.

[0083] In such cases, the control unit 24 lowers the voltage of the drive signal at the timing when the target particle P11 reaches the vicinity of the connecting channel 43, as shown by the broken line L11. More specifically, the control unit 24 gradually lowers the voltage of the drive signal, and after it drops to a predetermined voltage, it supplies the drive signal so that the voltage is maintained at that level.

[0084] 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.

[0085] When the voltage applied to the piezo actuator 103 (drive voltage) is reduced by this drive, the piezo actuator 103 contracts as shown by arrow Q12, and as a result the diaphragm 101 is pulled out upwards in the figure, that is, outwards from the pressure chamber 44.

[0086] As a result, the pressure inside the pressure chamber 44 decreases, and a certain amount of the sample liquid containing the target particles P11, such as cells, and the sheath liquid are drawn into the main channel 34, i.e., the inlet of the connecting channel 43, into the connecting channel 43. Then, as shown below arrow Q12, the drawn-in liquid (sample liquid and sheath liquid) gradually changes from a columnar shape to a spherical shape.

[0087] The connecting passage 43 is composed of an upstream connecting passage 71, which is a smaller cross-sectional area portion (first stage portion), and a downstream connecting passage 72, which is a larger cross-sectional area portion (second stage portion) to which the oil passage 38 is connected. Furthermore, the cross-sectional area of ​​the pressure chamber 44 connected to the downstream connecting passage 72 is even larger than the cross-sectional area of ​​the downstream connecting passage 72.

[0088] Therefore, the flow containing the target particles P11 that has been drawn into the connecting channel 43 (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 71, which has a smaller cross-sectional area, to the downstream connecting channel 72, which has a larger cross-sectional area, and further to the pressure chamber 44. At this time, the liquid consisting of the sample liquid and sheath liquid, which contains the target particles P11, separates from the walls of the channels through which the liquid passes, becoming a flow (jet).

[0089] The downstream side of the connecting channel 43 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 72 and beyond, is initially in the shape of a liquid column, but due to interfacial tension, it gradually changes into a spherical shape. In other words, droplets DP11 containing the target particles P11 are formed.

[0090] As shown in the broken line L11, after the period T12, the control unit 24 gradually increases the voltage of the drive signal to the normal voltage, returning the drive state of the piezo actuator 103, i.e., the operating state of the sorting unit 35, to the normal state.

[0091] 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 103 gradually extends, as shown by arrow Q13, and consequently, the diaphragm 101 is pushed inward into the pressure chamber 44 by the piezo actuator 103. Although this example describes a case where the drive voltage changes linearly, the drive voltage may also change sinusoidally.

[0092] Even during sorting, oil flows (is introduced) from the oil passage 38 to the connecting passage 43 at a constant flow rate. In addition, when the control unit 24 restores the drive voltage to the normal voltage and drives the piezo actuator 103 back to its normal extended state, a flow in the opposite direction to normal occurs near the connecting passage 43, as shown below arrow Q13. This reverse flow is in the opposite direction to the direction of liquid discharge from the connecting passage 43, that is, the direction toward the downstream side (the direction toward the pressure chamber 44).

[0093] 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.

[0094] Because the droplets DP11 are advected to the back of the pressure chamber 44 by the jet effect, and because oil is constantly supplied from the oil passage 38, the oil near the outlet of the connecting passage 43 is discharged (flows) downstream, the generated droplets DP11 do not flow back into the connecting passage 43. In other words, the generated droplets DP11 are not released from the connecting passage 43 into the main passage 34.

[0095] 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.

[0096] <Regarding the generation of droplets containing two microparticles> This section explains the generation of droplets containing two target particles (droplets containing two microparticles).

[0097] First, we will explain the target utilization rate, pair generation rate, and Purity, which are indicators for evaluating the performance of generating droplets containing two microparticles.

[0098] In particular, here, a predetermined type of particle to be recovered is designated as the target particle, and other particles of a different type to be recovered are designated as partner particles, and droplets containing the target particle and the partner particle are generated. In other words, the target particle and the partner particle are paired.

[0099] For example, after droplet generation, the microparticles that are to be used for analysis or other processing using those droplets, i.e., microparticles such as cells that are to be observed, are designated as target particles, and the reagents or microparticles such as cells that are to be acted upon by the target particles are designated as partner particles.

[0100] In this example, the objective is to generate a droplet containing one target particle and one or more partner particles. That is, a droplet containing two or more target particles is not permitted, but a droplet containing two or more partner particles is permitted. Therefore, if a droplet containing one target particle and one or more partner particles, but no other microparticles, is generated, droplet generation is considered successful. Hereafter, the generated droplet will also be referred to as a paired droplet.

[0101] The target utilization rate, used as an indicator for evaluating the performance of paired droplet generation, is calculated by the following equation (1). This target utilization rate indicates the utilization efficiency of target particles contained in the sample solution.

[0102]

[0103] In equation (1), the target utilization rate is obtained by dividing the number of target particles that successfully formed droplets (paired) and were recovered from the recovery channel 36 by the number of target particles introduced, i.e., the number of target particles contained in the sample solution.

[0104] Purity is obtained by dividing the number of successfully generated pair droplets recovered from the recovery channel 36—that is, the number of droplets containing one target particle and one or more partner particles—by the total number of droplets recovered from the recovery channel 36. Purity indicates how many successfully generated pair droplets are contained within the recovered droplets.

[0105] Furthermore, the pair generation rate, which indicates the rate at which paired droplets are formed, can be calculated using the following equation (2).

[0106]

[0107] In equation (2), the pair generation rate is the number of paired droplets generated per second. Here, the number of paired droplets generated is the number of paired droplets that were successfully generated.

[0108] The use of the above targets, pair generation rates, and Purity will be discussed later.

[0109] Next, with reference to Figures 5 and 6, the generation of droplets containing two microparticles by sorting will be explained.

[0110] In Figures 5 and 6, the upper section of the figures shows the flow path from the main flow path 34 to the pressure chamber 44, while the lower section of the figures shows the waveform of the drive signal (drive waveform) that drives the piezo actuator 103.

[0111] 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 timing shown in the upper part (upper section) of the figure for that section of the drive waveform. In other words, at the timing shown in the upper section 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 103 as a drive signal.

[0112] As shown on the left side of Figure 5, in a normal state where no sorting operation is being performed, the optical detection unit 23 (control unit 24) detects the particle P21 to be recovered, and then another particle P22 to be recovered is detected.

[0113] Here, we assume that the recovered particles P21 and P22 are of different types, such as a target particle and a partner particle.

[0114] 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. Furthermore, 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.

[0115] 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.

[0116] 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.

[0117] In the example shown in Figure 5, the control unit 24 drives the piezo actuator 103 as shown in the lower left panel of Figure 5 when the first target particle P21 has been advected to the vicinity of the connecting channel 43 (upstream connecting channel 71), thereby initiating the first sorting operation. In the lower left panel of Figure 5, the broken line L21 shows the drive waveform during the first sorting operation.

[0118] When the first sorting operation is performed, as shown on the right in Figure 5, the target particle P21 is drawn into the connecting channel 43 (recovered), and a droplet DP21 containing one minute particle, including the target particle P21, is generated. When the first droplet, droplet DP21, is generated, the control unit 24 controls the driving of the piezo actuator 103 so that the velocity (initial velocity) of droplet DP21 in the pressure chamber 44 is relatively slow, that is, slower than the velocity of droplet DP22, which will be described later.

[0119] Immediately after the formation of droplet DP21, when the second target particle to be recovered, P22, has advected to the vicinity of the upstream connecting channel 71, the control unit 24 drives the piezo actuator 103 to start the second sorting operation, as shown in the lower right panel of Figure 5. In the lower right panel of Figure 5, the broken line L22 shows the drive waveform during the second sorting operation.

[0120] Here, the drive waveform during the first sorting operation and the drive waveform during the second sorting operation are different. In other words, the control unit 24 performs different drive control on the piezo actuator 103 for the first sorting operation and the second sorting operation. In particular, comparing the broken lines L21 and L22, the voltage difference of the drive signal (the difference between the normal voltage and the holding voltage) is larger for broken line L22 than for broken line L21. In other words, the drive voltage (holding voltage) of the drive waveform during the first sorting operation shown by broken line L21 is greater than the drive voltage (holding voltage) of the drive waveform during the second sorting operation shown by broken line L22.

[0121] When the second sorting operation is performed, as shown on the left side of Figure 6, the particles to be recovered P22 are drawn into the connecting channel 43, and a droplet DP22 containing one minute particle, which includes the particles to be recovered P22, is generated. When the second droplet, droplet DP22, is generated, the control unit 24 controls the driving of the piezo actuator 103 so that the velocity (initial velocity) of droplet DP22 in the pressure chamber 44 is relatively fast.

[0122] Thus, after the slow-moving droplet DP21 (first droplet) is generated, when the fast-moving droplet DP22 (second droplet) is generated, droplet DP22 catches up to droplet DP21, and the two droplets DP21 and DP22 collide and merge. That is, droplet DP22 collides with droplet DP21, and the two droplets DP21 and DP22 merge, generating a single droplet DP23 (pair droplet) containing the target particles P21 and P22, as shown on the right side of Figure 6.

[0123] 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.

[0124] 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 (pair) a single droplet DP23.

[0125] The velocity difference between droplet DP21 and droplet DP22 can be achieved (controlled) by changing the waveform of the drive signal (drive waveform) that drives the piezo actuator 103.

[0126] Specifically, the drive waveform of the piezo actuator 103 is determined by the fall time, hold time, voltage difference, and rise time of the drive voltage described above. Therefore, by appropriately changing the fall time, hold time, voltage difference, and rise time of the drive waveform, it is possible to control the velocity of the generated droplets.

[0127] <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 103 is driven until the operation to generate droplets begins.

[0128] 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).

[0129] When the recovery for each delay time is plotted, the characteristics shown in Figure 7 are obtained. In Figure 7, the horizontal axis represents the delay time, and the vertical axis represents the recovery.

[0130] In the characteristics shown in Figure 7, 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%.

[0131] 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.

[0132] 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.

[0133] 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.

[0134] 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.

[0135] 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.

[0136] 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).

[0137] <Regarding the limit time> For example, in the example explained with reference to Figures 5 and 6, droplet DP21 containing the target particle P21 is slow to move downstream after its formation. Therefore, it has been found that after a certain amount of time has elapsed, even if a fast-moving droplet DP22 containing the target particle P22 is formed, the collision and coalescence will fail.

[0138] Figures 8 and 9 show examples of failed collision and merging. In Figures 8 and 9, parts corresponding to those in Figure 5 or Figure 6 are denoted by the same reference numerals, and their explanations are omitted as appropriate.

[0139] For example, as shown on the left side of Figure 8, suppose that in a normal state where no sorting operation is being performed, the optical detection unit 23 (control unit 24) detects the first particle to be recovered, which is the particle to be recovered P21, and then, after a predetermined time has elapsed, the second particle to be recovered, which is the particle to be recovered P22, is detected.

[0140] In this case, when the particles to be recovered P21 have been advected to the vicinity of the upstream connecting channel 71, the piezo actuator 103 is started to drive with the drive waveform shown by the broken line L21, as shown in the lower left of Figure 8, and the first sorting operation is performed.

[0141] The target particle P21 is then drawn into the connecting channel 43, and a droplet DP21 containing the target particle P21 is generated. This droplet DP21 is advected downstream at a relatively slow speed, as shown on the right side in Figure 8.

[0142] After some time has passed since the formation of droplet DP21, when the particles to be recovered P22 have been advected to the vicinity of the upstream connecting channel 71, the piezo actuator 103 is driven with the drive waveform shown by the broken line L22, as shown in the lower right panel of Figure 8, and the second sorting operation is started.

[0143] When the second sorting operation is performed, as shown on the left side of Figure 9, the particles to be recovered P22 are drawn into the connecting channel 43, and droplets DP22 containing the particles to be recovered P22 are generated.

[0144] Although the velocity (movement speed) of droplet DP22 is faster than that of droplet DP21, the timing of droplet DP22's generation was delayed, so droplet DP22 cannot catch up to droplet DP21, as shown on the right side of Figure 9. In other words, droplet DP22 does not collide with droplet DP21, and the generation of a droplet containing two microparticles fails.

[0145] Thus, if the generation of droplets containing two microparticles fails, droplets DP21 containing only the target particle P21 and droplet DP22 containing only the target particle P22 will be generated. As a result, these droplets containing only one target particle cannot be used for analysis or other processing. In other words, droplets containing only one target particle are wasted.

[0146] Therefore, the control unit 24 sets a limit time after the generation of the first droplet (a droplet containing one minute particle) that is still unable to collide and merge even if it waits any longer.

[0147] When generating droplets containing two microparticles, if the second target particle is not detected within the limit time after the generation of the first droplet (a droplet containing one microparticle) containing the first target particle, the waiting period until the second target particle is detected is canceled. In other words, the sorting operation to generate the second droplet, which uses the already generated first droplet (a droplet containing one microparticle), is canceled.

[0148] Then, the recovery of the next target particle, that is, the generation of a new first droplet (a droplet containing one microparticle), begins. In this way, the number of useless droplets containing only one target particle can be reduced.

[0149] <Regarding the collection order of fine particles> This section explains the collection order of particles of different types that are to be collected.

[0150] 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.

[0151] 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.

[0152] 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).

[0153] 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.

[0154] 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).

[0155] 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.

[0156] For example, suppose we have a sample solution containing a predetermined target particle (particle to be recovered 1) and a partner particle (particle to be recovered 2).

[0157] In this case, simulations have shown that the target utilization rate, pair generation rate, and Purity, which are aspects of pair droplet generation performance, change depending on the component concentration ratio of target particle 1 and target particle 2 in the sample solution, or the count ratio, which is the ratio of the number of each target particle detected by the optical detection unit 23 (control unit 24).

[0158] Figures 10 to 12 show the Monte Carlo simulation results, which reflect the device characteristics obtained from experimental results of the microfluidic chip 21, for target utilization rate, pair generation rate, and Purity when the optical detection rate of microparticles is fixed at 5000 eps and the component concentration ratio of target particle 1 (target particle) and target particle 2 (partner particle) is varied. In particular, this is an example of Monte Carlo simulation of pair droplet generation performance assuming that target particle 1 and target particle 2 follow a Poisson process. The optical detection rate is the number of events, i.e., microparticles, detected per second by the optical detection unit 23 (control unit 24) (unit: events / sec (eps)).

[0159] Figure 10 shows the target utilization rate when paired droplets are generated.

[0160] In Figure 10, the horizontal axis represents the concentration of target particles 1 (the particles to be recovered) in the sample solution, and the vertical axis represents the target utilization rate.

[0161] The concentration of target particle 1 in the sample solution is equivalent to the component concentration ratio of target particle 1 and partner particle 2.

[0162] Furthermore, the target utilization rate is given by equation (1) above, and is the ratio of the number of target particles recovered as paired droplets from the recovery channel 36 to the number of target particles contained in the sample solution. Here, a paired droplet is a droplet containing one target particle and one or more partner particles. The target utilization rate is an indicator of the utilization efficiency of target particles.

[0163] In Figure 10, line L51 shows the target utilization rate when paired droplets are generated using the fixed recovery order method, and line L52 shows the target utilization rate when paired droplets are generated using the variable recovery order method.

[0164] Comparing these line graphs L51 and L52, we can see that the superiority of the fixed-sequence-of-collection method versus the variable-sequence-of-collection method, i.e., the target utilization rate, reverses when the target particle concentration (target concentration) reaches 20%, but there is no significant difference in target utilization rate at any concentration.

[0165] Figure 11 shows the pair generation rate when paired droplets are generated.

[0166] In Figure 11, the horizontal axis represents the concentration of target particles 1 in the sample solution, and the vertical axis represents the pair generation rate. The pair generation rate is given by equation (2) above and represents the number of paired droplets generated per second. The pair generation rate is an indicator of the throughput of paired droplet generation.

[0167] In Figure 11, line L61 shows the pair generation rate when paired droplets are generated using the fixed retrieval order method, and line L62 shows the pair generation rate when paired droplets are generated using the variable retrieval order method.

[0168] Comparing these line graphs L61 and L62, we can see that as the target particle concentration exceeds 20%, the pair generation rate of the variable recovery order method gradually increases compared to the fixed recovery order method. Furthermore, when the target particle concentration is 90%, the pair generation rate of the variable recovery order method is more than 1.5 times better (higher performance) than that of the fixed recovery order method.

[0169] Figure 12 shows the Purity when paired droplets are generated.

[0170] In Figure 12, the horizontal axis represents the concentration of target particles 1 (the particles to be recovered) in the sample solution, and the vertical axis represents Purity.

[0171] Purity is the ratio of the number of successfully generated paired droplets to the total number of droplets recovered in the recovery channel 36. Purity is an indicator of how much of the recovered droplets were generated as intended (paired droplets).

[0172] In Figure 12, line L71 shows the Purity when paired droplets are generated using the fixed collection order method, and line L72 shows the Purity when paired droplets are generated using the variable collection order method.

[0173] Comparing these line graphs L71 and L72, it can be seen that when the target particle concentration (target concentration) is 50% or less, the fixed recovery order method performs better than the variable recovery order method, i.e., has higher purity.

[0174] This is because, in variable recovery order methods, partner particles, which make up a larger proportion of the sample solution, are detected first and formed into droplets, but the target particles are often not detected for a long time, causing the limit time to expire and the formation of paired droplets to be canceled. In this case, as a result, many droplets containing only partner particles are generated, leading to a decrease in purity.

[0175] On the other hand, when the concentration of target particles (target concentration) is 50% or higher, the variable recovery order method performs slightly better (higher Purity) than the fixed recovery order method.

[0176] From the results shown in Figures 10 to 12 above, it can be seen that when the concentration of target particles (component concentration ratio) is low, it is best to generate droplets using a fixed recovery order method, and conversely, when the concentration of target particles is high, it is best to generate droplets using a variable recovery order method.

[0177] 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.

[0178] 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.

[0179] As a specific example, the control unit 24 may perform the determination by threshold processing based on a predetermined ratio (threshold) "X%".

[0180] In other words, for example, the control unit 24 can generate droplets using a fixed recovery order method when the component concentration ratio or count ratio of the target particles is less than X%, and generate droplets using a variable recovery order method when the component concentration ratio or count ratio of the target particles is X% or more. It is desirable that the value of ratio X used as a threshold be 50 or less, or more specifically, a value between 20 and 50 (a value of 20 or more and 50 or less). For example, the value of ratio X can be set to 40.

[0181] Figures 13 and 14 show the droplet generation results when droplets were actually generated using the microfluidic chip 21.

[0182] Figure 13 shows the vicinity of the connecting channel 43 and pressure chamber 44 when two microparticle-containing droplets are generated using the microfluidic chip 21.

[0183] In the example shown in Figure 13, when droplets containing the first target particle are generated, the piezo actuator 103 is driven with a drive waveform having a fall time of 30 μsec, a voltage difference of 31 V, a holding time of 30 μsec, and a rise time of 30 μsec.

[0184] Furthermore, when droplets containing the second set of particles to be recovered are generated, the piezo actuator 103 is driven with a drive waveform that has a fall time of 60 μsec, a voltage difference of 48 V, a holding time of 120 μsec, and a rise time of 60 μsec.

[0185] In this example, the driving (sorting operation) of the piezo actuator 103 for generating the first droplet is started at a timing of "0 μsec".

[0186] Furthermore, it can be seen that 1000 μsec after the start of operation a first droplet DP41 containing the first target particle for recovery is generated, and that this first droplet DP41 is advected at a low speed until 4000 μsec after the start of operation.

[0187] Furthermore, immediately afterward, the piezo actuator 103 is driven, and the generation of a second droplet DP42 containing the second target particle for collection begins. The second droplet DP42 moves quickly, and 4350 μsec after the start of driving for the generation of the first droplet, the second droplet DP42 catches up with the first droplet DP41 and collides and merges, and 4560 μsec later, it can be confirmed that a single droplet DP43 containing the first target particle and the second target particle for collection has been generated.

[0188] Figure 14 shows the results of generating droplets containing two types of microparticles when using the microfluidic chip 21. Specifically, Figure 14 shows an optical microscope image of the collection liquid obtained by generating a large number of droplets containing a first target particle and a second target particle, and collecting these droplets in the collection channel 36.

[0189] In the example shown in Figure 14, when a droplet containing the first target particle is generated, the piezo actuator 103 is driven with a drive waveform that has a fall time of 30 μsec, a voltage difference of 32 V, a holding time of 25 μsec, and a rise time of 15 μsec.

[0190] Furthermore, when droplets containing the second set of particles to be recovered are generated, the piezo actuator 103 is driven with a drive waveform that has a fall time of 30 μsec, a voltage difference of 46 V, a holding time of 25 μsec, and a rise time of 15 μsec.

[0191] In this example, each circle represents a single droplet, and the points within those droplets represent minute particles. Here, many droplets containing two target particles are generated, confirming that droplets containing the first and second target particles can be generated with high accuracy.

[0192] <Explanation of the microparticle recovery process> Next, the operation of the microparticle sorting system 11 will be explained.

[0193] 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 15 is performed. The fine particle recovery process will be explained below with reference to the flowchart in Figure 15.

[0194] In step S11, a suspension of fine particles containing the fine particles to be recovered is prepared as a sample solution.

[0195] 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.

[0196] In step S12, a preliminary measurement is performed and the fractionation of particles to be recovered is specified.

[0197] 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.

[0198] 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.

[0199] In such cases, as shown in Figure 16, 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.

[0200] 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).

[0201] 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.

[0202] 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 16, then that minute particle is considered a target particle.

[0203] 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.

[0204] 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.

[0205] 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.

[0206] 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 16, 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.

[0207] 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.

[0208] Returning to the flowchart in Figure 15, in step S13, the control unit 24 selects a method for recovering the fine particles. In other words, the control unit 24 decides whether to carry out the process (process) of generating droplets containing two fine particles using a fixed recovery order method or a variable recovery order method.

[0209] 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.

[0210] 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 selects (determines) a recovery method based on the entered component concentration ratios.

[0211] Furthermore, if the count ratio of each target particle has been obtained through prior measurement, the control unit 24 will select (determine) a recovery method based on the count ratio.

[0212] Specifically, for example, the control unit 24 selects a fixed recovery order method as the recovery method when the component concentration ratio or count ratio of the target particles is less than X%, and selects a variable recovery order method as the recovery method when the component concentration ratio or count ratio of the target particles is X% or more.

[0213] Furthermore, it may be predetermined whether the recovery method is a fixed recovery order method or a variable recovery order method.

[0214] Once the retrieval method is selected, the actual measurement begins. Alternatively, the actual measurement may begin immediately after the preliminary measurement.

[0215] 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.

[0216] 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.

[0217] 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. 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.

[0218] 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.

[0219] 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.

[0220] 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.

[0221] 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.

[0222] 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.

[0223] <Explanation of droplet generation process> For example, in step S15 of Figure 15, the droplet generation process shown in Figure 17 is performed as a process to generate droplets containing two fine particles.

[0224] The droplet generation process by the microparticle sorting system 11 will be explained below with reference to the flowchart in Figure 17.

[0225] In step S51, the control unit 24 determines whether or not an event has been detected.

[0226] 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.

[0227] 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).

[0228] As a specific example, if the control unit 24 holds gate information as described with reference to Figure 16, 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.

[0229] 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.

[0230] If, in step S51, it is determined that an event has been detected, the process then proceeds to step S52.

[0231] 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.

[0232] Specifically, for example, suppose the fine particles to be recovered are a target particle and a partner particle, and in step S13 of Figure 15, 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.

[0233] 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 15. 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.

[0234] 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.

[0235] 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.

[0236] 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).

[0237] 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).

[0238] 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.

[0239] 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.

[0240] 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 particle to be recovered. The control unit 24 generates the first droplet by driving the piezo actuator 103 according to the output sort time.

[0241] For example, for the first target particle to be recovered, the driving conditions of the piezo actuator 103, namely the driving waveform determined from the fall time, voltage difference, holding time, and rise time, as well as the delay time DT until the piezo actuator 103 is started to operate, are predetermined as set conditions.

[0242] The control unit 24 supplies a drive signal to the piezo actuator 103 according to the setting conditions defined for the first target particle to be recovered, that is, with a predetermined drive waveform and drive start timing, thereby driving the piezo actuator 103. In other words, the sorting operation is performed by the sorting unit 35.

[0243] The piezo actuator 103 performs the drive described, for example with reference to Figure 5, according to the control of the control unit 24. That is, the piezo actuator 103 pulls the diaphragm 101 outward from the pressure chamber 44, and after the diaphragm 101 is held in a certain position, the diaphragm 101 is pushed inward from the pressure chamber 44.

[0244] This causes the pressure in the pressure chamber 44 to fluctuate, generating a relatively slow-moving first droplet containing the first target particles. 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) within the emulsion during emulsion formation.

[0245] As described above, in response to the detection result of an event (microparticle), the control unit 24 controls the drive of the piezo actuator 103 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.

[0246] After the process in step S54 is performed and the sorting time of the first droplet is determined, the process proceeds to step S55.

[0247] 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.

[0248] 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.

[0249] 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 time from the sorting time of the first droplet (the time when sorting was actually performed or the scheduled sorting time) to the arrival time of the microparticle (event) calculated based on the event detection time is less than or equal to a predetermined limit time. The limit time here refers to the time from the sorting time of the first droplet as described above until the generation of the two microparticle-containing droplet is canceled. In step S55, it is determined whether the time from the sorting time of the first droplet (the time when the first droplet is generated) to the scheduled generation time of the second droplet is less than or equal to the limit time.

[0250] If it is determined in step S56 that the time is not less than or equal to the limit time, that is, if the time from the generation of the first droplet to the generation of the second droplet is longer than the limit time, then even if the second droplet is generated, it cannot be made to collide and merge with the first droplet.

[0251] Therefore, if it is determined in step S56 that the time is not less than or equal to the limit time, that is, if the time has not reached the arrival time of the second target particle even after the limit time has elapsed since the generation of the first droplet, the control unit 24 cancels the execution of the sorting operation to generate the second droplet. In other words, if the time from the generation time of the first droplet to the generation time of the second droplet exceeds a predetermined time (limit time), the execution of the sorting operation to generate the second droplet is canceled. Then, the processing in steps S57 to S59 is performed, and the sorting time of the first droplet is output again. That is, a sorting operation to generate a new first droplet is performed according to the detection result of the event (fine particle). In this way, droplets containing two fine particles can be generated more efficiently.

[0252] Note that the processing in steps S57 to S59 is the same as the processing in steps S52 to S54, so the explanation is omitted. However, if it is determined in step S57 that the particle is not the first particle to be recovered, and if it is determined in step S58 that droplet formation is not possible, the process then returns to step S55. Also, once the processing in step S59 is performed, the process then returns to step S55.

[0253] If it is determined in step S56 that the elapsed time is less than or equal to the limit time, then it is possible to generate droplets containing two fine particles, and the process proceeds to step S60.

[0254] In step S60, 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.

[0255] Specifically, for example, suppose the fine particles to be recovered are a target particle and a partner particle, and in step S13 of Figure 15, 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 S60 that it is the second particle to be recovered.

[0256] 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 15. 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 S60 to be the second particle to be recovered.

[0257] If it is determined in step S60 that the particle is not the second target particle for recovery, the subsequent processing returns to step S55, and the above-described processing is repeated.

[0258] In contrast, if it is determined in step S60 that it is a second particle to be recovered, in step S61 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.

[0259] For example, in step S61, 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.

[0260] If it is determined in step S61 that droplet formation is not possible, the process returns to step S55, and the above-described process is repeated.

[0261] In contrast, if it is determined in step S61 that droplet formation is possible, in step S62 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 103 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.

[0262] For example, for the second set of particles to be recovered, different setting conditions are predetermined for driving the piezo actuator 103 than those for the first set of particles to be recovered. These settings include the drive waveform and delay time DT determined from the fall time, voltage difference, holding time, and rise time.

[0263] The control unit 24 drives the piezo actuator 103 by supplying a drive signal to the piezo actuator 103 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.

[0264] The piezo actuator 103 performs the drive described, for example, with reference to Figure 6, according to the control of the control unit 24. In this case, although the fall time, voltage difference, holding time, and rise time of the drive waveform are different, the drive is performed in the same way as in step S54.

[0265] This causes the pressure in the pressure chamber 44 to fluctuate, generating a second droplet with a relatively high velocity that contains the second target particle for recovery. 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.

[0266] As described above, in response to the detection result of the event (fine particles), the control unit 24 controls the drive of the piezo actuator 103 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 103 so that a different pressure fluctuation occurs in the pressure chamber 44 than in step S54 (first separation process) described above.

[0267] 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. In particular, in this case, a second droplet is generated that moves at a faster speed than the first droplet.

[0268] In step S63, the control unit 24 determines whether or not to terminate the process of generating droplets containing two fine particles.

[0269] If it is determined in step S63 that the process is complete, the droplet generation process is terminated.

[0270] On the other hand, if it is determined in step S63 that the process of generating droplets containing two fine particles has not yet been completed, the process then proceeds to step S64.

[0271] In step S64, the control unit 24 determines whether or not an event has been detected. In step S64, the same processing as in step S51 is performed.

[0272] If it is determined in step S64 that no event was detected, the process in step S64 is repeated until it is determined that an event was detected.

[0273] On the other hand, if it is determined in step S64 that an event has been detected, in step S65 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.

[0274] 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 72 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.

[0275] 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.

[0276] If it is determined in step S65 that the wait time has not elapsed, i.e., if the wait time has not yet elapsed, the process returns to step S64, and the process described above is repeated.

[0277] If, in step S65, it is determined that the time is longer than the wait time, then the processing in steps S66 to S68 is performed, and a new sort time for the first droplet is output.

[0278] Note that the processing in steps S66 to S68 is the same as the processing in steps S52 to S54, so the explanation is omitted. However, if it is determined in step S66 that the particle is not the first particle to be recovered, and if it is determined in step S67 that droplet formation is not possible, the process then returns to step S64. Also, once the processing in step S68 is performed and the first droplet is generated, the process then returns to step S55.

[0279] As described above, the microparticle sorting system 11 drives the piezo actuator 103 to generate a first droplet at a relatively slow speed, and then generates a second droplet at a relatively high speed, while simultaneously causing the first and second droplets to collide and merge to produce a two-microparticle-containing droplet.

[0280] In this way, droplets containing the desired fine particles, namely the first and second target particles, can be generated more efficiently.

[0281] <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).

[0282] 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.

[0283] 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.

[0284] At this time, droplet generation is performed such that the velocity (initial velocity) of the newly generated droplet containing one microparticle is faster than the velocity (initial velocity) of the droplet containing one microparticle generated immediately before it. Furthermore, the drive of the piezo actuator 103 during droplet generation is controlled so that the velocity difference between these two consecutively generated droplets containing one microparticle, or more specifically, the velocity difference between the droplet containing one microparticle and the droplet (multiple microparticle droplet) generated by collision and fusion immediately before the generation of that droplet, is sufficient to fuse the droplet interfaces when the droplets collide.

[0285] Referring to Figures 18 to 20, an example of generating a single droplet containing three target particles (a droplet containing three microparticles) will be explained.

[0286] In Figures 18 to 20, the upper section of the figures shows the flow of each channel from the main channel 34 to the pressure chamber 44, while the lower section of the figures shows the waveform of the drive signal (drive waveform) that drives the piezo actuator 103.

[0287] 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 timing shown in the upper part (top row) of the diagram for that section of the drive waveform.

[0288] As shown on the left side of Figure 18, in a normal state where no sorting operation is being performed, the optical detection unit 23 (control unit 24) detects the target particle P101 through event detection.

[0289] When the particles to be recovered P101 have been advected to the vicinity of the upstream connecting channel 71, the control unit 24 drives the piezo actuator 103 as shown in the lower left of Figure 18, and starts the first sorting operation. In this case, the first sorting operation is performed with the drive waveform shown by the broken line L91. In other words, the first sorting process described above is carried out.

[0290] When the first sorting operation is performed, as shown on the right side of Figure 18, the particles to be recovered P101 are drawn into the connecting channel 43 (recovered), and droplets DP101 containing the particles to be recovered P101 are generated. When droplets DP101 are generated, the drive of the piezo actuator 103 is controlled so that the velocity (initial velocity) of the droplets DP101 in the pressure chamber 44 is relatively slow.

[0291] After detecting the target particle P101, the event detection detects the target particle P102. Immediately after the formation of the droplet DP101, when the target particle P102 is advected to the vicinity of the upstream connecting channel 71, the piezo actuator 103 is driven, as shown in the lower right panel of Figure 18, and the second sorting operation is started. In other words, the second sorting process described above is performed. In the lower right panel of Figure 18, the broken line L92 shows the drive waveform during the second sorting operation.

[0292] In this case, the drive waveform during the first sorting operation and the drive waveform during the second sorting operation are different. That is, the control unit 24 performs different drive control on the piezo actuator 103 for the first sorting operation and the second sorting operation.

[0293] When the second sorting operation is performed, as shown on the left side of Figure 19, the particles to be recovered P102 are drawn into the connecting channel 43, and droplets DP102 containing the particles to be recovered P102 are generated. When droplets DP102 are generated, the drive of the piezo actuator 103 is controlled so that the velocity (initial velocity) of the droplets DP102 in the pressure chamber 44 is moderate.

[0294] Thus, after a slow-moving droplet DP101 is generated, if a faster-moving droplet DP102 is generated, droplet DP102 catches up to droplet DP101, and the two droplets DP102 and DP101 collide and merge through interfacial fusion. As a result, as shown on the right side of Figure 19, a single droplet DP103 (a droplet containing two microparticles) containing the target particles P101 and P102 is generated.

[0295] After detecting the target particle P102, the event detection detects the target particle P103, and immediately after the formation of the droplet DP103, when the target particle P103 is advected to the vicinity of the upstream connecting channel 71, the piezo actuator 103 is driven as shown in the lower right panel of Figure 19, and the third sorting operation is started. In the lower right panel of Figure 19, the broken line L93 shows the drive waveform during the third sorting operation.

[0296] In this case, the drive waveform during the third sorting operation is different from the drive waveforms during the first and second sorting operations. In other words, the control unit 24 performs different drive control on the piezo actuator 103 for the first, second, and third sorting operations.

[0297] When the third sorting operation is performed, as shown on the left side of Figure 20, the particles to be recovered P103 are drawn into the connecting channel 43, and droplets DP104 containing the particles to be recovered P103 are generated. When droplets DP104 are generated, the drive of the piezo actuator 103 is controlled so that the velocity (initial velocity) of the droplets DP104 in the pressure chamber 44 is fast.

[0298] Thus, after droplet DP103 is formed, if droplet DP104 is formed at a faster speed than droplet DP103, droplet DP104 will catch up to droplet DP103, and droplets DP104 and DP103 will collide and merge through interfacial fusion. As a result, as shown on the right side of Figure 20, a single droplet DP105 (a droplet containing three fine particles) containing the target particles P101, P102, and P103 is formed.

[0299] The process of generating droplets DP105 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 103 to fluctuate the pressure in the pressure chamber 44, thereby separating the target particles P103 flowing through the main channel 34 into the oil and generating droplets DP104. In particular, in the third separation step, the control unit 24 controls the drive of the piezo actuator 103 so that different pressure fluctuations occur in the pressure chamber 44 compared to the first and second separation steps.

[0300] In the third separation step, the particles to be recovered P103 are separated into oil while they are contained in a liquid consisting of the sample liquid and the sheath liquid, and droplets DP104 consisting of the sample liquid and the sheath liquid, containing the particles to be recovered P103, are generated. In particular, in this case, droplets DP104 are generated that move at a faster speed than droplets DP103.

[0301] In the sorting section 35, a velocity difference sufficient to cause interfacial fusion between two colliding droplets is generated. Specifically, the drive of the piezo actuator 103 is controlled so that droplet DP102 moves faster than droplet DP101, and droplet DP104 moves faster than droplet DP102.

[0302] 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 can be fused at the interface with a simple configuration, and a single droplet containing three microparticles can be efficiently generated.

[0303] The velocity difference of each droplet can be achieved by changing the drive waveform of the piezo actuator 103. Specifically, the velocity of the generated droplets can be controlled by appropriately changing the fall time, hold time, voltage difference, and rise time of the drive waveform described above.

[0304] According to this technology, it is possible to generate droplets containing multiple desired fine particles more efficiently.

[0305] 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).

[0306] 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.

[0307] 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).

[0308] 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.

[0309] 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.

[0310] Specifically, in this technology, for example, the optical detection unit 23 (control unit 24) detects minute particles, and droplet generation is performed according to the detection result, thereby suppressing the influence of chance and enabling the generation of target droplets with high accuracy (efficiency). Moreover, proximity detection can further improve the droplet generation efficiency.

[0311] 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.

[0312] 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.

[0313] <Regarding reagent conservation and stabilization of reactions within droplets> For example, when generating a droplet containing one cell and performing analysis by reacting that cell with a reagent within the droplet, it is conceivable to generate a droplet DP121 containing one cell and a reagent by passing a laminar flow consisting of sample solution SM11 and sheath solution SH11 through the main channel 34, as shown in Figure 21.

[0314] In such cases, for example, the sample solution SM11 containing the suspended cells (the particles to be recovered) is mixed with reagents that react with intracellular substances after cell lysis. As a specific example, when performing a reaction to synthesize cDNA from intracellular mRNA, oligoprimers and reverse transcriptase can be used as reagents.

[0315] Furthermore, the sheath solution SH11 is mixed with reagents that are desirable to come into contact with cells and react after droplet formation, such as cell lysate.

[0316] As the sample solution SM11 and sheath solution SH11 are drawn from the main channel 34 to the upstream connecting channel 71 and droplets are formed, the sample solution SM11 and sheath solution SH11 are mixed within the droplets. Then, as in droplet DP122, for example, cells are lysed and intracellular substances are eluted, initiating a reaction with the reagents mixed in the sample solution SM11.

[0317] In such examples, the sheath fluid SH11 is used to focus the sample flow, consisting of the sample fluid SM11, towards the center of the flow path. However, to improve optical detection accuracy (event detection accuracy), a larger volume of sheath fluid SH11 (sheath flow rate) is required to obtain a narrower sample flow.

[0318] For example, as shown in Figure 22, the cross-section of the main channel 34 is a 200 μm square, and the flow rate of the sample solution SM11 (sample flow rate) is 50 μl / min.

[0319] In this case, to converge the sample flow to 10 μm, that is, to form a sample flow with a cross-sectional length of 10 μm, 10 ml / min of liquid is required to flow through the main channel 34. Therefore, a large amount of sheath solution SH11 needs to be prepared, and a large amount of reagents to be mixed with the sheath solution SH11 are also required. Furthermore, since the sheath solution SH11 mixed with reagents that flows through the main channel 34 is almost entirely discarded, a lot of waste occurs.

[0320] Furthermore, the reagents mixed with the sample solution SM11 are mixed with the sheath solution SH11 during droplet formation and diluted with the sheath solution SH11. For example, under normal conditions, the sample solution SM11 is diluted by approximately 5 to 10 times with the sheath solution SH11.

[0321] Therefore, assuming that the reagent to be mixed with the sample solution SM11 will be diluted with the sheath solution SH11, it is necessary to mix it at a concentration 5 to 10 times the concentration required for the reaction.

[0322] Furthermore, the mixing ratio of sample solution SM11 and sheath solution SH11 varies depending on manufacturing variations of the microfluidic chip 21, sample flow rate, and droplet generation conditions (including the drive waveform of the piezo actuator 103, which includes the voltage difference). As a result, the concentration of the reagent added to sample solution SM11 after droplet mixing varies, leading to variations in reaction conditions and results. In other words, variations occur in the concentration of the cell reaction reagent within the droplet, resulting in variations in reaction results with each experiment.

[0323] Therefore, in this technology, as shown in Figure 23 for example, a sheath flow is generated by adding a layer of reagent flow around the sample liquid (sample flow), thereby saving reagents and suppressing variations in reaction conditions and reaction results.

[0324] In this example, as shown on the left side of the figure, a laminar flow consisting of a sample flow SM21 made up of the sample solution, a reagent flow RE21 made up of the reagent solution, and a sheath flow SH21 made up of the sheath solution flows through the main channel 34.

[0325] Specifically, the sample flow SM21 flows through the center of the main channel 34, and a layer of reagent flow RE21 is formed surrounding (enveloping) the sample flow SM21. Furthermore, a sheath flow SH21 is formed surrounding the reagent flow RE21. In this example, it can also be said that a two-layer sheath flow is formed from the reagent flow RE21 and the sheath flow SH21, enveloping the sample flow SM21.

[0326] In the figure, the right side shows an enlarged cross-section of the dashed-dotted line portion of the main channel 34 shown on the left side. Specifically, the right side of the figure shows the cross-sectional shapes of the sample flow SM21, reagent flow RE21, and sheath flow SH21. The right side of the figure also shows the relationship between the size of the cross-sections of each flow, such as the sample flow SM21, flowing through the main channel 34, and the size of the cross-section at the inlet (upstream end) of the upstream connecting channel 71.

[0327] In this example, the cross-section of the sample flow SM21 is smaller than the cross-section of the upstream connecting channel 71, while the cross-section of the reagent flow RE21 is larger than the cross-section of the upstream connecting channel 71.

[0328] Therefore, when the droplet generation operation (sorting operation) is performed, only the sample flow SM21 and the reagent flow RE21 are drawn into the upstream connection channel 71, and only the sample solution and reagent solution are dropletized, so that the dilution by the sheath solution described above does not occur. In other words, because the sample flow SM21 is surrounded by the reagent flow RE21 which has an appropriate cross-sectional size, the sheath solution does not flow into the upstream connection channel 71, and droplets consisting only of the sample solution and reagent solution are generated.

[0329] Similar to the example described with reference to Figure 22, let's assume, for example, that the cross-section of the main channel 34 is a square with a side length of 200 μm, the flow rate of the sheath flow SH21 is 10 ml / min, and the flow rate of the sample flow SM21 is 50 μl / min. Also, let's assume that the cross-section of the upstream connecting channel 71 is a square with a side length of 30 μm.

[0330] In such a case, the flow rate of the reagent stream RE21 is 800 μl / min, and the cross-sectional length (diameter) of the reagent stream RE21 is approximately 46.5 μm, satisfying the relationship described above. That is, the cross-sectional length (diameter) of the sample stream SM21 can be converged to 10 μm.

[0331] When the sorting operation is performed, for example as shown in Figure 24, only the sample flow SM21 and the reagent flow RE21 are drawn into the upstream connection channel 71, and droplets DP141 containing the particles to be recovered and consisting of the sample solution and the reagent solution are generated (formed). Furthermore, for example as in droplet DP142, when the sample solution and the reagent solution are mixed within droplet DP142, the cells, which are the particles to be recovered, dissolve, intracellular substances are eluted, and these intracellular substances begin to react with the reagent.

[0332] As a specific example, in a sample solution containing suspended cells, such as sample flow SM21, reaction reagents (e.g., oligo-primers and reverse transcriptase) are mixed in, adjusted to the required 1:1 concentration for the droplet reaction. These reaction reagents react with intracellular substances after cell lysis.

[0333] Furthermore, the reagent solution that becomes the reagent flow RE21 is, for example, a mixture of a reagent that comes into contact with cells and reacts after droplet formation (e.g., cell lysate) and a reaction reagent (e.g., oligoprimer and reverse transcriptase) that has been adjusted to a 1:1 concentration, similar to the sample solution.

[0334] In this way, the sample solution and reagent solution are formed into droplets, and even after these droplets are mixed, the reaction reagent is not diluted by the sheath solution. In other words, the droplets contain a 1:1 concentration of the reaction reagent.

[0335] Therefore, this technology allows for precise maintenance of the reagent concentration within the droplet, enabling the droplet reaction to be performed under the same conditions even when multiple experiments are conducted, regardless of individual differences (manufacturing variations) in the microfluidic chip 21 or droplet generation conditions. This suppresses variations in reaction conditions and reaction results. Moreover, since there is no need to add reagents (cell lysate) to the sheath liquid that becomes the sheath flow SH21, reagents can be saved.

[0336] When the two-layer sheath flow described above is passed through the main channel 34, in the microparticle sorting system 11 shown in Figure 1, the sample liquid channel 31, sheath liquid channel 32, and sheath flow forming section 33 of the microfluidic chip 21 are replaced with the configuration shown in Figure 25.

[0337] In the example shown in Figure 25, the microfluidic chip 21 has a sample liquid channel 31, a reagent liquid channel 301-1, a reagent liquid channel 301-2, a sheath liquid channel 302-1, a sheath liquid channel 302-2, a first sheath flow forming section 303, and a second sheath flow forming section 304.

[0338] In the following, when there is no need to distinguish between reagent solution channel 301-1 and reagent solution channel 301-2, they will simply be referred to as reagent solution channel 301, and when there is no need to distinguish between sheath solution channel 302-1 and sheath solution channel 302-2, they will simply be referred to as sheath solution channel 302.

[0339] A cell suspension containing reaction reagents and other particles to be recovered, such as cells, is introduced as the sample solution into the sample solution channel 31, and this sample solution flows through the sample solution channel 31 as a sample stream.

[0340] In the reagent flow path 301, a reagent solution containing reagents (such as cell lysate) or reaction reagents that come into contact with the particles to be recovered after droplet formation is introduced as a sheath liquid, and the reagent solution flows through the reagent flow path 301 as a reagent stream. For example, the reaction reagent is adjusted (mixed) so that it is of equal concentration in the sample solution and the reagent solution.

[0341] The first sheath flow forming section 303 is a sheath-forming channel where the sample liquid channel 31 and the two reagent liquid channels 301 merge. That is, the downstream end of the sample liquid channel 31 and the downstream ends of each of the two reagent liquid channels 301 are connected to the first sheath flow forming section 303, and the sample liquid and reagent liquid merge in the first sheath flow forming section 303.

[0342] In the first sheath flow forming section 303, the sample flow, which is the sample liquid flowing from the sample liquid channel 31, is surrounded by the reagent flow, which is the reagent liquid flowing from each reagent liquid channel 301, and the sample flow is converged to the center of the first sheath flow forming section 303 (channel). In other words, the first sheath flow forming section 303 merges the sample flow and the reagent flow so that the sample flow is surrounded by the reagent flow. As a result, a laminar flow in which the sample liquid is surrounded by the reagent liquid is formed as the first sheath flow.

[0343] The sheath fluid channel 302 is a channel through which a sheath fluid (second sheath fluid) flows, which mainly consists of, for example, physiological saline and does not contain reagents. For example, the sheath fluid introduced into the sheath fluid channel 302 is a liquid with a different composition from the reagent solution (sheath fluid) introduced into the reagent fluid channel 301.

[0344] The second sheath flow forming section 304 is a sheath forming channel where the channel forming the first sheath flow forming section 303 and the two sheath liquid channels 302 merge. That is, the downstream end of the first sheath flow forming section 303 and the downstream ends of each of the two sheath liquid channels 302 are connected to the upstream side of the second sheath flow forming section 304. In addition, the main channel 34 is connected to the downstream end of the second sheath flow forming section 304.

[0345] In the second sheath flow forming section 304, the laminar flow (first sheath liquid) consisting of the sample liquid and reagent liquid that has flowed from the first sheath flow forming section 303 and the sheath liquid (second sheath liquid) from each sheath liquid flow channel 302 merge. At this time, the first sheath liquid, consisting of the sample liquid and reagent liquid, is used as the sample liquid, and the first sheath liquid is surrounded by the second sheath liquid. The resulting laminar flow then flows into the main flow channel 34 as sheath flow.

[0346] In other words, the second sheath flow forming section 304 combines the first and second sheath liquids to form a sheath flow, such that the laminar flow (first sheath liquid) consisting of the sample liquid and reagent liquid is enveloped by the second sheath liquid. The sheath flow thus formed, i.e., the particles to be recovered contained in the sheath flow, is appropriately separated from the main flow channel 34 into the oil in the connecting flow channel 43.

[0347] Furthermore, the first sheath flow forming section 303 and the second sheath flow forming section 304 may be provided with a taper such that the cross-sectional area of ​​the flow path decreases as it moves from the upstream side to the downstream side. In other words, the first sheath flow forming section 303 and the second sheath flow forming section 304 may have tapered flow paths.

[0348] In particular, in the second sheath flow forming section 304, the flow rate of the sheath fluid is high, so there is a risk of secondary flow occurring after the first sheath fluid and the second sheath fluid merge. Therefore, by making the second sheath flow forming section 304 tapered, the occurrence of such secondary flow can be suppressed, and a sheath flow in which the first sheath fluid converges at the center of the flow path can be obtained with a simple configuration.

[0349] Furthermore, any method can be adopted as a method for suppressing secondary flow by tapering, such as the method described in Japanese Patent Application Publication No. 2011-179945. Also, for example, if the flow rate of the reagent liquid passing through the reagent liquid channel 301 is small, the first sheath flow forming section 303 may not have a tapered structure. Whether or not the first sheath flow forming section 303 has a tapered structure can be determined by various conditions.

[0350] Figure 26 shows the results of a fluid simulation when the first sheath flow forming section 303 has a non-tapered shape and the second sheath flow forming section 304 has a tapered shape. In Figure 26, the horizontal axis (Y-axis) and vertical axis (Z-axis) represent the horizontal and vertical positions in space, respectively. In particular, the center positions of the cross-sections of the main flow channel 34 and the upstream connecting flow channel 71 are set to Y=0 and Z=0.

[0351] In this example, the first sheath flow forming section 303, i.e., the confluence of the sample liquid channel 31 and the reagent liquid channel 301, does not have a taper, but the second sheath flow forming section 304, i.e., the confluence of the first sheath flow forming section 303 and the sheath liquid channel 302, does have a taper. Furthermore, the flow rate of the sample liquid is set to 100 μl / min, the flow rate of the reagent liquid is set to 800 μl / min, and the flow rate of the sheath liquid is set to 10 ml / min.

[0352] The roughly circular point group L201 shows the boundary between the sample liquid and the reagent liquid in the first sheath flow when viewed in cross-section of the main channel 34, or more specifically, the cross-section of the connection portion between the main channel 34 and the upstream connecting channel 71. The roughly circular point group L202 shows the boundary between the reagent liquid (reagent flow) and the sheath liquid (second sheath liquid) when viewed in cross-section of the main channel 34.

[0353] Furthermore, the square L203 shows the cross-section of the upstream connecting channel 71. Here, the cross-section of the upstream connecting channel 71 is a square with sides of 30 μm.

[0354] This graph shows that the cross-section of the laminar flow, consisting of the sample flow, reagent flow, and sheath flow, has a concentric shape perpendicular to the direction of flow. It also shows that the cross-section of the sample flow is smaller than the cross-section of the upstream connecting channel 71, and the cross-section of the reagent flow is larger than the cross-section of the upstream connecting channel 71.

[0355] If the microfluidic chip 21 has the configuration shown in Figure 25, the following may be used as the sample solution, reagent solution, and sheath solution.

[0356] In other words, for example, the sample solution contains a buffer containing cells (PBS (Phosphate-Buffered Saline) with BSA (Bovine Serum Albumin)), reagents necessary for reverse transcription of mRNA (Ribonucleic acid) to synthesize cDNA (Deoxyribonucleic acid) (dNTP (Nucleoside triphosphate), RNAse inhibitors (RNasin Plus, RNase OUT, etc.), reverse transcriptases (SuperScript III, SuperScript IV, Maxima H Minus, etc.), reverse transcription primers, or particles immobilized with reverse transcription primers), etc.

[0357] Furthermore, reagents necessary for cell lysis (such as NP-40 Surfact-Amps® Detergent Solution, Sarkosyl, and IGEPAL CA-630) and reagents necessary for reverse transcription (such as ThermoPol Reaction Buffer and First-strand buffer, which are necessary for reverse transcription but cannot be included in the sample solution because they have cell lysis properties) are added to the reagent solution.

[0358] Furthermore, physiological saline is used as a sheath solution (second sheath solution), for example. By generating droplets using such sample solution, reagent solution, and sheath solution, the sample solution and reagent solution are mixed within the generated droplet, cell lysis occurs, and mRNA is extracted into the droplet.

[0359] Subsequently, the mRNA priming and cDNA synthesis using reverse transcription primers are carried out within the droplet by adjusting the temperature appropriately, for example, by setting it to 35°C for 5 minutes, then 50°C for 50 minutes, and then 70°C for 15 minutes. Alternatively, by adding PCR (Polymerase Chain Reaction) primers to the sample solution and DNA polymerase to the reagent solution, PCR can be performed after cDNA synthesis within the droplet.

[0360] Furthermore, while the flow rate of the sheath fluid was set to 10 ml / min when the cell lysis reagent was added to the sheath fluid, the flow rate of the reagent solution can be reduced to 800 μl / min, thus reducing the amount of cell lysis reagent used to 1 / 12.5.

[0361] Alternatively, a buffer solution containing cells may be used as the sample solution, and drug candidates (small molecules, antibodies, antibody-drug conjugates, genetically modified cells, beads immobilized with drug candidates, etc.) may be added to the reagent solution. In this way, the cells and drug candidates are mixed when droplets are formed. In this case, drug candidates can be screened by detecting the reaction within the droplet. For detecting the reaction within the droplet, for example, bright-field microscopy, fluorescence microscopy, imaging flow cytometry, and flow cytometry can be used.

[0362] By using a buffer solution or culture medium containing cells in the sample solution, and adding a viral vector for gene transfer (retrovirus, lentivirus, etc.) to the reagent solution, gene transfer into cells can be performed within a droplet. Gene transfer can be performed on a single cell or in groups of cells within the uniform reaction environment of the droplet. In this case, the gene transfer efficiency of a single cell or in groups of cells can be evaluated.

[0363] By adding beads or cells supporting reagents or drugs to a sample solution, and by adding synthesis reagents to the reagent solution, chemical synthesis (chemical synthesis of reagents or drugs, chemical modification of the cell surface, etc.) can be carried out within the droplet.

[0364] Furthermore, by using a buffer or culture medium containing cells in the sample solution, and adding factors necessary for cell differentiation (such as hormones, steroids, cytokines, growth factors, etc.) to the reagent solution, cell differentiation can be carried out at the single-cell level within a droplet.

[0365] In addition, for example, by adding phages to a sample solution and E. coli such as E. coli to a reagent solution, phage growth can be induced within the droplet. Furthermore, by adding beads immobilized with targets that phages bind to to the reagent solution, the grown phages will bind to the targets on the surface of the beads. By collecting these beads, it is possible to select phages that have affinity for the target.

[0366] According to this technology, it becomes possible to precisely control the concentration of the reaction reagent used to react with intracellular substances within a droplet. This ensures that stable droplet reactions always occur under consistent conditions, regardless of variations in the manufacturing of the microfluidic chip 21, the flow rate of the sample solution, or the droplet generation conditions. Furthermore, it reduces the amount of reagent used that was previously mixed with the sheath solution, thereby achieving cost reductions.

[0367] 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).

[0368] 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.

[0369] <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.

[0370] Figure 27 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.

[0371] In a computer, the processing circuit 901, ROM (Read Only Memory) 902, and RAM (Random Access Memory) 903 are interconnected by a bus 904.

[0372] An input / output interface 905 is further connected to the bus 904. An input / output interface 905 is connected to an input unit 906, an output unit 907, a recording unit 908, a communication unit 909, and a drive 910.

[0373] 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.

[0374] 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.

[0375] 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).

[0376] 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.

[0377] 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.

[0378] 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.

[0379] 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.

[0380] 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.

[0381] 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.

[0382] 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).

[0383] 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.

[0384] 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.

[0385] 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.

[0386] 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.

[0387] Furthermore, the embodiments of this technology are not limited to those described above, and various modifications are possible without departing from the gist of this technology.

[0388] 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.

[0389] 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.

[0390] Furthermore, if a single step includes multiple processes, those processes can be executed by a single device or shared among multiple devices.

[0391] Furthermore, this technology can also be configured as follows:

[0392] (1) A method for separating fine particles of a fine particle separation mechanism having a main channel through which fine particles flow and a pressure chamber communicating with the main channel, comprising: a detection step of detecting the fine particles flowing through the main channel; a first separation step of separating the fine particles flowing through the main channel by varying the pressure in the pressure chamber according to the detection result of the fine particles and generating droplets containing the fine particles; and a second separation step of separating the fine particles flowing through the main channel by varying the pressure in the pressure chamber such that a different pressure fluctuation occurs than in the first separation step, according to the detection result of the fine particles and generating droplets containing the fine particles. (2) The fine particle sorting method according to (1), wherein the fine particle sorting mechanism further comprises a drive unit for varying the pressure in the pressure chamber, the drive unit is driven with a first drive waveform in the first sorting step, and the drive unit is driven with a second drive waveform different from the first drive waveform in the second sorting step. (3) The fine particle sorting method according to (2), 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. (4) The fine particle sorting method according to (3), wherein the drive voltage in the first drive waveform is greater than the drive voltage in the second drive waveform. (5) A method for separating fine particles according to any one of (1) to (4), 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 predetermined fine particles in the vicinity of the predetermined fine particles to be separated, and the first separation step or the second separation step is performed according to the result of the determination.(6) A method for separating fine particles according to any one of (1) to (5), wherein a first liquid containing the fine particles flows through the main channel, in the first separation step, the first target particles, which are fine particles, are separated into a second liquid that is immiscible with respect to the first liquid while the first target particles are contained in the first liquid, thereby generating a first droplet containing the first target particles, in the second separation step, the second target particles, which are fine particles, are separated into the second liquid while the second target particles are contained in the first liquid, thereby generating a second droplet containing the second target particles and having a faster moving speed than the first droplet, and the second droplet is combined with the first droplet to generate a third droplet containing the first target particles and the second target particles. (7) The fine particle sorting method according to (6), wherein if the time from the time of generation of the first droplet to the time of generation of the second droplet exceeds a predetermined time, the execution of the second sorting step is canceled. (8) The fine particle sorting method according to (7), wherein after the execution of the second sorting step is canceled, the first sorting step is newly performed according to the detection result of the fine particles. (9) The fine particle sorting method according to any one of (6) to (8), wherein the first target particle is a fine particle of a predetermined type, and the second target particle is a fine particle of a different type from the predetermined type, which is predetermined. (10) The fine particle sorting method according to any one of (6) to (8), wherein the first target particle is the first fine particle detected among two different types of fine particles, and the second target particle is a fine particle of a different type from the first fine particle detected among the two types of fine particles.(11) A method for separating fine particles according to any one of (6) to (8), wherein, based on at least one of the component concentration ratio of the first target particles and the second target particles in the first liquid, and the count ratio which is the ratio of the number of detected first target particles to the second target particles, the first separation step and the second separation step are carried out by a first method in which the fine particles of a predetermined type are designated as the first target particles and the fine particles of another predetermined type different from the predetermined type are designated as the second target particles, or by a second method in which the fine particles that are first detected from among two different types of fine particles are designated as the first target particles and the fine particles of a different type from the first detected fine particle are designated as the second target particles. (12) The fine particle separation method according to (11), wherein if the component concentration ratio or the count ratio is less than a predetermined value, the first separation step and the second separation step are performed by the first method, and if the component concentration ratio or the count ratio is equal to or greater than the predetermined value, the first separation step and the second separation step are performed by the second method. (13) The fine particle separation method according to (12), wherein the predetermined value is 50% or less. (14) A method for separating fine particles according to any one of (6) to (13), further comprising a third separation step of separating the third target particles, which are fine particles, into the second liquid while the third target particles are contained in the first liquid, in accordance with the detection result of the fine particles, thereby generating a fourth droplet containing the third target particles and having a faster movement speed than the third droplet, and combining 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) A method for separating fine particles according to (14), wherein the pressure fluctuations in the pressure chamber are different in the first separation step, the second separation step, and the third separation step. (16) 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, the method for separating fine particles according to any one of (6) to (15).(17) A microparticle separation system comprising: a main channel through which microparticles flow; a pressure chamber communicating with the main channel; a detection unit for detecting the microparticles flowing through the main channel; and a control unit for controlling a drive unit for varying the pressure in the pressure chamber, wherein the control unit controls the drive unit according to the detection result of the microparticles to vary the pressure in the pressure chamber, thereby separating the microparticles flowing through the main channel and generating droplets containing the microparticles; and controls the drive unit according to the detection result of the microparticles to vary the pressure in the pressure chamber so as to cause a different pressure variation than in the first separation step, thereby separating the microparticles flowing through the main channel and generating droplets containing the microparticles. (18) The microparticle separation system according to (17), further comprising: a first sheath flow forming unit that combines the first liquid and the second liquid to form a first sheath flow such that the first liquid containing the microparticles is surrounded by the second liquid; and a second sheath flow forming unit that combines the first sheath flow and the third liquid to form a second sheath flow such that the first sheath flow is surrounded by a third liquid having a different composition from the second liquid, wherein in the first separation step and the second separation step, droplets containing the microparticles are generated by separating the second sheath flow flowing through the main channel.

[0393] 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, 71 Upstream connecting channel, 72 Downstream connecting channel, 101 Vibrating plate, 103 Piezo actuator

Claims

1. A method for separating fine particles using a fine particle separation mechanism having a main channel through which fine particles flow and a pressure chamber communicating with the main channel, comprising: a detection step of detecting the fine particles flowing through the main channel; a first separation step of separating the fine particles flowing through the main channel by varying the pressure in the pressure chamber according to the detection result of the fine particles and generating droplets containing the fine particles; and a second separation step of separating the fine particles flowing through the main channel by varying the pressure in the pressure chamber such that a different pressure fluctuation occurs than in the first separation step, according to the detection result of the fine particles and generating droplets containing the fine particles.

2. The fine particle sorting method according to claim 1, wherein the fine particle sorting mechanism further comprises a drive unit for varying the pressure in the pressure chamber, the drive unit is driven with a first drive waveform in the first sorting step, and the drive unit is driven with a second drive waveform different from the first drive waveform in the second sorting step.

3. The method for separating fine particles according to claim 2, 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.

4. The method for separating fine particles according to claim 3, wherein the driving voltage in the first driving waveform is greater than the driving voltage in the second driving waveform.

5. A method for separating fine particles according to claim 1, 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 predetermined fine particles in the vicinity of the predetermined fine particles to be separated, and the first separation step or the second separation step is performed according to the result of the determination.

6. The method for separating fine particles according to claim 1, wherein a first liquid containing the fine particles flows through the main channel, in the first separation step, the first target particles, which are the fine particles, are separated into a second liquid that is immiscible with respect to the first liquid while the first target particles are contained in the first liquid, thereby generating a first droplet containing the first target particles, in the second separation step, the second target particles, which are the fine particles, are separated into the second liquid while the second target particles are contained in the first liquid, thereby generating a second droplet containing the second target particles and having a faster moving speed than the first droplet, and the second droplet is combined with the first droplet to generate a third droplet containing the first target particles and the second target particles.

7. The fine particle sorting method according to claim 6, wherein if the time from the time of generation of the first droplet to the time of generation of the second droplet exceeds a predetermined time, the execution of the second sorting step is canceled.

8. The method for separating fine particles according to claim 7, wherein, after canceling the execution of the second separation step, the first separation step is newly performed according to the detection result of the fine particles.

9. The method for separating fine particles according to claim 6, wherein the first target particles are fine particles of a predetermined type, and the second target particles are fine particles of a different type from the predetermined type, as determined in advance.

10. The method for separating fine particles according to claim 6, wherein the first target particle is the first fine particle detected from among two different types of fine particles, and the second target particle is a fine particle of a different type from the first fine particle detected from among the two types of fine particles.

11. A method for separating fine particles according to claim 6, wherein, based on at least one of the component concentration ratio of the first target particles and the second target particles in the first liquid, and the count ratio which is the ratio of the number of detected first target particles to the second target particles, the first separation step and the second separation step are performed by a first method in which a predetermined type of fine particles is designated as the first target particles and a predetermined other type of fine particles different from the predetermined type is designated as the second target particles, or by a second method in which the first detected fine particle among two different types of fine particles is designated as the first target particle and the second target particle is a different type of fine particle among the two types of fine particles different from the first detected fine particle.

12. A method for separating fine particles according to claim 11, wherein if the component concentration ratio or the count ratio is less than a predetermined value, the first separation step and the second separation step are performed by the first method, and if the component concentration ratio or the count ratio is equal to or greater than the predetermined value, the first separation step and the second separation step are performed by the second method.

13. The method for separating fine particles according to claim 12, wherein the predetermined value is 50% or less.

14. The method for separating fine particles according to claim 6, further comprising a third separation step, in which, depending on the detection result of the fine particles, the third target particles, which are the fine particles, are separated into the second liquid while the third target particles are contained in the first liquid, thereby generating a fourth droplet containing the third target particles and having a faster movement speed than the third droplet, and the fourth droplet is combined 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 fluctuations in the pressure chamber are different in the first separation step, the second separation step, and the third separation step.

16. The method for separating fine particles according to claim 6, 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. A microparticle separation system comprising: a main channel through which microparticles flow; a pressure chamber communicating with the main channel; a detection unit for detecting the microparticles flowing through the main channel; and a control unit for controlling a drive unit for varying the pressure in the pressure chamber, wherein the control unit controls the drive unit according to the detection result of the microparticles to vary the pressure in the pressure chamber, thereby separating the microparticles flowing through the main channel and generating droplets containing the microparticles; and controls the drive unit according to the detection result of the microparticles to vary the pressure in the pressure chamber so as to cause a different pressure fluctuation than in the first separation step, thereby separating the microparticles flowing through the main channel and generating droplets containing the microparticles.

18. The microparticle separation system according to claim 17, further comprising: a first sheath flow forming unit that combines the first liquid and the second liquid to form a first sheath flow such that the first liquid containing the microparticles is surrounded by the second liquid; and a second sheath flow forming unit that combines the first sheath flow and the third liquid to form a second sheath flow such that the first sheath flow is surrounded by a third liquid having a different composition from the second liquid, wherein in the first separation step and the second separation step, droplets containing the microparticles are generated by separating the second sheath flow flowing through the main channel.

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