Particle sorting method, particle sorting device, and particle sorting system

Abstract

The present invention provides a technique capable of generating a desired emulsion with high efficiency and high purity.　The present invention provides a particle sorting method and the like comprising: a flow step for causing a liquid containing first particles and second particles to flow through a flow path; a first detection step for detecting a plurality of particles flowing in the flow path; an identification step for identifying whether or not the plurality of detected particles are the first particles and the second particles; a calculation step for calculating a time interval between the first particles and the second particles flowing through the flow path; a determination step for determining whether or not to sort the first particles and the second particles according to the time interval; and a sorting step for sorting the first particles and the second particles into an emulsion if it is determined that the particles are to be sorted.

Description

Particle separation method, particle separation apparatus, and particle separation system

[0001] This technology relates to a particle separation method, a particle separation apparatus, and a particle separation system. More specifically, this technology relates to a particle separation method, a particle separation apparatus, and a particle separation system for separating multiple particles into an emulsion.

[0002] In emulsions containing particles, there is a need for techniques to generate and separate emulsions in which only specific particles and particles corresponding to those specific particles are co-encapsulated, in order to further increase the speed, accuracy, and sophistication of analysis.

[0003] Here, an example of a method for generating an emulsion containing specific particles is the technology disclosed in Patent Document 1. Specifically, Patent Document 1 discloses a fine particle sorting mechanism having a flow channel structure including a main flow channel through which fine particles are passed, a recovery flow channel for recovering target particles from the fine particles, a connecting flow channel connecting the main flow channel and the recovery flow channel, and a liquid supply flow channel connected to the connecting flow channel so as to be able to supply liquid, wherein the fine particle sorting mechanism includes a flow step of passing a first liquid containing fine particles through the main flow channel, a determination step of determining whether the fine particles flowing through the main flow channel are target particles, and a recovery step of recovering the target particles into the recovery flow channel, and in the recovery step, the target particles are recovered in a second liquid in the recovery flow channel that is immiscible with the first liquid while contained in the first liquid.

[0004] Furthermore, as a method for separating particles, for example, the technology disclosed in Patent Document 2 can be cited. Specifically, Patent Document 2 discloses a particle separation device comprising: an excitation light irradiation unit that irradiates particles flowing through a channel with excitation light; a velocity detection light irradiation unit that irradiates the particles with velocity detection light at a position different from that of the excitation light; a photodetection unit that detects light emitted from the particles; an arrival time calculation unit that individually calculates the time it takes for each particle to reach a separation unit communicating with the channel from the detection time difference between the light originating from the excitation light and the light originating from the velocity detection light; and a separation control unit that controls the separation of the particles, wherein the channel and the separation unit are provided within a microchip, and the separation control unit determines whether or not to collect the particles based on the data of each particle detected by the photodetection unit and the arrival time calculated by the arrival time calculation unit.

[0005] International Publication No. 2021 / 084814, Japanese Patent Publication No. 2014-202573

[0006] Conventional technologies aimed at separating specific particles, and therefore controlled the separation based on the time it took for those specific particles to reach the separation section connected to the flow path. However, with such separation control, when separating an emulsion in which a first particle and a second particle corresponding to the first particle are co-encapsulated, the time interval between the first particle and the second particle is limited, resulting in poor efficiency in generating the emulsion in which these particles are co-encapsulated. Furthermore, since the proximity of the first and second particles is probabilistic, emulsions other than those containing the desired first and second particles may be generated, leading to problems with purity.

[0007] In light of these circumstances, the primary objective of this technology is to provide a method that can generate desired emulsions with high efficiency and high purity.

[0008] In response to this, the inventors of the present invention have found that the above problem can be solved by a specific particle separation method. Specifically, the present invention provides a particle separation method that includes: first, a flow step of flowing a liquid containing first particles and second particles into a flow channel; first, a detection step of detecting a plurality of particles flowing in the flow channel; an identification step of identifying whether or not the detected plurality of particles are the first particles and the second particles; a calculation step of calculating the time interval between the first particles and the second particles flowing in the flow channel; a determination step of determining whether or not to separate the first particles and the second particles according to the time interval; and, if it is determined that separation is to be performed, a separation step of separating the first particles and the second particles into an emulsion.

[0009] Furthermore, this technology also provides a particle separation method comprising: a flow step of flowing a liquid containing first particles and second particles into a flow channel; a first detection step of detecting a plurality of particles flowing through the flow channel; an identification step of identifying whether the detected plurality of particles are the first particles and the second particles; a calculation step of calculating the time interval between the first particles and the second particles flowing through the flow channel; a determination step of determining whether or not to separate the first particles and the second particles according to the time interval; a separation step of separating the first particles and the second particles into an emulsion if separation is determined to be performed; and a bonding execution step of performing bonding between the first particles and / or secretions secreted from the first particles and the second particles within the emulsion.

[0010] Furthermore, the present technology also provides a particle separator comprising: a channel through which a liquid containing first particles and second particles flows; a detection unit for detecting a plurality of particles flowing through the channel; an information processing unit for processing information obtained from the detection unit; and a separator unit for separatory particles determined to be separatory into an emulsion, wherein the information processing unit performs an identification step of identifying whether the detected plurality of particles are the first particles and the second particles; a calculation step of calculating the time interval between the first particles and the second particles flowing through the channel; and a determination step of determining whether or not to separatory the first particles and the second particles according to the time interval.

[0011] In addition, the present technology also provides a particle separation system comprising: a channel through which a liquid containing first particles and second particles flows; a detection unit for detecting a plurality of particles flowing through the channel; an information processing unit for processing information obtained from the detection unit; and a separation unit for separating particles determined to be separated into an emulsion, wherein the information processing unit is configured to perform: an identification step for identifying whether the detected plurality of particles are the first particles and the second particles; a calculation step for calculating the time interval between the first particles and the second particles flowing through the channel; and a determination step for determining whether or not to separate the first particles and the second particles according to the time interval.

[0012] This figure shows the flow channel structure of a particle sorting microchip, which is an example of the configuration of the particle sorting mechanism used in the particle sorting method according to this embodiment. This figure shows an example of the flow of the particle sorting method according to the first embodiment. This is an enlarged view of an example of the sorting section. This is an enlarged view of the connecting flow channel section. This is an enlarged view of the connecting flow channel section. This is an enlarged view of the connecting flow channel section. This is an enlarged view of the connecting flow channel section. This figure shows an example of a basic drive waveform applied to sort the particles to be sorted. This is a schematic diagram showing the suction and discharge operations. This figure shows the relationship between the holding time Wm and the sorting margin time. This figure shows an example of the flow of the acquisition determination process. This figure shows an outline of the pair determination process and the calculation of the sorting timing Ts. This figure explains the effect of the pair determination process. This figure shows the determination conditions when generating an emulsion containing one first particle and one or more second particles. This is a schematic diagram of an emulsion when the number of first particles and second particles co-encapsulated is (first particle:second particle) = (single particle vs. multiple particles; single:multi). This figure shows the criteria for generating an emulsion containing only one first particle and one second particle. This is a schematic diagram of an emulsion when the number of co-encapsulated first particles and second particles is (first particle:second particle) = (single particle vs. single particle; single:single). This figure shows a part of the flow channel structure of the particle sorting microchip when the second detection step is performed. This figure shows an example of the flow until the acquisition judgment parameters are determined. This figure shows the relationship between various times Td and the number of particles sorted into the sorting channel at various times Td when there is one representative particle. This figure shows the relationship between various times Td and the number of particles sorted into the sorting channel at various times Td when there are two representative particles. This figure shows an outline of the pair determination process and the calculation of the sorting timing Ts in Modification 1. This figure explains an outline of Modification 2. This figure shows an example of the flow of the particle sorting method according to the second embodiment. This figure explains an example of an embodiment of the coupling execution step. This figure explains an example of an embodiment of the coupling execution step. This figure explains an example of an embodiment of the coupling execution step. This figure illustrates an application example of the second embodiment.This figure illustrates an application example of the second embodiment. This figure schematically shows the overall configuration of the particle sorting apparatus according to this embodiment. This is a block diagram illustrating an example of the configuration of the information processing unit. This figure illustrates the waveform of the electrical signal read by the event detection circuit. This figure illustrates an event data packet. This figure illustrates gating for histogram charts and 2D charts. This figure shows an example of an algorithm executed by the information processing unit. This figure shows an example of an algorithm executed by the information processing unit. This figure shows the results of comparing the sorting performance when sorting control is performed using this technology and the conventional technology by Monte Carlo simulation. This figure shows the relationship between the flow path and the sorting unit. This figure shows the relationship between event detection and time, actuator drive signal and time, and recovery rate and time. This figure shows the relationship between event detection and time, actuator drive signal and time, and recovery rate and time when co-encapsulation is performed using the conventional technology. This figure shows the results of co-encapsulation using the conventional technology, with 10 μm beads as the target particles for sorting and 5 μm beads as the paired sorting target particles. This figure shows an example of an embodiment of sequenced particles. This figure illustrates a method for identifying and analyzing S cells that produce molecules that react with E cells. This figure illustrates a method for identifying and analyzing S cells that produce molecules that react with E cells, which differs from Figure 41. This figure illustrates a method for identifying and analyzing S cells that produce molecules that react with E cells, which differs from Figures 41 and 42.

[0013] The following describes preferred embodiments for implementing this technology. Note that the embodiments described below are representative embodiments of this technology, and the scope of this technology is not limited to these embodiments. Furthermore, the description of this technology will proceed in the following order: 1. First Embodiment (Particle Separation Method) (1) Description of the First Embodiment (1-1) Description of the Prior Art (1-2) Overview of this Technology (2) Example of the First Embodiment (2-1) Flow Process S101 (2-2) First Detection Process S102 (2-3) Identification Process S103 (2-4) Calculation Process S104 (2-5) Determination Process S105 (2-6) Separation Process S106 (2-7) Second Detection Process S107 (3) Particle Separation Mechanism and Particles (4) Modified Examples (4-1) Modified Example 1 (4-2) Modified Example 2 2. 1. Second Embodiment (Particle Separation Method) (1) Description of the second embodiment (2) Example of the second embodiment (2-1) Combination execution step S108 (2-2) Application example of the second embodiment (2-3) Other steps 3. Third Embodiment (Particle Separation Apparatus 100) (1) Description of the third embodiment (2) Example of the third embodiment (2-1) Sample S (2-2) Flow path C (2-3) Irradiation unit 101 (2-4) Detection unit 102 (2-5) Information processing unit 103 (2-6) Separation unit 104 (2-7) Others (3) Example of information processing unit 103 and example of algorithm executed by the information processing unit 103 (3-1) Example of information processing unit 103 (3-2) Example of algorithm 4. Fourth Embodiment (Particle Separation System)

[0014] 1. First Embodiment (Particle Separation Method)

[0015] (1) Description of the first embodiment

[0016] The particle separation method according to this embodiment includes a flow step S101, a first detection step S102, an identification step S103, a calculation step S104, a determination step S105, and a separation step S106. A second detection step S107, etc., may also be included as needed.

[0017] (1-1) Explanation of the prior art

[0018] Conventional particle sorting devices and emulsion generators aim to sort target particles, and therefore the timing of sorting is based on the time it takes for the target particles to reach the sorting section. Figure 36 shows the relationship between the flow path and the sorting section. As shown in Figure 36, sorting is performed when the target particles reach point B, just before the orifice, after an event is detected in the detection unit. Sorting of target particles is performed by a hydrodynamically generated suction flow using an actuator in the pressure chamber of the sorting flow path. Particles that reach the sorting section within the time when negative pressure is generated in the sorting flow path and the suction flow is maintained are sucked in. In other words, even if particles are present between B and C or between B and A in Figure 36, sorting will occur, and there are regions where particle sorting is successful.

[0019] Figure 37 shows the relationship between event detection and time, actuator drive signal and time, and recovery rate and time. Typically, in order to stably and reliably separate the target particles, the delay time from event detection to execution of separation is determined so that separation is performed at the time when the recovery rate peaks. In this case, if a preceding particle exists between A and B and a succeeding particle exists between B and C, they are separated together (in the case of an emulsion generator, they are co-encapsulated). The time between A and B is the entrapment time with the preceding particle, and the time between B and C is the entrapment time with the succeeding particle.

[0020] In conventional technology, as described above, only the target particle was targeted for separation, and therefore the separation timing was determined based on the time it took for the target particle to reach the separation unit. Furthermore, there was no control to separate the target particle together with other specific particles. Here, by performing separation regardless of the presence or absence of adjacent particles before and after the target particle, if other adjacent particles are present, it is possible to separate them together and co-encapsulate them. However, whether the target particle and the pairing target particle to be co-encapsulated are present in close proximity within the time of co-encapsulation is probabilistic. In this specification, "pairing target particle" refers to a target particle corresponding to a specific target particle. Also, in this specification, "co-encapsulation" refers to the state in which the target particle and the pairing target particle are contained within the same emulsion.

[0021] Figure 38 shows the relationship between event detection and time, actuator drive signal and time, and recovery rate and time when co-encapsulation is performed using conventional technology. The recovery rate between A1 and C1 in Figure 38 is a time region in the characteristic curve that shows a high recovery rate (hereinafter defined as "sorting margin time"). Therefore, co-encapsulation is successful only when pairing sorting target particles exist within the sorting margin time between A1 and B, or between B and C1. However, the time interval between sorting target particles that successfully co-encapsulate and pairing sorting target particles is limited, resulting in poor co-encapsulation generation efficiency. Furthermore, even when there are no particles to be separated for pairing in close proximity, separation is performed based on the arrival of the particles to be separated at the separation unit. As a result, emulsions containing only the particles to be separated, or emulsions containing unwanted particles other than the particles to be separated for pairing, are also generated, reducing the proportion of the state in which only the desired pair of particles (particles to be separated and particles to be separated for pairing; target particles and partner particles) are separated (i.e., the purity decreases). Figure 39 shows the results of co-encapsulation using conventional technology, with 10 μm beads as the particles to be separated and 5 μm beads as the particles to be separated for pairing. In Figure 39, emulsions that have failed to co-encapsulate can be seen.

[0022] (1-2) Overview of this technology

[0023] In this technology, the sorting timing of at least one of the first and second particles is controlled based on the time it takes for the target particle (hereinafter also referred to as the "first particle") and a nearby partner particle (hereinafter also referred to as the "second particle") to reach the sorting section, using the time interval between the arrival time of the first particle and the arrival time of the second particle. Furthermore, the sorting of the paired first and second particles is controlled based on the particle spacing between the first and second particles, the time interval between the arrival time of the particles before and after the paired particle to the sorting section and the sorting timing, and the types of particles before and after the paired particle. By performing such control, it is possible to generate an emulsion containing only the desired paired particles with higher efficiency and purity compared to conventional technologies.

[0024] By using this technology, a time window exists in which adjacent particles are incorporated during a single sorting operation, and within that time window, there is a time region with a high recovery rate. By determining the sorting timing based on the arrival time of a second particle adjacent to the first particle at the sorting unit, the time region with a high recovery rate can be effectively utilized compared to when the timing is determined based on the first particle, and co-encapsulation of paired particles with greater spacing between them can also be successfully achieved. Furthermore, by determining the acquisition of only the first particle that forms a pair arriving within a time interval of the high recovery rate time window (hereinafter also referred to as "pair determination"), and by comparing the sorting timing of the paired particles with the time interval between the arrival times of the particles before and after the paired particle and the time window for incorporation to determine acquisition (hereinafter also referred to as "proximity determination"), sorting can be performed using only the conditions that successfully generate an emulsion containing only the target paired particles.

[0025] Based on the above, by using this technology, the time interval between the first and second particles that successfully co-encapsulate is increased, and co-encapsulation of first and second particles with a greater distance between them can be achieved. As a result, the number of particles that successfully co-encapsulate increases, and emulsions containing the desired pair of particles can be produced with high efficiency. Furthermore, by determining whether the first and second particles form a pair and determining the proximity of the particles before and after them, only particles that form the desired pair can be separated, thereby producing emulsions containing paired particles with high purity.

[0026] In this technology, the first and second particles are separated into the second liquid, which is immiscible with the first liquid, while still contained in the liquid that contained them (hereinafter also referred to as the "first liquid"), as described later. This allows for the formation of an emulsion in which the second liquid is the dispersion medium and the first liquid is the dispersed phase, with the first and second particles co-encapsulated within each emulsion.

[0027] The dispersed phase and dispersion medium constituting the emulsion may be appropriately selected depending on the type of emulsion to be produced. Furthermore, the emulsion may be a multiple emulsion, and examples of multiple emulsions include oil-in-oil ("o / w / o") and water-in-oil ("w / o / w") emulsions.

[0028] The kinematic viscosity of the second liquid is preferably 1 / 1000 to 1000 times, more preferably 1 / 100 to 100 times, even more preferably 1 / 10 to 10 times, even more preferably 1 / 5 to 5 times, and particularly preferably 1 / 2 to 2 times, the kinematic viscosity of the first liquid. In this technology, it is preferable that the kinematic viscosities of the first liquid and the second liquid are approximately the same. This facilitates the formation of an emulsion.

[0029] The densities of both the first and second liquids at 25°C are, for example, 0.5 g / cm³. 3 ~5g / cm 3 Preferably 0.6 g / cm³ 3 ~4g / cm 3 , more preferably 0.7 g / cm³ 3 ~3g / cm 3 This is possible. Furthermore, the density of the second liquid is preferably 1 / 100 to 100 times the density of the first liquid, more preferably 1 / 10 to 10 times, even more preferably 1 / 5 to 5 times, and particularly preferably 1 / 2 to 2 times. In this technology, it is preferable that the densities of the first liquid and the second liquid are similar. This facilitates the formation of an emulsion.

[0030] The kinematic viscosity of the first liquid and the second liquid at 25°C may be, for example, 0.3 cSt to 5 cSt, preferably 0.4 cSt to 4 cSt, and more preferably 0.5 cSt to 3 cSt.

[0031] The first and second liquids, having the aforementioned physical properties, facilitate the formation of emulsions within the preparative channel. Furthermore, these physical properties facilitate the flow of these liquids within the microchannel.

[0032] In this technology, in one embodiment, the first liquid may be a hydrophilic liquid and the second liquid may be a hydrophobic liquid. In this embodiment, an emulsion can be generated in the separation channel in which the hydrophobic liquid is the dispersion medium and the hydrophilic liquid is the dispersed phase. For example, biological particles such as cells are preferably contained in a hydrophilic liquid such as a buffer solution or culture medium. Therefore, this embodiment is suitable for separating particles that are preferably contained in a hydrophilic liquid, particularly biological particles, and more particularly cells.

[0033] The hydrophilic liquid includes, for example, water, water-miscible liquids, etc. For example, the hydrophilic liquid may be a liquid whose main component is a mixture of one or more selected from the group consisting of water, hydrophilic alcohol, hydrophilic ether, ketone, nitrile solvent, dimethyl sulfoxide, and N,N-dimethylformamide. In this specification, "main component" refers to a component that accounts for, for example, 50% by mass or more, more particularly 60% by mass or more, more particularly 70% by mass or more, 80% by mass or more, 85% by mass or more, or 90% by mass or more of the liquid. Examples of the hydrophilic alcohol include ethanol, methanol, propanol, glycerin, etc. Examples of the hydrophilic ether include tetrahydrofuran, polyethylene oxide, 1,4-dioxane, etc. Examples of the ketone include acetone, methyl ethyl ketone, etc. Examples of the nitrile solvent include acetonitrile, etc.

[0034] The hydrophilic liquid may preferably be a liquid mainly composed of water, and may be, for example, water, an aqueous solution, or a water dispersion. The hydrophilic liquid may be, for example, sheath fluid and / or sample fluid. The hydrophilic liquid is preferably a hydrophilic liquid that does not adversely affect particles (e.g., biological particles). Also, the hydrophilic liquid may be, for example, a liquid containing biomolecules. The biomolecules may be, for example, one or a combination of two or more selected from the group consisting of amino acids, peptides, and proteins. Further, the hydrophilic liquid may contain, for example, a surfactant, particularly a nonionic surfactant. Examples of nonionic surfactants include triblock copolymers of polyethylene oxide and polypropylene oxide, etc., and such triblock copolymers are also referred to as poloxamers or pluronic surfactants. Specifically, a pluronic surfactant is, for example, Pluronic (registered trademark) F68.

[0035] Examples of the hydrophilic liquid include, but are not limited to, culture solutions, buffer solutions, etc. in this embodiment. The buffer solution is preferably a Good buffer. By using a culture solution as the hydrophilic liquid, biological particles (particularly cells) separated as the particles to be separated can be cultured while being held in the emulsion. Also, when the hydrophilic liquid (particularly the sheath fluid) contains a biological particle stimulating component, the biological particles separated as the particles to be separated can be stimulated while being held in the emulsion. Further, characteristics (e.g., morphology, etc.) of the stimulated biological particles can also be observed by a microscope or the like. Furthermore, the hydrophilic liquid (e.g., sheath fluid, sample fluid, etc.) may contain an assay system capable of observing the response to biological particle stimulation. By this assay system, the response from the biological particles separated as the particles to be separated can be optically detected, for example, while being held in the emulsion. The assay system is preferably a wash-free assay system, and for example, a system utilizing fluorescence resonance energy transfer, bioluminescence resonance energy transfer, etc. is preferred.

[0036] The density of the hydrophilic liquid at 25°C is, for example, 0.5 g / cm 3 to 5 g / cm 3 , preferably 0.6 g / cm 3 to 4 g / cm 3 , more preferably 0.7 g / cm 3 to 3 g / cm 3 It can be. The kinematic viscosity of the hydrophilic liquid at 25°C can be, for example, 0.3 cSt to 5 cSt, preferably 0.4 cSt to 4 cSt, more preferably 0.5 cSt to 3 cSt. By having the above-described physical properties, the hydrophilic liquid easily flows in the microchannel, and an emulsion is easily generated in the fractionation channel.

[0037] The hydrophobic liquid may be any liquid selected from liquids immiscible with the hydrophilic liquid. The hydrophobic liquid is, for example, an aliphatic hydrocarbon, a fluorinated oil, a low molecular or high molecular weight compound containing a fluorine atom, a silicone oil, an aromatic hydrocarbon, an aliphatic monohydric alcohol (e.g., n-octanol, etc.), and a liquid mainly composed of one or more mixtures selected from the group consisting of fluorinated polysaccharides. The aliphatic hydrocarbon is preferably an aliphatic hydrocarbon having 7 or more and 30 or less carbon atoms. By having 7 or more and 30 or less carbon atoms, the kinematic viscosity of the hydrophobic liquid is suitable for flowing in the microchannel. Examples of the aliphatic hydrocarbon include mineral oil; oils derived from animals and plants such as squalane oil and olive oil; paraffinic hydrocarbons having 10 or more and 20 or less carbon atoms such as decane and hexadecane; olefinic hydrocarbons having 10 or more and 20 or less carbon atoms, and the like.

[0038] In this technology, from the perspective of good immiscibility with the hydrophilic liquid, the hydrophobic liquid is preferably a fluorinated oil. Examples of fluorinated oils include perfluorocarbons (PFCs), perfluoropolyethers (PFPEs), hydrofluoroethers (HFEs), and the like. Examples of perfluorocarbons include Fluorinert (registered trademark) FC40, Fluorinert FC-770 (manufactured by 3M), and the like. An example of a perfluoropolyether is Krytox (manufactured by DuPont). Examples of hydrofluoroethers include HFE7500 (manufactured by 3M), and the like.

[0039] The density of the hydrophobic liquid at 25°C can be, for example, 0.5 g / cm 3 to 5 g / cm 3 , preferably 0.6 g / cm 3 to 4 g / cm 3 , more preferably 0.7 g / cm 3 to 3 g / cm 3 It can be. The kinematic viscosity of the hydrophobic liquid at 25°C can be, for example, 0.3 cSt to 5 cSt, preferably 0.4 cSt to 4 cSt, more preferably 0.5 cSt to 3 cSt. When the hydrophobic liquid has the above-described physical properties, an emulsion is likely to be generated in the fractionation channel. For example, if the density or kinematic viscosity is too high, there is a possibility that the flow in the connection channel will not be smooth.

[0040] In this embodiment, one or both of the first liquid and the second liquid may contain a surfactant. For example, one or both of the hydrophobic liquid and the hydrophilic liquid may contain a surfactant. The surfactant facilitates the formation of an emulsion and also helps to maintain the emulsion stably. Examples of surfactants include nonionic surfactants and fluorinated surfactants. Examples of nonionic surfactants include Span 80 and Abil EM, but are not limited to these in this embodiment. The type of surfactant may be appropriately selected depending on the purpose. Examples of fluorinated surfactants include perfluoropolyether-based surfactants and pseudosurfactants. An example of the former is Krytox (manufactured by DuPont), and an example of the latter is perfluorooctanol.

[0041] The surfactant may be present, for example, in the hydrophobic liquid at a concentration equal to or greater than the critical micelle concentration of the surfactant. The critical micelle concentration is, for example, 1 μM to 1000 μM, preferably 10 μM to 100 mM. The interfacial tension of the surfactant is, for example, 40 mN / m or less, preferably 20 mN / m or less.

[0042] In other embodiments of this technology, the first liquid may be a hydrophobic liquid and the second liquid may be a hydrophilic liquid. In this embodiment, an emulsion can be formed in the preparative channel in which a hydrophilic liquid is used as the dispersion medium and a hydrophobic liquid is used as the dispersed phase. The hydrophobic liquid and the hydrophilic liquid are the same as those described above, so their explanation is omitted here. Furthermore, this embodiment may be applied, for example, when preparating only the target particles from an emulsion in which the dispersion medium and dispersed phase are a hydrophobic liquid and a hydrophilic liquid, respectively, and the emulsion contains particles. Moreover, as an assay system that can be used in this embodiment, not only a system in which particles emit fluorescence but also a system in which the emulsion emits fluorescence can be used.

[0043] (2) An example of the first embodiment

[0044] The particle separation method according to this embodiment is performed, for example, using a particle separation mechanism. Hereinafter, an example of the particle separation method according to this embodiment will be described with reference to Figure 1, which shows the flow channel structure of a particle separation microchip 150, an example of the configuration of the particle separation mechanism used in the particle separation method according to this embodiment, and Figure 2, which shows an example of the flow of the particle separation method according to the first embodiment.

[0045] As shown in Figure 1, the particle sorting microchip 150 includes a main channel 155 through which particles flow and a sorting channel 159 through which particles to be sorted are sorted. The particle sorting microchip 150 is also provided with a sorting unit 157.

[0046] An enlarged view of the particle separation section 157 is shown in Figure 3. As shown in Figure 3A, the particle separation section 157 includes a connecting channel 170 that connects the main channel 155 and the particle separation channel 159. A liquid supply channel 161 capable of supplying liquid to the connecting channel 170 is connected to the connecting channel 170. From the above, the particle separation microchip 150 has at least a channel structure including the main channel 155, the particle separation channel 159, the connecting channel 170, and the liquid supply channel 161.

[0047] Furthermore, as shown in Figure 1, the particle sorting microchip 150, in addition to the particle sorting microchip 150, constitutes part of the particle sorting device 100, which also includes the irradiation unit 101, the detection unit 102, and the information processing unit 103.

[0048] As shown in Figure 2, the particle separation method according to this embodiment includes at least the following steps in the particle separation microchip 150: a flow step S101 in which a liquid containing first particles and second particles is flowed through a main channel 155; a first detection step S102 in which a plurality of particles flowing through the main channel 155 are detected; an identification step S103 in which the detected plurality of particles are identified as the first particles and the second particles; a calculation step S104 in which the time interval between the first particles and the second particles flowing through the main channel 155 is calculated; a determination step S105 in which the first particles and the second particles are separated according to the time interval; and a separation step S106 in which, if it is determined that separation is to be performed, the first particles and the second particles are separated into an emulsion. Each step will be described in detail below.

[0049] (2-1) Flow process S101

[0050] In the flow passage step S101, a liquid containing the first and second particles is passed through the main flow passage 155. The liquid flows through the main flow passage 155 from the confluence section 162 towards the separation section 157. The liquid may be a laminar flow formed from a sample liquid containing the first and second particles and a sheath liquid, and in particular, a laminar flow in which the sample liquid is surrounded by the sheath liquid. The flow passage structure for forming the laminar flow will be described below.

[0051] The particle sorting microchip 150 is provided with a sample liquid inlet 151 and a sheath liquid inlet 153. A sample liquid containing first and second particles and a sheath liquid without particles are introduced from these inlets into the sample liquid channel 152 and the sheath liquid channel 154, respectively.

[0052] The particle sorting microchip 150 has a flow channel structure in which the sample channel 152 through which the sample liquid flows and the sheath liquid channel 154 through which the sheath liquid flows merge at a confluence 162 to form a main channel 155. The sample liquid and the sheath liquid merge at the confluence 162 to form a laminar flow in which, for example, the sample liquid is surrounded by the sheath liquid. Preferably, the first particles and the second particles are arranged in a substantially straight line in the laminar flow. Thus, in this embodiment, the flow channel structure forms a laminar flow containing the first particles and the second particles flowing in a substantially straight line.

[0053] The laminar flow flows through the main channel 155 toward the sorting section 157. Preferably, the first and second particles flow in a line within the main channel 155. This makes it easier to distinguish between the light generated by the irradiation of one particle and the light generated by the irradiation of other particles during the light irradiation in the detection section 102, which will be described below.

[0054] (2-2) First detection step S102

[0055] In the first detection step S102, multiple particles flowing through the main channel 155 are detected. This detection is based on light generated by the irradiation of particles flowing through the main channel 155 (particularly the detection region 156) by the irradiation unit 101. An example of the first detection step S102 will be described below.

[0056] In the first detection step S102, the irradiation unit 101 irradiates particles flowing through the main channel 155 (particularly the detection region 156) in the particle sorting microchip 150 with light (e.g., excitation light), and the detection unit 102 detects the light generated by the irradiation.

[0057] The irradiation unit 101 irradiates particles flowing in a channel within the particle sorting microchip 150 with light (e.g., excitation light). The irradiation unit 101 includes a light source unit that emits light and a light guide optical system that guides the light to the irradiation point. The light source unit includes one or more light sources. Examples of light sources include laser light sources and LEDs. The wavelength of the light emitted from each light source may be ultraviolet light, visible light, or infrared light. The light guide optical system includes optical components such as a beam splitter group, a mirror group, or an optical fiber. The light guide optical system may also include a lens group for focusing the light, for example, an objective lens. There may be one or more irradiation points where the sample S and the light intersect. The irradiation unit 101 may also be configured to focus light irradiated from one or more different light sources to a single irradiation point.

[0058] The detection by the detection unit 102 will be explained in "(2-3) Identification process S103".

[0059] (2-3) Identification step S103

[0060] In the identification step S103, it is determined whether the detected plurality of particles are the first particle and the second particle. This identification is performed based on the optical characteristics obtained in the first detection step S102. An example of the identification step S103 is described below.

[0061] Depending on the characteristics of the light detected by the detection unit 102, the information processing unit 103 identifies whether the detected particle is the target first particle or the target second particle. For example, the information processing unit 103 may perform identification based on scattered light signals, identification based on fluorescence signals, or identification based on images (e.g., dark-field images and / or bright-field images).

[0062] <Identification based on fluorescence signals and / or scattered light signals>

[0063] In this embodiment, the detection unit 102 detects fluorescence and / or scattered light generated from the particles by light irradiation by the irradiation unit 101. The detection unit 102 may include a focusing lens and a photodetector for focusing the fluorescence and / or scattered light generated from the particles. As the photodetector, PMT, photodiode, CCD, CMOS, etc. may be used, but this embodiment is not limited to these. In addition to the focusing lens and photodetector, the detection unit 102 may include other optical elements as needed. The detection unit 102 may further include, for example, a spectroscopic unit. Examples of optical components constituting the spectroscopic unit include a grating, prism, optical filter, etc. The spectroscopic unit can, for example, separate and detect light of a wavelength to be detected from light of other wavelengths. The detection unit 102 may convert the detected light into an analog electrical signal by photoelectric conversion. The detection unit 102 may further convert the analog electrical signal into a digital electrical signal by AD conversion.

[0064] The information processing unit 103 can process the waveform of the digital electrical signal obtained by the detection unit 102 to generate information (data) regarding the characteristics of the light used for identification. As information regarding the characteristics of the light, the information processing unit 103 may obtain one, two, or three of the following from the waveform of the digital electrical signal: the width of the waveform, the height of the waveform, and the area of ​​the waveform. The information regarding the characteristics of the light may also include, for example, the time when the light was detected. The processing by the information processing unit 103 can be performed in an embodiment in which fluorescence and / or scattered light is detected.

[0065] The information processing unit 103 identifies whether a particle is the target first particle or the target second particle based on the light generated by irradiating the particle with light flowing through the channel. Specifically, in an embodiment in which fluorescence and / or scattered light are detected, the waveform of the digital electrical signal obtained by the detection unit 102 is processed by the information processing unit 103, and based on the information regarding the characteristics of the light generated by this processing, it identifies whether the particle is the target first particle or the target second particle. For example, in the determination based on scattered light, the external shape and / or internal structure characteristics of the particle may be identified, and the particle may be identified based on these characteristics. Furthermore, for example, by pre-treating the particle, such as a cell, it is also possible to identify whether the particle is the target particle based on characteristics similar to those used in flow cytometry. In addition, for example, by labeling the particle, such as a cell, with an antibody or dye (particularly a fluorescent dye), it is also possible to identify whether the particle is the target particle based on the characteristics of the surface antigen of the particle.

[0066] <Identification based on bright-field images>

[0067] In this embodiment, the detection unit 102 may acquire a bright-field image generated by light irradiation by the irradiation unit 101. In this embodiment, the irradiation unit 101 may include, for example, a halogen lamp, and the detection unit 102 may include a CCD or CMOS. For example, light is irradiated onto particles by a halogen lamp, and a bright-field image of the irradiated particles may be acquired by the CCD or CMOS.

[0068] The information processing unit 103 identifies whether a particle is the target first particle or the target second particle based on the acquired bright-field image. Specifically, for example, whether a particle is the target particle can be identified based on one or more combinations of the particle's morphology, size, and color.

[0069] <Identification based on dark-field imaging>

[0070] In this embodiment, the detection unit 102 may acquire a dark-field image generated by light irradiation by the irradiation unit 101. In this embodiment, the irradiation unit 101 may include, for example, a laser light source, and the detection unit 102 may include a CCD or CMOS. For example, light is irradiated onto particles by a laser, and a dark-field image (e.g., a fluorescence image) of the irradiated particles may be acquired by the CCD or CMOS.

[0071] The information processing unit 103 determines, based on the acquired dark-field image, whether the particle is the target first particle or the target second particle. For example, whether the particle is the target particle can be identified based on one or more combinations of the particle's morphology, size, and color.

[0072] <Other>

[0073] In any of the above-mentioned "identification based on fluorescence signals and / or scattered light signals," "identification based on bright-field images," and "identification based on dark-field images," the detection unit 102 may be, for example, an image sensor in which a substrate incorporating a CMOS sensor and a substrate incorporating a DSP (Digital Signal Processor) are stacked. By operating the DSP of the image sensor as a machine learning unit, the image sensor can be operated as a so-called AI sensor. The detection unit 102 including the image sensor can identify whether or not a particle is a target particle, for example, based on a learning model. Furthermore, the learning model may be updated in real time while the particle sorting method according to this embodiment is being performed. For example, the DSP can perform machine learning processing during the reset of the pixel array in the CMOS sensor, during the exposure of the pixel array, or during the reading of pixel signals from each unit pixel of the pixel array. An example of an image sensor operating as an AI sensor is the imaging device described in International Publication No. 2018 / 051809. When using an AI sensor as an image sensor, the raw data acquired from the image array is directly used for learning, resulting in faster recognition processing.

[0074] The determination may be made, for example, by determining whether the information regarding the characteristics of the light meets a predetermined criterion. The criterion may be one that indicates that the fine particles are to be recovered. The criterion may be set appropriately by a person skilled in the art and may be a criterion relating to the characteristics of light, such as a criterion used in the art of flow cytometry.

[0075] (2-4) Calculation step S104

[0076] In calculation step S104, the time interval between the first particle and the second particle flowing through the main channel 155 is calculated. This calculation is performed based on the detection time of light detected by the detection unit 102 upon light irradiation of the first and second particles. An example of calculation step S104 is described below.

[0077] Based on the light (e.g., fluorescence and / or scattered light) generated by irradiating a particle at one location within the detection region 156, it is possible to identify whether the particle is the target first particle or the second particle, as described in the identification step S103. Furthermore, based on the detection time of the light generated by the irradiation at that location (event detection time), the time interval between the arrival of the first particle and the second particle can be calculated from the difference between the detection time of the light for the first particle and the detection time of the light for the second particle.

[0078] The information processing unit 103 may control light irradiation by the irradiation unit 101 and / or light detection by the detection unit 102. The information processing unit 103 may also control the drive of a pump for supplying fluid into the particle separation microchip 150. The information processing unit 103 may consist of, for example, a hard disk, CPU, and memory containing a program and OS for causing the particle separation device 100 to execute the particle separation method according to this embodiment. For example, the functions of the information processing unit 103 can be realized in a general-purpose computer. The program may be recorded on a recording medium such as a microSD memory card, SD memory card, or flash memory. A drive in the particle separation device 100 may read the program recorded on the recording medium, and the information processing unit 103 may cause the particle separation device 100 to execute the particle separation method according to this embodiment in accordance with the read program.

[0079] (2-5) Judgment step S105

[0080] In the determination step S105, it is determined whether or not to separate the first particle and the second particle according to the time interval calculated in the calculation step S104. Hereinafter, the determination of whether or not to separate the first particle and the second particle will also be called the "acquisition determination." The first particle and the second particle will also be collectively called the "pair particle." An example of the determination step S105 will be explained below.

[0081] In the determination step S105, in order to emulsify the first and second particles with high efficiency and purity, the acquisition determination process shown in Figure 9 is performed, and then the separation operation is controlled. The acquisition determination process consists of pair determination S1051 and proximity determination S1052, and according to the acquisition determination result, separation control such as outputting the separation timing is performed (S1053).

[0082] Figure 10 shows an overview of the pair determination process and the calculation of the sorting timing Ts. In Figure 10, T(n) is the time it takes for the nth particle to reach the sorting unit 157, calculated based on the event detection time. In the determination step S105, it is further determined whether the first particle and the second particle are continuous. If the first particle and the second particle are continuous in the main flow path 155 and the time interval calculated in the calculation step S104 is less than or equal to a predetermined threshold, it is determined that the first particle and the second particle should be sorted.

[0083] Specifically, the conditions for acquiring the first particle are: the first and second particles are consecutive but not in any particular order, and the time interval ΔTpair between the first and second particles is less than or equal to the sorting margin time. These are referred to as the pair determination process. Furthermore, the sorting timing Ts for the first particle is set to a timing midway between the arrival time of the first particle and the arrival time of the adjacent second particle, so that the sorting timing Ts is (T(n) + T(n+1)) / 2.

[0084] In the following description, the separation timing Ts of the first particle is controlled based on the time interval ΔTpair. However, this embodiment is not limited to this, and the separation timing of at least one of the first particle and the second particle may be controlled based on the time interval ΔTpair.

[0085] In the determination step S105, the sorting timing Ts is changed according to the order in which the first and second particles flow through the main channel 155. Specifically, in the case of a pair of particles in which the second particle precedes the first particle, the sorting timing Ts of the first particle is corrected by -1 / 2ΔTpair relative to the conventional sorting timing (time to reach the sorting unit 157) T(n+1). Also, in the case of a pair of particles in which the first particle precedes the first particle, the sorting timing Ts of the first particle is corrected by +1 / 2ΔTpair relative to the conventional sorting timing (time to reach the sorting unit 157) T(n).

[0086] The effects of the pair determination process will be explained with reference to Figure 11. In the conventional technology, sorting control based on the arrival time of the target particles to be sorted to the sorting unit allowed for successful co-encapsulation of particles between A1 and B, or between B and C1. On the other hand, in this embodiment, based on the arrival times of consecutive first and second particles, the sorting timing Ts of the first particle is corrected to the (+) side when the first particle is consecutive to the second particle, and to the (-) side when the second particle is consecutive to the first particle. This makes it possible to co-encapsulate paired particles that are separated by up to the particle spacing between A1 and C1. This indicates that it is possible to co-encapsulate paired particles separated by twice the particle spacing compared to the conventional technology.

[0087] In the determination step S105, after the pair determination process, the proximity determination process shown in Figure 9 is performed. By determining acquisition based on the spacing between particles before and after the paired particles and the types of particles, it is prevented that particles other than the target paired particles are encapsulated together. Here, the determination conditions when generating an emulsion containing one first particle and one or more second particles (single particle vs. multiple particles; single:multi) are shown in Figure 12. In these determination conditions, it is determined whether or not to perform the separation based on the types of preceding and succeeding particles, and the proximity situation between the preceding and succeeding particles and the first and second particles, for the first and second particles that are flowing continuously. As also shown in the recovery rate characteristics in Figure 12, the time during which the preceding particle is incorporated into the paired particle is ΔT2, and the time during which the succeeding particle is incorporated into the paired particle is ΔT1.

[0088] Specifically, the determination is made based on the sorting timing Ts of the first particle forming the pair, and the type and time interval of the preceding particle that precedes the pair particle (n, n+1). That is, if particle (n-1) is the second particle, acquisition is determined regardless of the time interval. On the other hand, if particle (n-1) is anything other than the second particle, acquisition is not determined if the time interval Ts - T(n-1) is less than or equal to ΔT2, and acquisition is determined only if it is greater than ΔT2.

[0089] Next, the sorting timing Ts of the first particle forming the pair is determined based on the type and time interval of the subsequent particle that follows the pair particle (n, n+1). That is, if particle (n+2) is the second particle, acquisition is determined regardless of the time interval. On the other hand, if particle (n+2) is anything other than the second particle, acquisition is not determined if the time interval T(n+2)-Ts is less than or equal to ΔT1, and acquisition is determined only if it is greater than ΔT1.

[0090] By performing this acquisition determination process, separation is only performed when the number of first and second particles to be co-encapsulated is (first particle:second particle) = (single particle vs. multiple particles; single:multi), as shown in Figure 13. This makes it possible to generate emulsions containing high-purity paired particles.

[0091] In this specification, the mode in which separation is performed in the case described above (first particle:second particle) = (single particle vs. multiple particles; single:multi) is referred to as the "single particle vs. multiple particle mode".

[0092] Furthermore, as a modified example, Figure 14 shows the determination conditions for generating an emulsion containing only one first particle and one second particle (single particle to single particle; single:single). In these determination conditions, the decision of whether or not to perform the separation is made based on the proximity of the preceding and succeeding particles that precede the continuously flowing first and second particles to the first and second particles.

[0093] Specifically, in the single-particle versus multi-particle mode described above, the determination conditions were changed depending on the type of preceding particle and succeeding particle that precedes the paired particle. However, in the current determination conditions, if the (n-1) particle has a time interval Ts - T(n-1) of ΔT2 or less, it is not acquired, and it is only acquired if it is greater than ΔT2. Similarly, if the (n+2) particle has a time interval T(n+2) - Ts of ΔT1 or less, it is not acquired, and it is only acquired if it is greater than ΔT1.

[0094] By performing this acquisition determination process, it is possible to generate emulsions where the number of first and second particles co-encapsulated is (first particle:second particle) = (single particle vs. single particle; single:single), as shown in Figure 15.

[0095] In this specification, the mode in which separation is performed when (first particle:second particle) = (single particle vs. single particle; single:single) is referred to as the "single particle vs. single particle mode". In the determination step S105, the single particle vs. multiple particle mode and the single particle vs. single particle mode can be appropriately selected depending on the purpose.

[0096] (2-6) Preparation step S106

[0097] In the separation step S106, if it is determined in the determination step S105 that separation is necessary, the first and second particles are separated into the emulsion. In the separation step S106, the first and second particles are separated into the second liquid, which is immiscible with the first liquid in the separation channel 159, while still contained in the liquid that contained them (first liquid). This allows for the formation of an emulsion in the separation channel 159 with the second liquid as the dispersion medium and the first liquid as the dispersed phase, and each emulsion contains the first and second particles co-encapsulated. An example of the separation step S106 will be described below.

[0098] The sorting process S106 is performed in the sorting section 157 of the particle sorting microchip 150. In the sorting section 157, the laminar flow that has flowed through the main channel 155 splits and flows into two waste channels 158. The sorting section 157 shown in Figure 1 has two waste channels 158, but in this embodiment the number of branch channels is not limited to two. The sorting section 157 may be provided with one or more (for example, two, three, or four) branch channels. The branch channels may be configured to branch in a Y-shape on a single plane, as shown in Figure 1, or they may be configured to branch in three dimensions.

[0099] In this embodiment, in the sorting section 157, a flow is formed from the main flow path 155 through the connecting flow path 170 to the sorting flow path 159 only when the first and second particles to be sorted are flowing in, and both particles to be sorted are collected into the sorting flow path 159. As shown in Figure 3A, the main flow path 155 and the sorting flow path 159 are connected via the connecting flow path 170, which is coaxial with the main flow path 155. As shown in Figure 3B, both particles to be sorted flow through the connecting flow path 170 to the sorting flow path 159. Particles that are not to be sorted flow to the waste flow path 158, as shown in Figure 3C.

[0100] Enlarged views of the vicinity of the connecting channel 170 are shown in Figures 4A and 4B. Figure 4A is a schematic perspective view of the vicinity of the connecting channel 170. Figure 4B is a schematic cross-sectional view in a plane passing through the center line of the liquid supply channel 161 and the center line of the connecting channel 170. The connecting channel 170 includes a channel 170a on the detection region 156 side (hereinafter also referred to as the "upstream connecting channel 170a"), a channel 170b on the dispensing channel 159 side (hereinafter also referred to as the "downstream connecting channel 170b"), and a connection portion 170c between the connecting channel 170 and the liquid supply channel 161. The liquid supply channel 161 is provided so as to be substantially perpendicular to the axis of the channel of the connecting channel 170. In Figures 4A and 4B, two liquid supply channels 161 are provided so as to face each other at approximately the center position of the connecting channel 170, but only one liquid supply channel 161 may be provided.

[0101] The shape and dimensions of the cross-section of the upstream connecting channel 170a may be the same as those of the downstream connecting channel 170b. For example, as shown in Figures 4A and 4B, both the cross-section of the upstream connecting channel 120a and the cross-section of the downstream connecting channel 120b may be approximately circular with the same dimensions. Alternatively, both of these cross-sections may be rectangular (e.g., square, rectangle, etc.) with the same dimensions.

[0102] From the two liquid supply channels 161, the second liquid is supplied to the connecting channel 170 as shown by the arrows in B of Figure 4. This second liquid flows from the connection part 170c to both the upstream connecting channel 170a and the downstream connecting channel 170b.

[0103] If the preparative step S106 is not performed, the second liquid flows as follows: The second liquid that flows into the upstream connecting channel 170a exits the connection surface between the connecting channel 170 and the main channel 155, and then splits and flows into the two waste channels 158. By having the second liquid exit from this connection surface, it is possible to prevent the first liquid and particles that do not need to be preparated into the preparative channel 159 from entering the preparative channel 159 through the connecting channel 170. The second liquid that flows into the downstream connecting channel 170b flows into the preparative channel 159. As a result, the preparative channel 159 is filled with the second liquid, and the second liquid becomes a dispersion medium for emulsion formation.

[0104] Even when the preparative step S106 is performed, the second liquid can be supplied from the two liquid supply channels 161 to the connecting channel 170. However, pressure fluctuations within the preparative channel 159, particularly by generating negative pressure within the preparative channel 159, create a flow from the main channel 155 through the connecting channel 170 to the preparative channel 159. That is, a flow is created from the main channel 155, passing through the upstream connecting channel 170a, the connection section 170c, and the downstream connecting channel 170b in that order to the preparative channel 159. As a result, the first and second particles, which are the particles to be preparated, are prepared into the second liquid within the preparative channel 159 while encased in the first liquid. By performing the preparative step S106, an emulsion can be generated within the preparative channel 159 or in a container connected to the end of the preparative channel 1621, for example, via a channel.

[0105] The cross-sectional shape and / or dimensions of the upstream connecting channel 120a may differ from those of the downstream connecting channel 120b. An example of these two channels having different dimensions is shown in Figures 5A and 5B. As shown in Figures 5A and 5B, the connecting channel 180 includes a channel 180a on the detection area 156 side (hereinafter also referred to as the "upstream connecting channel 180a"), a channel 180b on the dispensing channel 159 side (hereinafter also referred to as the "downstream connecting channel 180b"), and a connection portion 180c between the connecting channel 180 and the liquid supply channel 161. Both the cross-sections of the upstream connecting channel 180a and the downstream connecting channel 180b have a substantially circular shape, but the diameter of the latter cross-section is larger than the diameter of the former cross-section. By making the diameter of the latter cross-section larger than that of the former, it is possible to more effectively prevent the particles already separated into the separation channel 159 from being released into the main channel 155 through the connecting channel 180 immediately after the particle separation operation by negative pressure described above, compared to the case where the diameters of both are the same. For example, if both the cross-section of the upstream connecting channel 180a and the cross-section of the downstream connecting channel 180b are rectangular, by making the area of ​​the latter cross-section larger than the area of ​​the former cross-section, it is possible to more effectively prevent the particles already separated from being released into the main channel 155 through the connecting channel 180, as described above.

[0106] In the separation process S106, the particles to be separated are collected into the separation channel 159 through the connecting channel 170 due to pressure fluctuations within the separation channel 159 (particularly the pressure chamber provided in the separation channel 159; this is a region whose volume fluctuates due to an actuator described later, and the cross-sectional area of ​​the surface perpendicular to the direction of liquid flow is larger than that of other parts of the separation channel 159). This separation may be performed, for example, by generating negative pressure within the separation channel 159 as described above. This negative pressure is generated, for example, by driving an actuator attached to the outside of the particle separation microchip 150 to cause volume fluctuations in the pressure chamber. In particular, the actuator is driven to increase the volume of the pressure chamber, thereby lowering the pressure inside the pressure chamber. This pressure reduction can create a flow of particles to be separated into the separation channel 159. When the particles to be separated are drawn into the separation channel 159, the sample liquid constituting the laminar flow, or the sample liquid and sheath liquid constituting the laminar flow, may also flow into the separation channel 159. In this way, the particles to be separated are separated in the separation unit 157 and separated into the separation channel 159.

[0107] The particles to be separated are collected in the second liquid, which is immiscible with the first liquid, within the separation channel 159, while still encased in the first liquid. As a result, as described above, an emulsion is formed within the separation channel 159, with the second liquid as the dispersion medium and the first liquid as the dispersed phase.

[0108] To prevent particles that are not the target of separation from entering the separation channel 159 through the connecting channel 170, the connecting channel 170 is equipped with a liquid supply channel 161. A second liquid, which is immiscible with the liquid (sample liquid and sheath liquid) flowing through the main channel 155, is introduced into the connecting channel 170 from the liquid supply channel 161. A portion of the second liquid introduced into the connecting channel 170 forms a flow from the connecting channel 170 toward the main channel 155, thereby preventing particles other than the target of separation from entering the separation channel 159. The second liquid formed by the flow from the connecting channel 170 toward the main channel 155 flows through the waste channel 158, without flowing through the main channel 155, just like the first liquid, due to the flow of the first liquid flowing through the main channel 155 toward the waste channel 158. The remainder of the second liquid introduced into the connecting channel 170 flows into the separation channel 159. As a result, the separation channel 159 can be filled with the second liquid.

[0109] The preparative channel 159 may be filled with a second liquid that is immiscible with the first liquid. In order to fill the preparative channel 159 with the second liquid, the second liquid may be supplied from the liquid supply channel 161 to the connecting channel 170. Upon this supply, the second liquid flows from the connecting channel 170 to the preparative channel 159, thereby filling the preparative channel 159 with the second liquid.

[0110] The laminar flow that flows into the waste channel 158 can be discharged to the outside of the particle sorting microchip 150 at the end of the waste channel 160. Furthermore, the particles to be sorted, which are sorted into the sorting channel 159, can be discharged to the outside of the particle sorting microchip 150 at the end of the sorting channel 1621.

[0111] A container may be connected to the end of the dispersion channel 1621, for example, via a channel such as a tube. An emulsion in which the first liquid containing the particles to be dispersed is used as the dispersion phase and the second liquid as the dispersion medium is dispersed into the container. Thus, in this embodiment, the particle dispersion device 100 may be equipped with a channel for dispersing the emulsion containing the particles to be dispersed into a container. Furthermore, by closing the end of the dispersion channel 1621 and performing the dispersion operation, multiple emulsions can be held in the dispersion channel 159. After the completion of the dispersion operation, the assay can be continued in the dispersion channel 159.

[0112] As described above, in this embodiment, the main flow path 155 may branch into the connecting flow path 170 and the at least one waste flow path 158. The at least one waste flow path 158 is a flow path through which particles other than the particles to be separated flow.

[0113] Furthermore, as shown in Figure 1, in the particle sorting microchip 150, the main channel 155, the connecting channel 170, and the sorting channel 159 may be arranged in a straight line. When these three channels are arranged in a straight line (particularly coaxially), the sorting process S106 can be performed more efficiently compared to, for example, when the connecting channel 170 and the sorting channel 159 are arranged at an angle to the main channel 155. For example, the amount of suction required to guide the particles to be sorted to the connecting channel 170 can be reduced. Also, as shown in Figure 1, in the particle sorting microchip 150, the particles flow in a roughly straight line through the main channel 155 toward the connecting channel 170. Therefore, the amount of suction in the sorting process S106 can also be reduced.

[0114] Furthermore, in the particle separation method according to this embodiment, the liquid supply channel 161 supplies liquid (particularly the second liquid) to the connecting channel 170. This creates a flow within the connecting channel 170 that flows from the connection point between the liquid supply channel 161 and the connecting channel 170 toward the main channel 155, preventing the liquid flowing through the main channel 155 from entering the connecting channel 170, and also preventing particles other than the particles to be separated from flowing through the connecting channel 170 toward the separation channel 159. When performing the separation step S106, as described above, for example, the negative pressure generated in the separation channel 159 causes the first liquid containing the particles to be separated to pass through the connecting channel 170 and be separated into the second liquid in the separation channel 159. As a result, an emulsion containing the particles to be separated is generated in the second liquid.

[0115] Furthermore, in this embodiment, if a particle is determined to be a particle to be separated in the determination step S105, for example, by driving the actuator at an appropriate timing (for example, when it reaches the separation unit 157), the hydrophilic solution containing the particle to be separated is separated into the separation channel 159 and an emulsion is generated. In the determination step S105, for example, by using the peak signal and area signal to determine whether or not a particle is a particle to be separated, it is also possible to determine whether it is a single particle (singlet), a doublet of two particles combined, or a triplet of three particles combined. Therefore, it is possible to avoid generating an emulsion that contains a combination of two or more particles in one emulsion, and to generate an emulsion containing the first particle and the second particle with high probability and high efficiency. In other words, it is also possible to determine the characteristics of two or more particles that are close enough to be simultaneously drawn into the connecting channel 170 in a single separation operation. For example, two or more particles having the desired characteristics can be confined within a single emulsion.

[0116] In this embodiment, the actuator is positioned on the surface of the particle sorting microchip 150 so as to be in contact with the sorting channel 159 (particularly the portion where the pressure chamber is located). The actuator may be a piezoelectric element, such as a piezo element. The intake of particles to be sorted into the sorting channel 159 is performed by the actuator generating negative pressure in the pressure chamber within the sorting channel 159, thereby attracting the particles to be sorted into the sorting channel 159. To generate this negative pressure, for example, a voltage that causes piezoelectric contraction is applied to the piezo element. The application of this voltage deforms the pressure chamber in a direction that increases its volume, thereby generating negative pressure.

[0117] Figure 6 shows an example of a basic drive waveform applied to separate particles. Figure 6 is a graph of the drive voltage applied to the actuator over time. As shown in Figure 6, the drive waveform has a falling waveform section Wf and a rising waveform section Wr. There may also be a holding time Wm between the falling waveform section Wf and the rising waveform section Wr during which the voltage is kept constant. The falling waveform section Wf corresponds to the suction operation and is applied to deform the pressure chamber (in particular, to increase the volume inside the pressure chamber) and generate negative pressure. The rising waveform section Wr, which corresponds to the discharge operation, is applied to return the deformed pressure chamber to its original state. In this case, when the rising waveform section Wr is applied, positive pressure is generated because it is accompanied by a decrease in the volume of the pressure chamber.

[0118] Figure 7 is a schematic diagram showing the suction and discharge operations. As shown in Figure 7A, the application of the falling edge Wf of the drive waveform generates a negative pressure NP in the separation channel 159, thereby collecting the particles into the separation channel 159 (particularly the pressure chamber). Subsequently, as shown in Figure 7B, the application of the rising edge Wr of the drive waveform generates a positive pressure PP in the separation channel 159, thereby applying a force to the particles to move in the direction of the arrow in Figure 7B. However, if the particles have flowed downstream to a certain extent from the particle intake port 171, they will not return to the main channel 155 through the connecting channel 170 again. This basic drive waveform is called a pulse drive waveform, and with a pulse drive waveform, the suction and discharge operations are performed continuously.

[0119] In the separation step S106, the separation is performed by a pressure change in a pressure chamber communicating with the flow path (particularly the main flow path 155) due to the application of a pulse voltage to an actuator, and it is preferable that the value of the pulse voltage or the drive waveform is determined based on information about the first particle and the second particle. The information about the first particle and the second particle can be, for example, the size of the particles, the time interval, etc.

[0120] Specifically, in a series of sorting operations of a pulse-driven waveform, a holding time Wm is provided between the suction operation that contracts the piezoelectric element and the discharge operation that expands the piezoelectric element and restores pressure, as described above, during which the displacement (voltage) of the piezoelectric element is kept constant. Based on information regarding the first and second particles, this holding time Wm is set according to the time corresponding to the sorting margin time (= the time required to maintain the pressure change necessary for sorting the first and second particles).

[0121] In other words, setting the holding time Wm to be longer than usual increases the time during which the flow in the suction direction is maintained, thereby increasing the total amount of material aspirated. Therefore, by determining the holding time Wm during the preparative operation based on information regarding the first and second particles, and then setting the holding time Wm to be longer than usual, the volume of the first liquid (dispersed phase) aspirated into the preparative channel 159 increases, making it possible to generate larger emulsions.

[0122] Figure 8 shows the relationship between the holding time Wm and the preparative time. As shown in Figure 8 II, setting the holding time of the pulsed drive waveform to be longer than in Figure 8 I increases the time during which the flow in the suction direction is maintained, and the preparative time, which is a recovery rate characteristic, increases. Therefore, by appropriately setting the holding time Wm of the pulsed drive waveform based on information about the first and second particles, it becomes possible to co-encapsulate the first and second particles with a larger time interval, and the generation efficiency of the emulsion containing the first and second particles can be improved.

[0123] Alternatively, instead of the pulse drive waveform described above, a piezo drive with a so-called "pulse drive waveform with falling edge," which has a falling edge waveform after the pulse voltage, may be used. In other words, with a pulse drive waveform with falling edge, a suction operation is performed after the suction and discharge operations. With a pulse drive waveform, the discharge operation in the preparative operation generates a flow (discharge flow) directed towards the main flow path 155 and unwanted flow vibrations, which hinders continuous high-speed preparative operation. Therefore, by using a pulse wave with falling edge, the discharge flow can be weakened and unwanted flow vibrations can be reduced. Accordingly, as shown in Figure 8 III, similar to the pulse drive waveform, by appropriately setting the holding time Wm of the pulse drive waveform with falling edge based on information about the first and second particles, it is possible to suppress the discharge flow and unwanted flow vibrations while maintaining a longer preparative margin time, enabling faster and more efficient preparative operation.

[0124] (2-7) Second detection step S107

[0125] In the second detection step S107, the first particles and the second particles separated in the separation step S106 are detected. By performing the second detection step S107, a stable emulsion can be generated even if there are variations in the recovery rate characteristics due to individual or lot-to-lot variations in the particle separation microchip 150. An example of the second detection step S107 is described below.

[0126] Figure 16 shows a part of the flow channel structure of the particle sorting microchip 150 when the second detection step S107 is performed. In the second detection step S107, light from the particles contained in the sorted emulsion and / or from the emulsion itself is detected using an optical detection system different from the detection unit 102 (i.e., a second detection unit 109).

[0127] As shown in Figure 16, the particle sorting microchip 150 has a second detection region 164 located downstream of the sorting channel 159. In this second detection region 164, a second irradiation unit 108 irradiates light onto the emulsion flowing through the sorting channel 159, and the second detection unit 109 detects the light generated by this irradiation. The second irradiation unit 108 can be configured similarly to the irradiation unit 101 described above, and the second detection unit 109 can be configured similarly to the detection unit 102 described above.

[0128] The second detection unit 109 can detect at least scattered light. The scattered light may be forward scattered light, back scattered light, or side scattered light. The second detection unit 109 may also detect at least fluorescence. The detected fluorescence may have the same or multiple wavelengths.

[0129] The second detection unit 109 may detect information regarding the presence or absence of the emulsion, the shape of the emulsion, the number of emulsion particles, etc., based on the forward scattered light, or the second detection unit 109 may detect information regarding the presence or absence of the first and second particles in the emulsion, the shape of the emulsion, the number of emulsion particles, etc., based on the fluorescence, the backscattered light, or the side scattered light.

[0130] In the second detection step S107, the conditions related to the sorting can also be adjusted based on the information obtained in the detection step. Specifically, the acquisition determination parameters used in the determination step S105 are automatically adjusted. For this automatic adjustment, the acquisition determination parameters are determined by measuring the recovery rate characteristics using representative particles (particularly the first particle or the second particle). The acquisition determination parameters to be determined are the time Td from particle detection in the detection unit 102 to the start of actuator driving, the sorting margin time, and the entanglement time (ΔT1, ΔT2).

[0131] Figure 17 shows an example of the flow up to determining the acquisition judgment parameters. First, the drive waveform is set (S201). Next, in S202, when the sorting operation is performed with the time from particle detection in the detection unit 102 to the start of actuator driving as time Td0, the number of particles that passed through the second detection area 164 downstream of the sorting channel 159 is counted. Here, the sorting operation is performed for a known number of particles (for example, 10 to 1000 particles), and the number of sorted particles is counted.

[0132] Next, in S203, the particle sorting operation is performed under the same conditions except that time Td0 is changed to a longer or shorter time Td, and the number of sorted particles is counted. As a result, as shown in Figure 18, the number of particles sorted into the sorting channel 159 at various times Td is obtained. The various times Td may be times increased or decreased by a predetermined percentage relative to time Td0 (for example, times increased or decreased by 0.01% to 5%).

[0133] Next, the sorting buffer time and sorting timing are determined (S204). In S204, the sorting buffer time is defined as the time interval Td during which the number of particles exceeds the threshold for determining the sorting buffer time. The threshold here can be determined, for example, as 80% or 90% of the number of sorting operations at each time Td. The sorting timing can be determined as the average or median value within the time Td during which the threshold for determining the sorting buffer time is exceeded.

[0134] Next, the entrapment times (ΔT1, ΔT2) are determined (S205). The entrapment times are determined by selecting the time Td and the sorting time, with the entrapment time ΔT2 for the preceding particle and the entrapment time ΔT1 for the succeeding particle being the time Td that is below and closest to the threshold for determining the entrapment time. The threshold here can be determined as, for example, 10%, 20%, etc., of the number of sorting operations at each time Td.

[0135] In steps S201 to S205, the acquisition determination parameters were determined using representative particles (particularly the first particle or the second particle). However, if the recovery rate characteristics of the first and second particles to be co-encapsulated differ, for example, due to particle size, the acquisition determination parameters may be determined using two types of representative particles (particularly the first particle and the second particle) used for the automatic adjustment described above. In this case, representative particle a and representative particle b are used, and steps S202 and S203 are performed for each particle to obtain the particle counts at various times Td for each particle. As a result, as shown in Figure 19, the parsing margin time corresponding to both particles can be determined from the particle count results for both particles based on the time Td at which both particles exceed the threshold for determining the parsing margin time. Furthermore, the entrapment times (ΔT1, ΔT2) corresponding to both particles can be determined from the particle count results for both particles by the combination of time Td that has the maximum time width among the time Td that is below the threshold for determining the entrapment times (ΔT1, ΔT2) and is closest to the threshold. In this way, by determining the acquisition judgment parameters, it is possible to generate emulsions containing high-purity first and second particles, even with two particles that have different recovery rate characteristics.

[0136] (3) Particle separation mechanism and particles

[0137] The particle separation mechanism used in the particle separation method according to this embodiment may be a structure or device having the above-described flow channel structure, for example, a chip having a microchannel, and in particular, a particle separation microchip 150.

[0138] In this specification, "micro" means that at least a portion of the channels included in the particle sorting microchip 150 has dimensions on the order of μm, and more particularly, has cross-sectional dimensions on the order of μm. That is, "microchip" means a chip that includes channels on the order of μm, and more particularly, a chip that includes channels having cross-sectional dimensions on the order of μm. For example, a chip that includes a sorting section 157 composed of channels having cross-sectional dimensions on the order of μm may be called a microchip in this embodiment. For example, the cross-section of the main channel 155 in the sorting section 157 is, for example, rectangular, and the width of the main channel 155 within the sorting section 157 is, for example, 100 μm to 500 μm, and more particularly, 100 μm to 300 μm. The width of the branch channels branching from the main channel 155 may be smaller than the width of the main channel 155. The cross-section of the connecting channel 170 is, for example, circular, and the diameter of the connecting channel 170 at the connection point between the connecting channel 170 and the main channel 155 may be, for example, 10 μm to 60 μm, and more particularly, 20 μm to 50 μm. These dimensions of the channel may be appropriately changed depending on the size of the particles, in particular the size of the particles to be separated.

[0139] The particle separation microchip 150 can be manufactured by conventionally known methods. For example, the particle separation microchip 150 can be manufactured by bonding together two or more substrates on which predetermined channels are formed. The channels may be formed on all of the two or more substrates (particularly two substrates), or on only some of the substrates (particularly one of the two substrates). In this embodiment, it is preferable that the channels are formed on only one substrate in order to facilitate adjustment of the position when bonding the substrates.

[0140] The material used to form the particle sorting microchip 150 can be any conventionally known material. Examples include polycarbonate, cycloolefin polymer, polypropylene, PDMS (polydimethylsiloxane), polymethyl methacrylate (PMMA), polyethylene, polystyrene, glass, silicon, etc., but this embodiment is not limited to these. In this embodiment, polymer materials such as polycarbonate, cycloolefin polymer, and polypropylene are preferred because they have excellent processability and allow for inexpensive manufacturing of the microchip using a molding apparatus.

[0141] The particle sorting microchip 150 is preferably transparent. For example, the particle sorting microchip 150 is transparent in at least the portion through which light (particularly laser light and scattered light) passes, and for example, the detection area 156 may be transparent. The particle sorting microchip 150 may also be transparent as a whole.

[0142] The above describes an embodiment in which the group of channels is formed on a disposable particle sorting microchip 150. However, in this embodiment, the group of channels does not have to be formed on the particle sorting microchip 150. For example, the group of channels may be formed in a substrate such as plastic or glass. Furthermore, the group of channels may have a two-dimensional or three-dimensional structure.

[0143] In this embodiment, the particles may be particles having dimensions that allow them to flow through the channels in a particle sorting mechanism (e.g., a particle sorting microchip 150), and may be appropriately selected depending on the purpose. The particles may include biological particles such as cells, cell aggregates, microorganisms, and liposomes, as well as synthetic particles such as gel particles, beads, latex particles, polymer particles, and industrial particles.

[0144] Biological particles (hereinafter also referred to as "biological particles") may include chromosomes, ribosomes, mitochondria, organelles, etc., which constitute various types of cells. Cells may include animal cells (e.g., hematopoietic cells, tissue cells, etc.) and plant cells. Hematopoietic cells may be suspension cells such as T cells and B cells. Tissue cells may be adherent culture cells, adherent cells separated from tissue, etc. Cell aggregates may include spheroids, organoids, etc. Biological factors may include growth factors, activators, stimulating factors, etc. Growth factors may include bacteria such as E. coli, viruses such as tobacco mosaic virus, and fungi such as yeast. Activators may include cytokines, chemokines, cofactors, effector molecules, transactivators, etc. Stimulating factors may include inflammation-inducing factors, immunostimulators, cytokines, antigens, signaling factors, ligands, trigger molecules, etc. Biological particles may also include biological macromolecules such as nucleic acids, proteins, and complexes thereof. These biological macromolecules may, for example, be extracted from cells, or they may be contained in blood samples or other liquid samples.

[0145] Synthetic particles may be particles made of, for example, organic polymer materials, inorganic polymer materials, metals, etc. Organic polymer materials may include polystyrene, styrene-divinylbenzene, polymethyl methacrylate, etc. Inorganic polymer materials may include glass, silica, magnetic materials, etc. Metals may include gold colloid, aluminum, etc. Figure 40 is a diagram showing an example of an embodiment of a sequenced particle. The synthetic particles may be, for example, gel particles, beads, etc., and in particular, gel particles or beads to which one or more combinations selected from the group consisting of oligonucleotides, peptides, proteins, and enzymes are bound. Furthermore, oligonucleotides, peptides, proteins, and enzymes may constitute a unique sequence for each synthetic particle in the form of a base sequence, amino acid sequence, peptide sequence, etc. In addition, porous beads may be used to increase the number of molecules on the beads. Moreover, the beads may be covered with a gel, and one or more combinations selected from the group consisting of oligonucleotides, peptides, proteins, and enzymes may be bound to the gel portion. Examples of the gel covering the beads include polymers, and specifically, for example, dendrimers.

[0146] Here, a cleavable site may be introduced between the gel particles or beads and the unique sequence so that the unique sequence can be released from the synthetic particles. Examples of cleavable sites include disulfide bonds, photocatalytically cleavable linkers, and enzymatically digestible sequences.

[0147] For cleavage of the disulfide bond, reducing agents such as Tris(2-carboxyethyl)phosphine (TCEP), Dithiothreitol (DTT), and 2-Mercaptoethanol may be used. The cleavable linker may include one or more selected from the group consisting of, for example, arylcarbonylmethyl groups, nitroaryl groups, coumarin-4-ylmethyl groups, arylmethyl groups, metal-containing groups, and other groups. The cleavable linker may be cleaved by light in the short-wavelength region, specifically light in the wavelength region of 360 nm to 410 nm, or it may be cleaved by light in the near-infrared or infrared region (for example, light in the wavelength region of 800 nm or more). Figure 40 specifically shows an example in which a disulfide bond is introduced between a dendrimer and a unique sequence, but this embodiment is not limited thereto.

[0148] In this embodiment, the first particle and / or the second particle is preferably one selected from the group consisting of cells, cell aggregates, synthetic particles, and bio-derived factors.

[0149] The particle shape may be spherical, nearly spherical, non-spherical, etc. The particle size and mass may be appropriately selected according to the size of the microchip's channel. On the other hand, the size of the microchip's channel may also be appropriately selected according to the particle size and mass. In this embodiment, chemical or biological labels, such as fluorescent dyes or fluorescent proteins, may be attached to the particles as needed. Such labels may make the detection of the particles easier. The labels to be attached may be appropriately selected according to the purpose. Molecules that react specifically with the particles (e.g., antibodies, aptamers, DNA, RNA, etc.) may be bound to the labels.

[0150] (4) Variant

[0151] Here, as an example of a modified method, we will describe the case of co-encapsulating three different types of particles. These three types of particles are the first particle (target particle), and the second particle a (partner particle a) and second particle b (partner particle b), which are the pairing targets. This modified method can also be used when co-encapsulating four or more different types of particles.

[0152] (4-1) Modification example 1

[0153] When co-encapsulating three different types of particles, the acquisition determination process is also performed in the determination step S105, but the content of the pair determination process differs. Figure 20 shows an overview of the pair determination process and the calculation of the sorting timing Ts in this modified example 1. In Figure 20, T(n) is the time it takes for the nth particle to reach the sorting unit 157, calculated based on the event detection time.

[0154] In this modified example 1, the determination step S105 further determines whether the first particle and two or more of the second particles are continuous, and determines whether to separate them based on the time interval between the earliest particle and the latest particle in the group consisting of the first particle and two or more of the second particles.

[0155] Specifically, the acquisition conditions for the first particle in this modified example 1 are: - The first particle, second particle a, and second particle b are consecutive in any order. - The maximum particle spacing among the three particles is ΔTpair, and ΔTpair is less than or equal to the preservation time. In other words, the particle spacing used for pair determination among the three particles is determined based on the time interval between the arrival times of the earliest and latest particles among the three particles, regardless of the arrival order of the first particle and the other two particles. Furthermore, the preservation timing of the first particle is also calculated based on the arrival times of the earliest and latest particles among the three particles, and the preservation timing Ts can be determined by (T(n-1) + T(n+1)) / 2. In this way, by determining the pair determination and the preservation timing Ts, an emulsion containing three different types of particles can be generated with high efficiency.

[0156] (4-2) Modification 2

[0157] This modified example 2 describes a co-encapsulation method different from that of this modified example 1. Figure 21 shows an overview of this modified example 2. In this modified example 2, when co-encapsulating three different types of particles, with respect to the first particle, the second particle a, and the second particle b, acquisition determination processing is performed based on the detection signal only for the first particle and the second particle a, and sorting is performed at sorting timing Ts.

[0158] In this modified example 2, in the determination step S105, if the first particle and two or more of the second particles are in close proximity, a determination is made as to whether or not to separate them based on the time interval between the first particle and some of the second particles among the two or more of the second particles.

[0159] Specifically, in the case described above, the second particle b is not subjected to acquisition determination processing. However, as shown in Figure 21, when the first particle and the second particle a are in close proximity, the three particles are co-encapsulated. In this case, if the second particle b has a detection signal, the threshold for determining that it is a signal is raised so that the detection signal of the second particle b is not used in the acquisition determination processing.

[0160] Furthermore, when the first particle, second particle a, and second particle b are detected based on their detection signals and sorted at sorting timing Ts, by setting the detection signal of the second particle c (partner particle c) below the threshold for determining it as a signal, it is possible to co-encapsulate the adjacent second particle c as well, thereby generating an emulsion containing four different types of particles.

[0161] 2. Second Embodiment (Particle Separation Method)

[0162] (1) Description of the second embodiment

[0163] Figure 22 shows an example of the flow of the particle separation method according to the second embodiment. The particle separation method according to this embodiment includes a flow step S101, a first detection step S102, an identification step S103, a calculation step S104, a determination step S105, a separation step S106, and a combination execution step S108. A second detection step S107, etc., may also be included as needed. Each step will be described in detail below.

[0164] (2) An example of a second embodiment

[0165] In the particle separation method according to this embodiment, steps S101 to S107 are the same as those described in "1. First Embodiment (Particle Separation Method)," and therefore their explanation is omitted here. The second embodiment may be adopted as an example of an application after co-encapsulation of the first and second particles.

[0166] (2-1) Combination execution step S108

[0167] In the bonding execution step S108, bonding occurs between the first particle and / or secretions secreted from the first particle and the second particle within the emulsion. By evaluating the degree of this bonding, the degree of affinity between them can also be evaluated. An example of the bonding execution step S108 will be explained below with specific examples.

[0168] Here, methods for obtaining a second particle (partner particle) that has a high affinity for the first particle (target particle) include, for example, phage display, single B cell screening, and single plasma cell screening.

[0169] When using phage display or single-plasma cell screening methods, identical phages (phage clones) or identical antibodies (antibody clones) can be obtained in a fraction by allowing phage proliferation or antibody production within the emulsion. When selecting high-affinity phages from a phage library, the phage library is designated as the first particle (target particle), the target molecule-immobilized particle as the second particle a (partner particle a), and the growth factor necessary for phage proliferation (e.g., E. coli) as the second particle b (partner particle b), and co-encapsulation is performed (see Figure 23). In this case, the first particle b (target particle b) may be set below a threshold so that it is not included in the detection signal. After co-encapsulation, the emulsion contains one type of phage, one or more target molecule-immobilized particles (the target molecule is the same for all particles), and one or more phage growth factors. When phages are grown within the emulsion, the grown phages bind to the target molecule-immobilized particles.

[0170] Furthermore, when selecting molecules with high affinity for phage clones from a molecular library, the target molecule-fixed particle can be designated as the first particle (target particle), and the phage clone as the second particle a (partner particle a).

[0171] On the other hand, if there is no affinity between the first particle and the second particle, no bonding will occur in the emulsion during the bonding execution step S108.

[0172] In this embodiment, in the binding execution step S108, the binding affinity between the first particle and / or the secretion secreted from the first particle and the second particle is measured. When encapsulating phages and plasma cells, the affinity of phage clones and antibody clones to the target molecule can be measured by co-encapsulating particles on which the first particle (especially an antigen, etc.) is immobilized or cells expressing the target molecule using the co-encapsulation method described above.

[0173] When measuring affinity, possible methods include measuring binding affinity, measuring dissociation affinity, or measuring both.

[0174] When measuring binding affinity, phages can be grown in an emulsion and then bound to target molecule-immobilized particles. The bound phages can then be measured using a phage-specific antibody (particularly a phage-specific fluorescent antibody) to which a fluorescent molecule is bound (see the left side of Figure 24). In this case, if the affinity is high, the phage-specific fluorescent antibody will aggregate with the phage bound to the target molecule-immobilized particles, and a high fluorescence signal will be detected. Therefore, phages with high affinity can be selected by sorting the emulsion with a high fluorescence signal as is, or by sorting the particles with a high fluorescence signal after emulsion disruption (the "emulsion disruption step" will be described later). The phage-specific fluorescent antibody should be added to the solution when co-encapsulating.

[0175] Furthermore, when measuring dissociability, phages that have grown in the emulsion are bound to target molecule-immobilized particles, and the emulsion is broken and diluted. Then, after a certain period of time, the fluorescence intensity of the phage-specific fluorescent antibody is measured. By sorting particles with a low decrease in fluorescence intensity, or particles with relatively high fluorescence intensity, phages with high affinity can be selected (see the right side of Figure 24). In this case, the phage-specific fluorescent antibody may be added at the time of co-encapsulation or reacted after emulsion breakdown. The phages that have been bound to the particles and selected can be subjected to base sequence analysis, for example, by NGS, to obtain genetic information of phages with high affinity for the target molecule.

[0176] When selecting high-affinity antibodies and high-affinity antibody-producing cells using plasma cells, which are antibody-producing cells, the plasma cells are designated as the first particle (target particle), and the target molecule-immobilized particle or target molecule-expressing cell is designated as the second particle a (partner particle a), and co-encapsulation is performed (see Figure 25).

[0177] Antibodies produced by plasma cells within the emulsion (secretions secreted from the first particle) bind to the target molecule-immobilized particle (second particle a). Therefore, by adding antibody-specific antibodies (fluorescent antibodies) to which fluorescent molecules are bound, if the antibody has high affinity, the fluorescent antibody will aggregate with the antibody bound to the target molecule-immobilized particle, and a high fluorescence signal will be detected. By sorting the emulsion with a high fluorescence signal as is, or by performing dilution or solution replacement after emulsion disruption and then sorting the particles that maintain a high fluorescence signal after a certain period of time, cells (plasma cells) that produce antibodies with high affinity can be selected.

[0178] When sorting after emulsion disruption, it is preferable to link the plasma cells, which are antibody-producing cells, with the target molecule-immobilized particles or target molecule-expressing cells using a linker before co-encapsulation or within the emulsion. Examples of linkers include bifunctional antibodies (e.g., BiTE) and oligo-conjugated antibodies (e.g., TotalSeq) hybridized with complementary oligos. This ensures that when particles maintaining a high fluorescence signal are sorted after emulsion disruption, antibody-producing cells that produce high-affinity antibodies are also sorted. By culturing the obtained high-affinity antibody-producing cells (the "culturing process" will be described later), a large number of antibody clones can be obtained, and antibody characterization can be performed. Furthermore, antibody information can be obtained by genetic analysis.

[0179] If bonding with a linker or similar device is difficult, a gelling agent can be mixed in during emulsion formation, and gelation can occur due to changes in temperature or other factors. For example, in the case of a 1.5% (w / v) agarose gel (gel point 8°C to 17°C), maintaining the temperature at 37°C allows the particles to maintain their freedom within the emulsion, enabling contact reactions between particles. Subsequently, lowering the temperature below the aforementioned gel point causes gelation, and the particles can be retained within the gel even after emulsion breakdown, allowing the gel containing a large number of particles to be sorted using a cell sorter or similar device.

[0180] Alternatively, the particles can be gelled after co-encapsulation, and then reacted within the gel. In this case, molecules and / or substances produced from the particles move within the gel and react with other particles (e.g., binding, irritation, etc.). After the reaction, the emulsion is similarly broken down, and the gel containing the target particles is sorted using a cell sorter or similar device.

[0181] Furthermore, by gelling, the emulsion can be re-emulsified by replacing the buffer after breakdown, which is suitable for long-term culture, for example. To re-emulsify the gel, this technique may be used, or the gel may be stirred with oil. Examples of gelling agents include temperature-responsive gels (e.g., agarose gel, extracellular matrix, etc.) and gels that respond to two reagents (e.g., a combination of sodium alginate and calcium carbonate, etc.).

[0182] When measuring dissociation properties, dilution or solution replacement is effective, but by flowing particles bound to phages or antibodies through a microchannel, the fluorescence intensity at point A (Ia) and the fluorescence intensity at point B downstream (Ib) can be measured, and the dissociation property can be determined from the difference (Ia - Ib). In this case, a smaller difference in fluorescence intensity between point A and point B indicates higher affinity. Examples of channel structures include straight structures, curved structures, and loop structures, but this embodiment is not limited to these.

[0183] Furthermore, when measuring dissociability, a comparative phage or antibody may be competitively reacted within the emulsion or after emulsion disruption. In this case, since higher affinity maintains binding to the target molecule-immobilized particle, the phage or antibody that shows less decrease in fluorescence intensity before and after addition of the comparative phage or antibody can be determined to have high affinity and sorted accordingly. If comparison before and after addition of the comparative phage or antibody is not possible, the phage or antibody (including antibody-producing cells) with high affinity can be sorted by sorting the particles with high fluorescence intensity based on relative evaluation after addition.

[0184] Furthermore, when measuring dissociability, in addition to disrupting the emulsion and diluting or replacing the solution, dilution or solution replacement may be performed by fusing an emulsion that does not contain phages or antibodies (antibody-producing cells). In this case, fusing may be performed using electrowetting or by controlling the speed of the capsule in a microfluidic channel.

[0185] In addition, when measuring binding or dissociation properties, non-target molecule immobilized particles may be included in the target molecule immobilized particles. In this case, target binding (specific binding) and non-target binding (non-specific binding) can be evaluated. By changing the color, particle size, etc., of the target molecule immobilized particles and non-target molecule immobilized particles, it is also possible to identify which type of particle is being measured during particle detection.

[0186] Furthermore, if it is difficult to detect a single phage as the first particle (target particle), the phage can be grown in a state where it is co-encapsulated with the particle to which it binds, thereby generating a particle to which a phage clone is bound. In this case, the phage may be nonspecifically bound to the particle. After emulsion disruption, the particle to which the phage clone is bound is recovered. Next, the signal is enhanced by binding a phage-specific fluorescent antibody to this particle. This particle can be co-encapsulated as the first particle (target particle), and the target molecule-immobilized particle as the second particle a (partner particle a). The subsequent steps are the same as the method shown in Figure 24.

[0187] (2-2) Application examples of the second embodiment

[0188] One application of co-encapsulation of multiple particles is the generation of spheroids and organoids (see Figure 26). By using stem cells, which are essential for the generation of spheroids and organoids, as the first particle (target particle), and other cells as the second particle (partner particle), the cells essential for the generation of spheroids and organoids can be reliably co-encapsulated. Since the second particle (partner particle) is co-encapsulated approximately equally in each emulsion according to its concentration, it is possible to perform spheroid standardized manufacturing.

[0189] Furthermore, it can also be used for potency testing of genetically modified cells (see Figure 27). By co-encapsulating genetically modified cells as the first particle (target particle) and cancer cells recognized by the genetically modified cells as the second particle (partner particle), killing assays can be performed within the emulsion. Additionally, by co-encapsulating genetically modified cells as the first particle (target particle) and beads that capture secretions such as cytokines secreted from the genetically modified cells as the second particle (partner particle), cytokine production capacity tests and cytokine-producing cell counts become possible. In this case, a sandwich assay is performed using a fluorescent dye-conjugated antibody that binds to cytokines on the beads that capture secretions, and the fluorescence intensity is measured to efficiently evaluate secretion production capacity and the number of secretion-producing cells (the number of capsules containing cells that produced secretions).

[0190] Furthermore, by co-encapsulating cells modified with CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) as the first particle (target particle) and various other cells as the second particle (partner particle), it becomes possible to efficiently evaluate receptors to which molecules expressed with CRISPR bind.

[0191] (2-3) Other processes

[0192] The particle separation method according to this embodiment may further include other steps. An example of such other steps will be described below with specific examples.

[0193] <Culture process>

[0194] In this embodiment, the first particles and / or the second particles may be cells or cell aggregates, in which case the first liquid may be a culture medium for the particles. In this embodiment, an emulsion can be generated in which the culture medium is the dispersed phase and the second liquid, which is immiscible with the culture medium, is the dispersion medium. This allows the first particles and / or the second particles to be cultured within each emulsion.

[0195] Furthermore, in this embodiment, the particle separation method may include a culture step in which the particles to be separated in the separation step S106 (i.e., cells or cell aggregates) are cultured in emulsion particles generated from the culture medium. The type of culture medium may be appropriately selected depending on the cells to be cultured.

[0196] <Emulsion Destruction Process>

[0197] In this embodiment, the emulsion may be destroyed after the preparative step S106 or the bonding step S108. In this embodiment, the emulsion generated by the preparative step S106 and containing the first and second particles may be destroyed. Emulsion destruction can be achieved by chemical methods such as adding a 20% (v / v) perfluorooctanol, 80% HFE-7500 solution, or by physical methods such as electrocoalesce using Zerostat 3 milty.

[0198] In another embodiment, each particle within the emulsion may be destroyed. In this case, the destruction can be carried out while maintaining the emulsion. This allows, for example, cellular components (e.g., intracellular components, cell membrane components, cell wall components, etc.) or components of the synthetic particles to be released into the emulsion, and these cellular components or components can be treated separately from the components of other particles.

[0199] <Detection Process>

[0200] In this embodiment, after the preparation step S106 or the binding execution step S108, the components of the co-encapsulated emulsion may be detected, analyzed, or reacted with other components. To perform the detection or reaction, for example, the emulsion may be merged with another emulsion. After the merging, the components of the emulsion may be detected, analyzed, or reacted with other components.

[0201] Furthermore, as a method other than merging, in "2. Second Embodiment (Particle Separation Method)" "(2-1) Binding Execution Step S108", a method of binding with linker 601 when sorting after emulsion disruption was described. More specifically, as shown in Figures 41A and B, paired S cells (cells to be analyzed, the same applies hereinafter) 501 and E cells (cells whose reaction is to be observed, the same applies hereinafter) 502 are encapsulated, or the S cells 501 and E cells 502 paired in the emulsion are labeled with a production antibody 504 and a capture fluorescent antibody label 505 to pair them. Next, the emulsion 503a is disrupted to sort the paired cells (see Figure 41C), and as shown in Figure 41D, they are re-encapsulated with barcode beads 506 and reacted with the barcode beads 506. Then, the paired particles with unique sequences are prepared as a library. Specifically, detection, analysis, and reactions of components in emulsion 503b may be performed.

[0202] Note that the emulsions shown in Figure 41B and Figure 41D have different molecular production conditions (e.g., culture) and molecular detection conditions (e.g., cell lysis, enzyme reaction). Therefore, it is necessary to either add the required reagents by emulsion merging or to re-emulsify after emulsion disruption.

[0203] In this method, since the S cells and E cells maintain their pair even after emulsion disruption, the response of the E cells can be identified and used to analyze the target S cells.

[0204] Furthermore, the method is not limited to binding via a linker within the emulsion; after gelling the emulsion containing target particles such as S cells and E cells, the emulsion may be disrupted, and the gel and barcode beads may be co-encapsulated again. More specifically, as shown in Figures 42A and B, a pair of S cells 501 and E cells 502 are encapsulated, and as shown in Figures 42B and C, they are labeled with a production antibody 504 and a capture fluorescent antibody label 505, gelled, and the emulsion 503a is disrupted to sort the gel 602. Next, as shown in Figure 42D, the target particles are re-co-encapsulated with barcode beads 506 and reacted with the barcode beads 506. The target particles with unique sequences are then prepared as a library. Specifically, PCR ligation, adapter addition, PCR, etc., may be performed.

[0205] Note that the emulsions shown in Figure 42B and Figure 42D have different molecular production conditions (e.g., culture) and molecular detection conditions (e.g., cell lysis (mRNA may be captured by barcode RT), enzymatic reaction, etc.). Therefore, it is necessary to either add the required reagents by emulsion merging or to break down the emulsion and then re-emulsify it.

[0206] In the linker-based method described above, if the pairing efficiency is 50%, then 50% of the input particles can be analyzed. However, this method has the advantage of not being affected by the pairing efficiency provided by the linker.

[0207] This merging or the re-co-encapsulation described above enables, for example, cellular indexing of transcriptomes and epitopes by sequencing. For example, consider a case where the first particle and / or the second particle are cells bound to an antibody to which an oligo barcode having a poly-A sequence is bound. In the preparation step S106, an emulsion containing these cells is generated. This emulsion is then merged with an emulsion containing beads or a gel having a barcode sequence. Alternatively, after the emulsion is disrupted, it is re-co-encapsulated with the barcode beads or gel. These methods allow for the detection of cell surface proteins or intracellular mRNA of these cells.

[0208] In addition, by co-encapsulating the first particle and / or the second particle with the synthetic particle, the same barcode can be assigned to the first particle and / or the second particle. The first particle and / or the second particle are bound to an oligo-conjugated antibody having a Poly-T sequence, and this Poly-T sequence binds to an oligo barcode having a Poly-A sequence released from the synthetic particle, allowing the particles in the emulsion to share the same barcode.

[0209] Furthermore, the reaction does not have to be between Poly-A and Poly-T; it is sufficient that the synthetic particles have a sequence complementary to the sequence of the oligo-bound antibody. To ensure specificity, the length of the complementary binding sequence is preferably 3 or more, and more preferably 10 or more. Because the particles in the emulsion have the same barcode, even if the particles separate after the emulsion is broken down, they can be identified as cells and molecules derived from those cells by classifying them according to their barcodes through subsequent NGS analysis.

[0210] Furthermore, the substance having a sequence complementary to Poly-T or the synthetic particle is not limited to oligo-bound antibodies; any substance that binds to the particle, such as a lipid molecule or aptamer, can be used instead of an antibody. Lipid molecules can be simple lipids, complex lipids, or derived lipids. Examples of simple lipids include single-chain lipids, which are esters of fatty acids and alcohols. Examples of complex lipids include double-chain phospholipids and glycolipids. Examples of derived lipids include fatty acids such as saturated fatty acids and unsaturated fatty acids, cholesterol, and steroids such as steroid hormones. In this embodiment, one or more of these can also be used in combination.

[0211] Alternatively, the oligo barcode having a Poly-A sequence released from the synthetic particles may be modified with lipid molecules, aptamers, or antibodies, and the barcode may be directly applied to the first and second particles within the emulsion.

[0212] If the first particle is a cell whose components are to be analyzed, and the second particle is a cell whose reactivity with the first particle is to be detected, the reaction between the first and second particles is carried out in the emulsion using the method described above, and furthermore, the same barcode is assigned to them. The second particle that reacted with the first particle is labeled with a fluorescent antibody or the like and co-encapsulated with barcode beads for NGS. In this case, the second particle that does not react is not co-encapsulated. By classifying molecules with the same barcode through NGS analysis, the first particle and molecules derived from the first particle that have the same barcode as the reacted second particle can be identified. Note that the first and second particles (target particles) may not be cells, but may be, for example, cell nuclei, extracellular vesicles, etc.

[0213] More specifically, as shown in Figures 43A and 43B, paired S cells 501 and E cells 502 are encapsulated, or paired in an emulsion. Then, as shown in Figures 43B and 43C, the same barcode (B1-1 or B1-2) is applied to the S cells to be NGS analyzed and the E cells to be reaction analyzed using barcode beads (506a, 506b). The barcode here refers to the oligo barcode-binding antibody, oligo barcode-binding lipid, etc., on the cell surface, as described above. Next, the emulsion is disrupted and each cell is sorted. Here, as shown in Figures 43C and 43D, E cells 502 that have been assigned barcode B1-2 do not show reactivity with S cells 501 and are therefore excluded from co-encapsulation. Next, fluorescently labeled antibodies are attached to all S cells 501 and all cells are co-encapsulated, while only the E cells 502 that show a reaction are co-encapsulated.

[0214] Then, as shown in Figure 43E, cell lysis is performed, and after barcoding is assigned to the mRNA, NGS analysis is performed to identify S cell 501 and molecules derived from S cell 501 that have the same barcode as E cell 502. On the other hand, E cell 502, which did not show a reaction, was not co-encapsulated as described above, and its barcode was not analyzed by NGS.

[0215] As described above, by attaching the same barcode to the cell surface of S cells and E cells, and then performing NGS analysis on the reacted E cells to identify cell S and cell S-derived molecules with the same barcode, the effect of not being affected by the pairing efficiency by linkers is achieved.

[0216] Furthermore, the emulsion of cells or cell aggregates (e.g., spheroids, organoids, etc.) may react with the drug. This allows for the detection or analysis of the response of the cells or cell aggregates to the drug. Biomolecules may also be detected during this detection process.

[0217] <Synthesis process>

[0218] In this embodiment, after the preparation step S106 or the binding execution step S108, the synthesis of a chemical substance using the first and / or second particles may be carried out. For example, this synthesis may be carried out within the emulsion produced in the preparation step S106. For example, by encapsulating a cell-free expression reagent within the emulsion, in vitro antibody production becomes possible. Examples of the cell-free expression reagent include linear DNA, promega (E. coli S30 Extract system for linear DNA), etc. Alternatively, for example, protein synthesis may be carried out within the emulsion using a cell-free protein synthesis system.

[0219] 3. Third Embodiment (Particle Separation Device 100)

[0220] (1) Description of the third embodiment

[0221] Figure 28 schematically shows the overall configuration of the particle sorting device 100 according to this embodiment. The particle sorting device 100 according to this embodiment comprises at least a flow path C, a detection unit 102, an information processing unit 103, and a sorting unit 104. It may also include an irradiation unit 101, etc., as needed. The irradiation unit 101, detection unit 102, information processing unit 103, and sorting unit 104 may be located in a single device, or they may be distributed and mounted in multiple devices.

[0222] The particle sorting device 100 can specifically include a flow cytometer and an imaging cytometer. Furthermore, the particle sorting device including the sorting unit 104 is specifically referred to as a cell sorter.

[0223] (2) An example of a third embodiment

[0224] (2-1) Sample S

[0225] Sample S may be a liquid sample containing particles. The particles are the same as those described in "(3) Particle separation mechanism and particles" of "1. First embodiment (Particle separation method)," so their explanation is omitted here.

[0226] (2-2) Flow channel C

[0227] The channel C is configured to allow the sample S to flow through. In particular, the channel C may be configured to form a flow in which the particles contained in the sample S are arranged in approximately a single line. The channel C is the same as that described in "1. First Embodiment (Particle Separation Method)" under "Channel Structure" and "(3) Particle Separation Mechanism and Particles," so its explanation is omitted here.

[0228] The flow channel C may be formed within a flow channel structure such as a flow cell. The particle sorting device 100 is configured such that light from the irradiation unit 101 is irradiated onto the sample S flowing through the flow channel C, particularly onto the particles in the sample S. The particle sorting device 100 may be configured such that the light irradiation point (interrogation point) for the sample S is located within the flow channel structure in which the flow channel C is formed. Specifically, a configuration in which the light is irradiated onto the flow channel C within a microchip or a flow cell can be mentioned.

[0229] (2-3) Irradiation section 101

[0230] The irradiation unit 101 irradiates light onto the sample S flowing through the channel C. The irradiation unit 101 is the same as the one described in "(2-2) First detection step S102" of "1. First embodiment (particle sorting method)," so its explanation is omitted here.

[0231] (2-4) Detection unit 102

[0232] The detection unit 102 detects multiple particles flowing through the channel C by detecting the light generated when light is irradiated onto the sample S. The detection unit 102 is the same as the one described in "(2-3) Identification step S103" of "1. First embodiment (particle sorting method)," so its explanation is omitted here.

[0233] Furthermore, the detection unit 102 may include a signal processing unit that converts the electrical signal obtained by the photodetector into a digital signal. The signal processing unit may include an A / D converter as the device that performs the conversion. The digital signal obtained by the conversion by the signal processing unit may be transmitted to the information processing unit 103. The digital signal may be treated by the information processing unit 103 as data related to light (hereinafter also referred to as "optical data"). The optical data may be, for example, optical data that includes fluorescence data. Specifically, the optical data may be light intensity data, and the light intensity may be light intensity data of light that includes fluorescence (which may include feature quantities such as Area, Height, Width).

[0234] (2-5) Information Processing Unit 103

[0235] The information processing unit 103 processes the information obtained from the detection unit 102. The information processing unit 103 is the same as that described in "(2-3) Identification step S103" and "(2-4) Calculation step S104" of "1. First Embodiment (Particle Separation Method)," but will be explained again here.

[0236] The information processing unit 103 includes, for example, a processing unit that performs processing of various data (e.g., optical data) and a storage unit that stores the various data. When the processing unit obtains optical data corresponding to a fluorescent dye from the detection unit 102, it may perform fluorescence leakage correction (compensation processing) on ​​the optical intensity data. In the case of a spectral flow cytometer, the processing unit also performs fluorescence separation processing on the optical data to obtain optical intensity data corresponding to a fluorescent dye. The fluorescence separation processing may be performed, for example, according to the unmixing method described in Japanese Patent Application Publication No. 2011-232259. If the detection unit 102 includes an image sensor, the processing unit may obtain particle morphology information based on the image obtained by the image sensor. The storage unit may be configured to store the acquired optical data. The storage unit may further be configured to store spectral reference data used in the unmixing processing.

[0237] Furthermore, the information processing unit 103 may determine whether or not to separate the particles based on the optical data and / or morphological information. Based on the result of this determination, the information processing unit 103 may control the separation unit 104, and the separation unit 104 may perform particle separation.

[0238] The information processing unit 103 may be configured to output various types of data (e.g., optical data, image data, etc.). For example, the information processing unit 103 may output various types of data (e.g., two-dimensional plots, spectral plots, etc.) generated based on the optical data. The information processing unit 103 may also be configured to accept input of various types of data, for example, by accepting gating processing on a plot by a user. The information processing unit 103 may include an output unit (e.g., a display, a portable information terminal, a wearable terminal, etc.) or an input unit (e.g., a keyboard, a voice input device, etc.) for executing such output or input.

[0239] The information processing unit 103 may be contained within the housing that houses the irradiation unit 101 and the detection unit 102, or it may be located outside the housing. Furthermore, the various processing or functions performed by the information processing unit 103 may be implemented by a server computer or cloud connected via a network.

[0240] (2-6) Preparation section 104

[0241] The separation unit 104 separates the particles that have been determined to be separated into the emulsion. The separation unit 104 performs particle separation according to the determination result by the information processing unit 103. The separation method may be a method in which the direction of particle movement is controlled within the flow channel structure to perform separation. The flow channel structure and the specific method of separation are the same as those described in "1. First Embodiment (Particle Separation Method)," so the explanation is omitted here.

[0242] (2-7) Others

[0243] Furthermore, the particle sorting microchip 150 described in "1. First Embodiment (Particle Sorting Method)" may be used in the particle sorting device 100 according to this embodiment and may be detachable from the particle sorting device 100. By making the particle sorting microchip 150 detachable from the particle sorting device 100, a new particle sorting microchip 150 can be used for each sample S, thereby preventing contamination between samples S.

[0244] (3) An example of an information processing unit 103 and an example of an algorithm executed by the information processing unit 103

[0245] (3-1) An example of an information processing unit 103

[0246] A more specific configuration example of the information processing unit 103 will be described below.

[0247] [Overall Configuration of Information Processing Unit 103] Figure 29 is a block diagram showing an example configuration of the information processing unit 103. The information processing unit 103 in Figure 29 has a plurality of circuits 2302 to 2309 connected to the bus. The information processing unit 103 is also referred to as the drive unit or control unit.

[0248] Specifically, the circuit indicated by reference numeral 2302 in Figure 29 is an analog-to-digital conversion circuit 2302. The circuit indicated by reference numeral 2303 is an event detection circuit 2303. Furthermore, the circuit indicated by reference numeral 2304 is an arrival time calculation circuit 2304. In addition, the circuit indicated by reference numeral 2305 is a gating circuit 2305. The circuit indicated by reference numeral 2306 is an output queuing circuit 2306. Furthermore, the circuit indicated by reference numeral 2307 is an output timing generation circuit 2307. In addition, the circuit indicated by reference numeral 2308 is an output signal generation circuit 2308. Finally, the circuit indicated by reference numeral 2309 is an MPU (microprocessing unit) 2309. Note that the analog-to-digital conversion circuit 2302 is represented as "A / D" in Figure 29.

[0249] Furthermore, as shown in Figure 29, the information processing unit 103 has a clock counter 2310. The clock counter 2310 is connected to an event detection circuit 2303, an arrival time calculation circuit 2304, a gating circuit 2305, an output queue circuit 2306, an output timing generation circuit 2307, and an output signal generation circuit 2308.

[0250] Furthermore, as shown in Figure 29, the information processing unit 103 includes a PCI / O unit (Input / Output Interface circuit for personal computer connection) 2311 connected to the MPU 2309, and a control PC 2312 connected to the PCI / O unit 2311.

[0251] In addition, as shown in Figure 29, the information processing unit 103 has a digital-to-analog conversion circuit 2313 connected to the output signal generation circuit 2308. The digital-to-analog conversion circuit 2313 is represented as "D / A" in Figure 29.

[0252] [Details of the Information Processing Unit 103] [Analog-to-Digital Conversion Circuit] The analog-to-digital conversion circuit 2302 is a circuit located downstream (output side) of the detection unit 102 and is connected to the detection unit 102. Multiple analog-to-digital conversion circuits 2302 are arranged. Here, the number of analog-to-digital conversion circuits 2302 may be the same as the number of channels in the detection unit 102, so that each corresponds to one of the multiple lights (wavelength ranges) detected by the detection unit 102. Alternatively, the number of analog-to-digital conversion circuits 2302 may be the same as the number of sensors in the detection unit 102.

[0253] Each analog-to-digital conversion circuit 2302 receives an electrical signal corresponding to it, which is output from the detection unit 102. This electrical signal is an analog signal obtained by the detection unit 102 from light (particularly fluorescence and scattered light) detected by the detection unit 102 through photoelectric conversion. Each analog-to-digital conversion circuit 2302 then converts the input electrical signal from an analog signal to a digital signal. Furthermore, each analog-to-digital conversion circuit 2302 outputs the converted digital electrical signal to a subsequent stage.

[0254] [Event Detection Circuit] The event detection circuit 2303 is a downstream circuit of each analog-to-digital conversion circuit 2302 and is connected to each analog-to-digital conversion circuit 2302.

[0255] The event detection circuit 2303 receives electrical signals output from each analog-to-digital conversion circuit 2302. The event detection circuit 2303 then uses these signals as trigger signals to recognize particles that possess a specific signal from among the input electrical signals. That is, the event detection circuit 2303 recognizes that each electrical signal was detected from a particle when the value of the trigger signal satisfies predetermined conditions. The trigger signal may be the electrical signal of the light with the highest intensity among the multiple lights detected by the detection unit 102 (for example, forward scattered light), but is not limited to this in this embodiment.

[0256] Furthermore, as shown in Figure 30, the event detection circuit 2303 reads the waveform of each input electrical signal and calculates the width, height, and area of ​​the read waveform. Then, using the calculated values ​​of the waveform, the event detection circuit 2303 creates an event data packet, as shown in Figure 31, associating each electrical signal with a corresponding particle. This event data packet is an example of measurement data for a single particle. Finally, the event detection circuit 2303 outputs the created event data packet to a subsequent stage.

[0257] Here, the event data packet includes items whose data recording is completed at the time the packet is created (hereinafter also referred to as the "first item"). The event data packet also includes items that are updated as processing of the electrical signal corresponding to the packet progresses after the packet is created (hereinafter also referred to as the "second item").

[0258] The first item includes, for example, the following: - Width, height, and area of ​​the electrical signal waveform - Number of the recognized particle (event number) - Number of the electrical signal that triggered the signal - Time of detection of the trigger signal Note that the number of the electrical signal that triggered the signal may be the channel number. In addition, a signal input from the clock counter 2310 may be used to record the time of detection of the trigger signal. This signal may be the result of the clock counter 2310 counting the clock signal from a clock generation circuit (not shown) that is input to it.

[0259] On the other hand, the second item includes, for example, the following items: • The time at which the particle should be taken in • A first flag indicating whether the particle is a target for acquisition • A second flag indicating whether the particle is a target for combination • A third flag indicating whether or not to take in the particle. The first and second flags are set by the gating circuit 2305. The third flag, on the other hand, is set by the output queue circuit 2306. These first, second, and third flags are basically set to 1 or 0 and may be used to determine whether or not to take in the corresponding particle. Details of each flag will be described later.

[0260] [Arrival Time Calculation Circuit] As shown in Figure 29, the arrival time calculation circuit 2304 is a downstream circuit of the event detection circuit 2303 and is connected to the event detection circuit 2303.

[0261] The arrival time calculation circuit 2304 receives event data packets output from the event detection circuit 2303. Based on the input event data packets, the arrival time calculation circuit 2304 calculates the arrival time at which the particles (particles to be separated) reach the communication port (the main channel 155 side inlet of the connecting channel 170), which is the time at which the particles included in the second item should be taken in. Hereafter, "time at which the particles should be taken in" will be replaced with "arrival time of the particles". The arrival time calculation circuit 2304 records the arrival time calculated by this calculation in an event data packet and outputs this recorded event data packet to the next stage.

[0262] The arrival time may be calculated by adding the time required (delay time) for the particles to be separated from the detection area 156 to the communication port to the detection time of the trigger signal included in the second item. Alternatively, the arrival time may be calculated using a clock counter value.

[0263] [Gating Circuit] The gating circuit 2305 is a downstream circuit of the event detection circuit 2303 and is connected to the event detection circuit 2303.

[0264] The gating circuit 2305 receives event data packets output from the event detection circuit 2303. The gating circuit 2305 then sets a first flag and a second flag on the input event data packets. Furthermore, the gating circuit 2305 outputs the event data packets with the first and second flags set to the next stage.

[0265] The first flag may be set based on a preset threshold for the parameters of each electrical signal included in the event data packet. In this case, the threshold may be at least one of the waveform width, height, and area. If the parameter satisfies the threshold, the value of the first flag may be set to a value indicating that particle capture will be performed (e.g., "1"). On the other hand, if the parameter does not satisfy the threshold, the value of the first flag may be set to a value indicating that particle capture will not be performed (e.g., "0"). Note that this embodiment is not limited to this flag setting method.

[0266] The setting of the second flag may be based on a predetermined threshold for the parameters of each electrical signal included in the event data packet. In this case, the threshold may be at least one of the waveform width, height, and area. If the parameter satisfies the threshold, the value of the second flag may be set to a value indicating that the particle is a target for combination (e.g., "1"). On the other hand, if the parameter does not satisfy the threshold, the value of the second flag may be set to a value indicating that the particle is not a target for combination (e.g., "0"). Note that this embodiment is not limited to such flag setting methods.

[0267] Furthermore, the threshold may be a range predetermined by gating. Here, gating is a process of enclosing and specifying the range corresponding to the particles to be separated on a distribution diagram that represents the characteristic distribution of particles in a particle group. This gating is performed before the start of the particle acquisition operation. The distribution diagram may be created by a GUI (Graphical User Interface) on the control PC 2312. Also, gating may be performed by a gating circuit 2305.

[0268] Here, Figure 32A shows the result of gating to a histogram chart as an example of a distribution map. In this histogram chart, the horizontal axis represents the parameter and the vertical axis represents the number of particles. The parameter shown in Figure 32 is the area of ​​the waveform of the electrical signal corresponding to channel number 1 (Ch1), but other parameters may also be used. The rectangular frame shown in Figure 32 is a gate that specifies the range corresponding to the particles to be separated, and this range may be used as a threshold for setting the first flag and / or the second flag.

[0269] On the other hand, Figure 32B shows the result of gating to a 2D (two-dimensional) chart as another example of a distribution map. In this 2D chart, different parameters are assigned to the horizontal and vertical axes. The parameter on the horizontal axis in Figure 32 is the area of ​​the waveform of the electrical signal corresponding to channel number 2 (Ch2), and the parameter on the vertical axis is the area of ​​the waveform of the electrical signal corresponding to channel number 3 (Ch3). However, parameters other than the area of ​​these two electrical signal waveforms may also be used. The rectangular frame shown in Figure 32 is a gate that specifies the range corresponding to the particles to be separated, and this range may be used as a threshold for setting the first flag and / or the second flag.

[0270] [Output Queue Circuit] Returning to Figure 29, the output queue circuit 2306 is a downstream circuit of the arrival time calculation circuit 2304 and the gating circuit 2305, and is connected to these arrival time calculation circuits 2304 and 2305.

[0271] The output queuing circuit 2306 receives event data packets output from the arrival time calculation circuit 2304 and event data packets output from the gating circuit 2305. The output queuing circuit 2306 then integrates (combines) the two input event data packets that are identical to each other, i.e., those indicating the same event number, into a single event data packet. The integrated event data packet has the delay time, a first flag, and a second flag written to it. The integration of the event data packets may be performed by either the arrival time calculation circuit 2304 or the gating circuit 2305 through communication between the two circuits. The arrival time calculation circuit 2304 and the gating circuit 2305 may also be connected in series.

[0272] Furthermore, the output queuing circuit 2306 arranges the sequentially input event data packets of different particles in order of arrival time contained in the event data packets, from earliest to latest. Here, the event data packets input to the output queuing circuit 2306 that are waiting for the output of the drive waveform for capturing the corresponding minute particle are defined as the "output queue". The output queue is updated in response to the input of new event data packets to the output queuing circuit 2306.

[0273] The single-particle versus multiple-particle mode is a separation mode in which, when multiple target particles (i.e., a first particle and a second particle) are captured within the same emulsion, the number of first particles is one, while there is no condition on the number of second particles. In the single-particle versus multiple-particle mode, the determination of whether or not to perform separation is made based on the types of preceding and succeeding particles, and the proximity of the preceding and succeeding particles to the first and second particles, for the first and second particles flowing continuously. Specifically, when the first and second particles are flowing in close proximity to each other to the extent that they can be co-encapsulated, and the second particle is also close before and after the first and second particles, the target first and second particles are considered to be "acquired." However, if particles other than the second particle are close and there is a high probability that they will be captured together with the two particles and contained in the same emulsion, the target first and second particles are considered "not acquired" and are not separated. The single-particle versus multiple-particle mode does not limit the number of second particles trapped within the same emulsion, thus increasing the rate at which emulsions in which the first and second particles are co-encapsulated can be obtained.

[0274] The single-particle-to-single-particle mode is a separation mode in which, when capturing a first particle and a second particle within the same emulsion, the number of both the first and second particles is limited to one. In the single-particle-to-single-particle mode, the decision of whether or not to perform separation is made based on the proximity of preceding and succeeding particles to the continuously flowing first and second particles, and the first and second particles themselves. Specifically, if the first and second particles have flowed in close proximity to each other to the extent that they can be co-encapsulated, and there is a high probability that particles are further close before and after these two particles and will be captured together with them and encapsulated in the same emulsion, the target first and second particles are considered "not acquired." Otherwise, the target first and second particles are considered "acquired" and separated.

[0275] The output queue circuit 2306 then sets a third flag based on the result of determining whether or not particles have been taken in and combined according to the set mode. At this time, if the output queue circuit 2306 determines that it will take in the target particles, it may set the third flag to "1". On the other hand, if the output queue circuit 2306 determines that it will not take in the particles, it may set the third flag to "0". Note that in this embodiment, the flag setting method is not limited to this.

[0276] In addition, the output queue circuit 2306 writes the application timing of the drive waveform to be applied to the actuator to memory. This memory may be RAM (Random Access Memory). This memory may also be connected to a bus, or it may be built into the circuit of the information processing unit 103 or the control PC 2312. Furthermore, when writing out the application timing, the settings of the first flag, the second flag, and the third flag may be referenced. In this case, if all flags are set to values ​​indicating capture execution, the application timing that captures the corresponding particle may be written out. In addition, the drive waveform may be a drive voltage.

[0277] Here, the output queuing circuit 2306 writes the application timing of the drive waveform to be used into memory. When a pulse drive waveform is applied to the drive waveform, as shown in Figure 6, the pulse waveform consists of a falling edge Wf and a rising edge Wr, with the falling edge Wf being the beginning of the pulse waveform and the rising edge Wr being the end. The height (amplitude) of the falling edge Wf and the height of the rising edge Wr are made the same.

[0278] The falling waveform Wf is applied to deform the pressure chamber in a direction that increases its volume and to generate a negative pressure change immediately after the falling waveform is applied. It may also be a force that weakens the pressing force in order to increase the volume inside the pressure chamber. If the actuator is a piezoelectric element, it may also be a waveform that reduces the driving voltage applied to the stretched piezoelectric element, thereby causing the piezoelectric element to contract.

[0279] On the other hand, the rising waveform Wr is applied to deform the pressure chamber so as to reduce its volume and generate a positive pressure change immediately after the application of the drive waveform. This may also be a force that increases the pressing pressure to reduce the volume inside the pressure chamber. If the actuator is a piezoelectric element, this waveform may also be one that increases the drive voltage applied to the contracted piezoelectric element, thereby extending the piezoelectric element.

[0280] When the output queuing circuit 2306 writes the application timing of the pulse drive waveform, which is composed of the falling-edge waveform section Wf and the rising-edge waveform section Wr described above, to memory, it may write the arrival time included in the event data packet as the application timing of the pulse drive waveform.

[0281] [Output Timing Generation Circuit] As shown in Figure 29, the output timing generation circuit 2307 is connected to the output queue circuit 2306. The output timing generation circuit 2307 reads from the RAM the arrival time of the event data packet that is at the very front of the output queue written to the RAM by the output queue circuit 2306. The output timing generation circuit 2307 then compares the read arrival time with the value of the signal from the clock counter 2310 and generates an output timing signal at that arrival time. Here, the output timing signal is a signal for assigning the output timing of the drive waveform. The output timing generation circuit 2307 outputs the generated output timing signal to the next stage. Furthermore, after outputting the output timing signal, the output queue circuit 2306 may send a completion signal to the output queue circuit 2306 to prompt the update of the output queue.

[0282] When the drive waveform is a pulse waveform, the output timing generation circuit 2307 refers to the memory where the application timings of the falling edge waveform Wf and rising edge waveform Wr have been written by the output queuing circuit 2306. The output timing generation circuit 2307 then compares the application timings of the falling edge waveform Wf and rising edge waveform Wr in the memory with the signal values ​​from the clock counter 2310 to generate an output timing signal for each waveform section. Furthermore, the output timing generation circuit 2307 outputs the generated output timing signals to the next stage.

[0283] [Output Signal Generation Circuit] The output signal generation circuit 2308 is a downstream circuit of the output timing generation circuit 2307 and is connected to the output timing generation circuit 2307.

[0284] The output signal generation circuit 2308 receives the output timing signal output from the output timing generation circuit 2307. The output signal generation circuit 2308 then generates a drive waveform (output signal) corresponding to the input output timing signal and outputs it to the next stage. Furthermore, after outputting the drive waveform, the output signal generation circuit 2308 updates the step counter and the output status signal. The output status signal indicates the waveform is stopped / outputting (output enabled / disabled).

[0285] When the drive waveform is a pulse waveform, the output signal generation circuit 2308 generates and outputs the falling edge waveform Wf and the rising edge waveform Wr separately.

[0286] [Digital-to-Analog Conversion Circuit] The drive waveform output from the output signal generation circuit 2308 is input to the digital-to-analog conversion circuit 2313. The digital-to-analog conversion circuit 2313 then converts the input drive waveform from a digital signal to an analog signal and outputs it to the drive circuit of the actuator 31.

[0287] Furthermore, the information processing unit 103 shown in Figure 29 may also be applied to apply drive waveforms other than pulse waveforms.

[0288] (3-2) An example of an algorithm

[0289] An example of the algorithm for the sorting process executed by the information processing unit 103 is described below.

[0290] An example of the algorithm executed by the information processing unit 103 is shown in Figures 33 and 34. In the flowchart shown in Figure 33, the following first to third processes are performed individually and in parallel.

[0291] [First Processing] In the first processing, first, in step S171-1, it is determined whether or not an event (event data packet) has been input. For this determination, the event data packet input from the preceding circuit (for example, the arrival time calculation circuit 2304 and the gating circuit 2305) is used. If a positive determination result is obtained in step S171-1, the process proceeds to step S171-2; if a negative determination result is obtained, step S171-1 is repeated. Next, in step S171-2, the output queue is updated by adding the new event, which was determined to have "input present" in step S171-1, to the output queue. Finally, in step S171-3, based on the output queue updated in step S171-2, the decision of whether or not to capture the particle (acquisition or non-acquisition) is re-evaluated according to the flowchart shown in Figure 33, and the process returns to step S171-1. The reason we use the term "re-evaluation" here is that the evaluation in this step involves re-evaluating events that are already in the queue (i.e., events that have already been evaluated and have a third flag set). This step is effective because events added to the queue later may be close to events added earlier.

[0292] The flowchart shown in Figure 34 determines whether the nth event stored in the output queue is acquired or not. In step S181-2, a determination is made as to whether the updated target event in step S181-1 is a particle to be separated. If it is not a particle to be separated, the process returns to step S181-1 and the target event is updated to the next event. If it is a particle to be separated, the process proceeds to the following steps, and in steps S182-1 to S182-8, an acquisition determination process is performed with the (n-1)th event, which is the preceding event of the target event, including the pair determination and proximity determination described above. Subsequently, in steps S183-1 to S183-9, an acquisition determination process is performed with the (n+1)th event, which is the subsequent event of the target event, and the acquisition determination of the target event is made.

[0293] In steps S182-1 and S182-2, a pair determination is made with the preceding event. If the pair determination is positive, in S182-3, the sorting timing Ts is calculated, corrected from the arrival time Tn of the event being determined and the arrival time T(n-1) of the preceding event. If the pair determination is negative, the process proceeds to step S183-1, where the acquisition determination process with the subsequent event is performed. In steps S182-4 to S182-8, proximity determination is performed based on the calculated sorting timing Ts. In step S182-4, it is determined whether the (n-2)th event, which is the preceding event of the paired event and is selected as the acquisition mode in single-particle versus multi-particle mode, is the second particle. If the determination is positive, the process proceeds to step S182-6 without performing proximity determination on the (n-2)th event; if the determination is negative, the process proceeds to step S182-5.

[0294] In step S182-5, the time interval between the calculated sorting timing Ts and the arrival time T(n-2) of the preceding event is compared with the set value ΔT2 of the preceding event's involvement time. If the time interval is greater than or equal to the set value of the involvement time, the process proceeds to step S182-6. If the time interval is less than the set value of the involvement time, the proximity determination result with the preceding event is set to negative, and the process proceeds to step S183-1. In step S182-5, a determination is made as to whether the (n+1)th event, which is a subsequent event of the paired event while the acquisition mode is selected as single-particle versus multi-particle mode, is the second particle. If the determination is positive, the process proceeds to step S182-8. If the determination is negative, the process proceeds to step S182-7.

[0295] In step S182-7, the time interval between the calculated sorting timing Ts and the arrival time T(n+1) of the subsequent event is compared with the set value ΔT1 of the subsequent event's involvement time. If the time interval is greater than or equal to the set value of the involvement time, the process proceeds to step S182-8. If the time interval is less than the set value of the involvement time, the proximity determination result with the subsequent event is set to negative, and the process proceeds to step S183-1. In step S182-8, the target event for which both the pair determination and proximity determination result were positive is designated as the acquisition determination, and the calculated sorting timing Ts is set.

[0296] In steps S183-1 and S183-2, a pair determination is made with the subsequent event. If the pair determination is positive, the process proceeds to step S183-3. If the pair determination is negative, the process proceeds to step S181-1 and the target particle is updated. In step S183-3, if the target particle has been acquired with a preceding event, the time interval ΔTpair(n-1) between the target event and the preceding event is compared with the time interval ΔTpair(n+1) between the target event and the subsequent event. If ΔTpair(n-1) is less than or equal to ΔTpair(n+1), priority is given to acquiring the pair event with the shorter time interval between events, and the acquisition determination process between the target particle and the subsequent event is not performed. The process proceeds to step S181-1 and the target particle is updated. If ΔTpair(n-1) is greater than ΔTpair(n+1), proceed to step S183-3 and calculate the allocation timing Ts calculated from the arrival time Tn of the event to be judged and the arrival time T(n+1) of the subsequent event.

[0297] In steps S183-5 to S183-9, proximity determination is performed based on the calculated sorting timing Ts. In step S182-4, it is determined whether the (n-1)th event, which is the preceding event of the paired event and is selected as the acquisition mode (single-particle vs. multi-particle mode), is the second particle. If the determination is positive, the process proceeds to step S183-7 without performing proximity determination for the (n-1)th event. If the determination is negative, the process proceeds to step S183-6. In step S183-6, the time interval between the calculated sorting timing Ts and the arrival time T(n-1) of the preceding event is compared with the set value ΔT2 of the preceding event's involvement time. If the time interval is greater than or equal to the set value of the involvement time, the process proceeds to step S183-7. If the time interval is less than the set value of the involvement time, the proximity determination result with the preceding event is negative, and the process proceeds to step S181-1.

[0298] In step S183-7, it is determined whether the (n+2)th event, which is a subsequent event of the paired event and is selected as the acquisition mode in single-particle versus multi-particle mode, is the second particle. If the result of this determination is positive, the process proceeds to step S183-9; if the result of this determination is negative, the process proceeds to step S183-8. In step S183-8, the time interval between the calculated sorting timing Ts and the arrival time T(n+2) of the subsequent event is compared with the set value ΔT1 of the subsequent event's involvement time. If the time interval is greater than or equal to the set value of the involvement time, the process proceeds to step S182-9. If the time interval is less than the set value of the involvement time, the proximity determination result with the subsequent event is negative, and the process proceeds to step S181-1.

[0299] In step S183-9, the target event for which both the pairing and proximity checks yielded positive results is designated as the acquisition check, and the calculated allocation timing Ts is set. Returning to step S181-1, the target event for the check is updated to the next event.

[0300] [Second Processing] In the second processing, in step S172-1, the falling timing of the falling waveform section Wf for the next event to be acquired (the particle to be separated) is written to memory. This written applied timing will be referenced by the output timing generation circuit 2307. Next, in step S172-2, it is determined whether or not the falling trigger output of the falling waveform section Wf has been completed. The falling trigger output of the falling waveform section Wf means that the falling timing of the falling waveform section Wf generated by the output timing generation circuit 2307 has been output to the output signal generation circuit 2308. Finally, in step S172-3, the output queue is updated by removing the event for which the falling trigger output of the falling waveform section Wf has been completed from the output queue, and the process returns to step S173-1.

[0301] [Third Process] In the third process, first, in step S173-1, the rising timing of the rising waveform section Wr is calculated. In the case of a pulse-driven waveform, the rising timing of the rising waveform section Wr is obtained by adding the falling time and holding time to the falling timing of the falling waveform section Wf. Next, the rising timing of the rising waveform section Wr calculated in step S173-2 is written to memory. This written-out applied timing will be referenced by the output timing generation circuit 2307. Next, in step S173-3, it is determined whether or not the rising trigger output of the rising waveform section Wr has been completed. The rising trigger output of the rising waveform section Wr means that the rising timing of the rising waveform section Wr generated by the output timing generation circuit 2307 has been output to the output signal generation circuit 2308. Then, if a positive result is obtained in step S173-3, the process returns to step S173-1; if a negative result is obtained, step S173-3 is repeated.

[0302] 4. Fourth Embodiment (Particle Separation System)

[0303] The particle sorting system according to this embodiment comprises at least a flow path C, a detection unit 102, an information processing unit 103, and a sorting unit 104. It may also include an irradiation unit 101, etc., as needed.

[0304] The particle sorting system according to this embodiment is configured to perform the particle sorting device 100 described above, and each part is the same as that described in "3. Third Embodiment (Particle Sorting Device 100)," so a detailed explanation is omitted here.

[0305] Furthermore, the following configurations can also be adopted in this technology: [1] A particle separation method comprising: a flow step of flowing a liquid containing a first particle and a second particle into a flow channel; a first detection step of detecting a plurality of particles flowing in the flow channel; an identification step of identifying whether the detected plurality of particles are the first particle and the second particle; a calculation step of calculating the time interval between the first particle and the second particle flowing in the flow channel; a determination step of determining whether or not to separate the first particle and the second particle according to the time interval; and, if it is determined that separation is to be taken, a separation step of separating the first particle and the second particle into an emulsion. [2] The particle separation method according to [1], wherein the determination step further determines whether or not the first particle and the second particle are continuous. [3] The particle separation method according to [2], wherein the determination step determines that the first particle and the second particle should be separated if the time interval is less than or equal to a predetermined threshold. [4] The particle sorting method according to [3], wherein in the determination step, the sorting timing of at least one of the first particle and the second particle is controlled based on the time interval. [5] The particle sorting method according to [4], wherein in the determination step, the sorting timing is changed according to the order in which the first particle and the second particle flow through the flow path. [6] The particle sorting method according to any one of [1] to [6], wherein, in a single particle versus multiple particle mode, the sorting is determined based on the types of preceding particles and succeeding particles, and the proximity of the preceding particles and the succeeding particles to the first particle and the second particle. [7] A particle separation method according to any one of [1] to [7], wherein, in a single-particle-to-single-particle mode, it is determined whether or not to perform the separation based on the proximity of the preceding particle and subsequent particle that precede the first particle and the second particle, which are continuously flowing, to the first particle and the second particle.[8] The particle separation method according to any one of [1] to [7], wherein the separation is performed by a pressure change in a pressure chamber communicating with the flow path due to the application of a pulse voltage to an actuator, and the value of the pulse voltage or the drive waveform is specified based on information relating to the first particle and the second particle. [9] The particle separation method according to [8], wherein the holding time during which the voltage of the drive waveform is kept constant is set according to a time corresponding to the separation margin time.

[10] The particle separation method according to [9], wherein the separation margin time is the time for maintaining the pressure change required for the separation of the first particle and the second particle.

[11] The particle separation method according to any one of [8] to

[10] , wherein the drive waveform further has a falling waveform after the pulse voltage.

[12] The particle sorting method according to any one of [1] to

[11] , wherein the determination step further determines whether the first particle and two or more of the second particles are continuous, and determines whether to sort based on the time interval between the earliest particle and the latest particle in the group consisting of the first particle and two or more of the second particles.

[13] The particle sorting method according to any one of [1] to

[12] , wherein the determination step further determines whether to sort based on the time interval between the first particle and some of the second particles among the two or more of the second particles if the first particle and two or more of the second particles are in close proximity.

[14] The particle sorting method according to any one of [1] to

[13] , further comprising a second detection step of detecting the first particle and the second particles sorted in the sorting step.

[15] The particle sorting method according to

[14] , wherein the conditions related to sorting are adjusted based on the information obtained in the detection.

[16] The particle separation method according to any one of [1] to

[15] , wherein the first particle and / or the second particle is selected from the group consisting of cells, cell aggregates, synthetic particles, and biologically derived factors.

[17] A particle separation method comprising: a flow step of flowing a liquid containing a first particle and a second particle into a flow channel; a first detection step of detecting a plurality of particles flowing through the flow channel; an identification step of identifying whether the detected plurality of particles are the first particle and the second particle; a calculation step of calculating the time interval between the first particle and the second particle flowing through the flow channel; a determination step of determining whether or not to separate the first particle and the second particle according to the time interval; a separation step of separating the first particle and the second particle into an emulsion if it is determined that separation is to be performed; and a binding execution step of performing binding between the first particle and / or a secretion secreted from the first particle and the second particle in the emulsion.

[18] The particle separation method according to

[17] , wherein the binding execution step measures the binding affinity between the first particle and / or a secretion secreted from the first particle and the second particle.

[19] A particle separator comprising: a channel through which a liquid containing first particles and second particles flows; a detection unit for detecting a plurality of particles flowing through the channel; an information processing unit for processing information obtained from the detection unit; and a separator unit for separatory particles determined to be separatory into an emulsion, wherein the information processing unit performs: an identification step for identifying whether the detected plurality of particles are the first particles and the second particles; a calculation step for calculating the time interval between the first particles and the second particles flowing through the channel; and a determination step for determining whether to separatory the first particles and the second particles according to the time interval.

[20] A particle separation system comprising: a channel through which a liquid containing first particles and second particles flows; a detection unit for detecting a plurality of particles flowing through the channel; an information processing unit for processing information obtained from the detection unit; and a separation unit for separating particles determined to be separated into an emulsion, wherein the information processing unit is configured to perform: an identification step for identifying whether the detected plurality of particles are the first particles and the second particles; a calculation step for calculating the time interval between the first particles and the second particles flowing through the channel; and a determination step for determining whether or not to separate the first particles and the second particles according to the time interval.

[0306] The present technology will be described in more detail below based on the following examples. The examples described below are representative examples of the present technology and should not be interpreted as narrowing the scope of the present technology.

[0307] Figure 35 shows a comparison of the preparative performance of this technology and the conventional preparative control technology, using Monte Carlo simulation, assuming that the arrival of particles in the preparative section follows a Poisson distribution.

[0308] Figure 35 shows the results under conditions where the event rate was 10 keps to 26 keps, and the concentrations of the first particle (target concentration) and the second particle (partner concentration) were (5%, 95%) and (10%, 90%), respectively. Here, the following three indicators were calculated as indicators of preparative performance: (i) Target utilization rate: The ratio of the number of first particles that successfully formed emulsions containing the target first and second particles (pair particles) to the number of first particles introduced. (ii) Emulsion formation rate containing the first and second particles: The number of emulsions containing the target first and second particles formed per second. (iii) Purity: The ratio of the number of emulsions containing the target first and second particles to the number of emulsions formed.

[0309] In conventional technology, the target utilization rate was around 30% to 50%, but with this technology, the target utilization rate was 50% to 70%. This is because this technology enables co-encapsulation of the first and second particles at a larger time interval. Furthermore, the improved target utilization rate has increased the rate of emulsion formation containing the first and second particles. In addition, while conventional technology produces emulsions with a purity of around 30% to 60% that do not contain the desired first and second particles, this technology achieves a purity of nearly 100% because emulsion formation is performed only when the first and second particles form a pair, based on proximity determination using the corrected separation timing of the first particle.

[0310] 100 Particle sorting device 101 Irradiation unit 102 Detection unit 103 Information processing unit 104 Sorting unit 108 Second irradiation unit 109 Second detection unit 150 Microchip for particle sorting 151 Sample liquid inlet 152 Sample liquid flow path 153 Sheath liquid inlet 154 Sheath liquid flow path 155 Main flow path 156 Detection area 157 Sorting unit 158 ​​Disposal flow path 159 Sorting flow path 162 Confluence unit 1621 End of sorting flow path 164 Second detection area 170, 180 Connecting flow paths

Claims

1. A particle separation method comprising: a flow step of flowing a liquid containing first particles and second particles into a flow path; a first detection step of detecting a plurality of particles flowing in the flow path; an identification step of identifying whether the detected plurality of particles are the first particles and the second particles; a calculation step of calculating the time interval between the first particles and the second particles flowing in the flow path; a determination step of determining whether or not to separate the first particles and the second particles according to the time interval; and, if it is determined that separation is to be performed, a separation step of separating the first particles and the second particles into an emulsion.

2. The particle sorting method according to claim 1, wherein the determination step further determines whether the first particle and the second particle are continuous.

3. The particle separation method according to claim 2, wherein in the determination step, if the time interval is less than or equal to a predetermined threshold, it is determined that the first particle and the second particle are separated.

4. The particle sorting method according to claim 3, wherein the determination step controls the sorting timing of at least one of the first particle and the second particle based on the time interval.

5. The particle sorting method according to claim 4, wherein in the determination step, the sorting timing is changed according to the order in which the first particles and the second particles flow through the flow path.

6. The particle separation method according to claim 1, wherein, in a single-particle versus multi-particle mode, a determination is made whether or not to perform the separation based on the types of preceding particles and succeeding particles, and the proximity of the preceding particles and succeeding particles to the first particles and second particles, with respect to the first particles and second particles that are continuously flowing through the system.

7. The particle separation method according to claim 1, wherein, in a single-particle-to-single-particle mode, a determination is made whether or not to perform the separation based on the proximity of the preceding particle and subsequent particle that precede the first particle and the second particle, which are continuously flowing, to the first particle and the second particle.

8. The particle separation method according to claim 1, wherein the separation is performed by a change in pressure in a pressure chamber communicating with the flow path due to the application of a pulse voltage to an actuator, and the value of the pulse voltage or the drive waveform is specified based on information relating to the first particle and the second particle.

9. The particle sorting method according to claim 8, wherein the holding time during which the voltage of the drive waveform is kept constant is set according to the time corresponding to the sorting margin time.

10. The particle separation method according to claim 9, wherein the separation allowance time is the time required to maintain the pressure change required for the separation of the first particle and the second particle.

11. The particle sorting method according to claim 8, wherein the drive waveform further has a falling edge waveform after the pulse voltage.

12. The particle sorting method according to claim 1, wherein the determination step further determines whether the first particle and two or more of the second particles are continuous, and determines whether to sort based on the time interval between the earliest particle and the latest particle in the group consisting of the first particle and two or more of the second particles.

13. The particle separation method according to claim 1, wherein in the determination step, if the first particle and two or more of the second particles are in close proximity, a determination is made whether or not to separate the particles based on the time interval between the first particle and some of the second particles among the two or more of the second particles.

14. The particle separation method according to claim 1, further comprising a second detection step of detecting the first particles and the second particles separated in the separation step.

15. The particle sorting method according to claim 14, wherein the conditions related to sorting are adjusted based on the information obtained from the detection.

16. The particle separation method according to claim 1, wherein the first particle and / or the second particle is selected from the group consisting of cells, cell aggregates, synthetic particles, and biologically derived factors.

17. A particle separation method comprising: a flow step of flowing a liquid containing first particles and second particles into a flow path; a first detection step of detecting a plurality of particles flowing through the flow path; an identification step of identifying whether the detected plurality of particles are the first particles and the second particles; a calculation step of calculating the time interval between the first particles and the second particles flowing through the flow path; a determination step of determining whether or not to separate the first particles and the second particles according to the time interval; a separation step of separating the first particles and the second particles into an emulsion if separation is determined to be performed; and a bonding execution step of performing bonding between the first particles and / or secretions secreted from the first particles and the second particles within the emulsion.

18. The particle separation method according to claim 17, wherein the binding step involves measuring the binding affinity between the first particle and / or the secretion secreted from the first particle and the second particle.

19. A particle separator comprising: a channel through which a liquid containing first particles and second particles flows; a detection unit for detecting a plurality of particles flowing through the channel; an information processing unit for processing information obtained from the detection unit; and a separator unit for separatory particles determined to be separatory into an emulsion, wherein the information processing unit performs: an identification step for identifying whether the detected plurality of particles are the first particles and the second particles; a calculation step for calculating the time interval between the first particles and the second particles flowing through the channel; and a determination step for determining whether to separatory the first particles and the second particles according to the time interval.

20. A particle separation system comprising: a channel through which a liquid containing first particles and second particles flows; a detection unit for detecting a plurality of particles flowing through the channel; an information processing unit for processing information obtained from the detection unit; and a separation unit for separating particles determined to be separated into an emulsion, wherein the information processing unit is configured to perform: an identification step for identifying whether the detected plurality of particles are the first particles and the second particles; a calculation step for calculating the time interval between the first particles and the second particles flowing through the channel; and a determination step for determining whether or not to separate the first particles and the second particles according to the time interval.

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