Method for collecting fine particles, and system for collecting fine particles

Abstract

To provide a novel method for more efficiently generating an emulsion particle including a single microparticle.SOLUTION: A microparticle recovery method includes, in a microparticle separation mechanism having a flow passage structure including a main flow passage through which a microparticle flows, a recovery flow passage in which a recovery object particle of the microparticle is recovered, a connection flow passage connecting the main flow passage and the recovery flow passage, and a liquid supply flow passage connected to the connection flow passage so as to enable supplying a liquid: a flow step of flowing a first liquid including the microparticle to the main flow passage; a determination step of determining whether the microparticle flowing through the main flow passage is the recovery object particle; and a recovery step of recovering the recovery object particle into the recovery flow passage, in which in the recover step, the recover object particle is recovered into a second liquid immiscible with the first liquid in the recovery flow passage, in a state where the recover object particle is included in the first liquid.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] This technology relates to a method for recovering fine particles, a microchip for separating fine particles, a fine particle recovery apparatus, a method for manufacturing an emulsion, and an emulsion. More specifically, this technology relates to a method for recovering fine particles in an emulsion, a microchip for separating fine particles, a fine particle recovery apparatus, a method for manufacturing an emulsion including the steps performed in the recovery method, and an emulsion. [Background technology]

[0002] To perform single-cell analysis, it is being considered to use emulsions in which each emulsion particle contains a single cell. Several techniques for forming such emulsions have been developed to date.

[0003] For example, Non-Patent Document 1 below describes a method for randomly capturing particles in an emulsion. In this method, the cell-containing solution is diluted to ~100 cells / μl or less so that each emulsion contains one cell. In this method, the efficiency of generating emulsion particles containing one cell follows a Poisson distribution, resulting in a high proportion of empty emulsion particles (emulsion particles that do not contain cells), making it inefficient. Therefore, Non-Patent Document 1 below proposes sorting the emulsion by controlling the particles or fluid, for example, by an electric field, dielectrophoresis, or local heating, in order to increase the proportion of cell-containing emulsion particles to empty emulsion particles. Furthermore, to increase the proportion of single-cell-containing emulsion particles in the generated emulsion, it has been proposed, for example, to utilize the Plateau-Rayleigh instability induced by cells, as described in Non-Patent Document 1 below, or to utilize the self-assembly that occurs when a high-density suspension is rapidly passed through a microchannel. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Agata Rakszewska et al., One drop at a time: toward droplet microfluidics as a versatile tool for single-cell analysis, NPG Asia Materials (2014) 6, e133 [Non-Patent Document 2] Linas Mazutis et al., Single-cell analysis and sorting using droplet-based microfluidic, Nat Protoc. 2013 May; 8(5): 870-891 [Overview of the project] [Problems that the invention aims to solve]

[0005] To perform single-cell analysis using emulsions, it is desirable to increase the proportion of emulsion particles containing a single cell within the emulsion. This technology aims to provide a novel method for more efficiently generating emulsion particles containing a single microparticle. [Means for solving the problem]

[0006] The inventors of this invention have found that the above problem can be solved by a specific method for recovering fine particles. In other words, this technology involves a main channel through which fine particles are passed, A collection channel from which the particles to be collected are collected, A connecting channel that connects the main channel and the recovery channel, A fine particle sorting mechanism having a flow channel structure including a liquid supply channel connected to the aforementioned connecting channel so as to be able to supply liquid, comprising a flow step of flowing a first liquid containing fine particles into the main channel, A determination step of determining whether the fine particles flowing through the main channel are particles to be collected, The process includes a recovery step of recovering the particles to be recovered into the recovery channel, and In the recovery process, the particles to be recovered are recovered in a second liquid that is immiscible with the first liquid in the recovery channel, while still contained in the first liquid. This invention provides a method for collecting fine particles. By carrying out the above-described fine particle recovery method, an emulsion can be formed in the recovery channel in which the second liquid is used as the dispersion medium and the first liquid is used as the dispersed phase. By carrying out the above-described fine particle recovery method, an emulsion is formed, and at least a portion of the droplets constituting the emulsion may contain one of the particles to be recovered. The first liquid may be hydrophilic and the second liquid may be hydrophobic. The kinematic viscosity of the second liquid may be 1 / 100 to 100 times that of the first liquid. The flow channel step, the determination step, and the recovery step may be performed while supplying the second liquid from the liquid supply channel to the connecting channel. The main channel may branch off to the connecting channel and at least one waste channel through which fine particles other than the particles to be recovered flow. The liquid supply channel can supply liquid to the connecting channel. The connecting channel may be provided with a valve to prevent the first liquid from proceeding to the recovery channel. In the flow process, the fine particles can flow through the main channel in a substantially straight line toward the connecting channel. The aforementioned fine particle sorting mechanism may have a flow path structure in which a sample flow path through which a liquid containing fine particles flows and a sheath flow path through which a liquid not containing fine particles flows are connected to a main flow path at a confluence, and fine particles flow in the main flow path after the confluence in a substantially straight line. This channel structure can create a laminar flow containing minute particles flowing in approximately a single line. In the determination step, light is irradiated onto the fine particles flowing through the main channel, and it can be determined whether the fine particles are to be recovered based on the light generated by the irradiation. In the recovery step, due to the pressure fluctuation in the recovery flow path, the particles to be recovered can be recovered into the recovery flow path through the connection flow path. The main flow path, the connection flow path, and the recovery flow path may be arranged linearly side by side. The microparticles may be cells or cell aggregates, and the first liquid may be a culture solution of the microparticles. The microparticles may be cells, cell aggregates, or synthetic particles, and the microparticles may be destroyed after the recovery step. The recovered microparticles in the recovery flow path may be subjected to further microparticle separation processing. The microchip for microparticle separation may include one or more of the flow path structures.

[0007] Further, the present technology includes a main flow path through which microparticles flow, a recovery flow path in which particles to be recovered among the microparticles are recovered, a connection flow path connecting the main flow path and the recovery flow path, and a liquid supply flow path connected to the connection flow path so as to be able to supply a liquid, and has a flow path structure including the above, The main flow path has a determination region used for determining whether the microparticles flowing in the first liquid are particles to be recovered, The microparticles determined to be particles to be recovered are recovered in a second liquid that is immiscible with the first liquid in the recovery flow path while being contained in the first liquid. A microchip for microparticle separation is also provided.

[0008] Further, the present technology includes a main flow path through which microparticles flow, a recovery flow path in which particles to be recovered among the microparticles are recovered, a connection flow path connecting the main flow path and the recovery flow path, a liquid supply flow path connected to the connection flow path so as to be able to supply a liquid, a microchip for microparticle separation having a flow path structure including the above, a first liquid supply unit that supplies a first liquid containing microparticles to the main flow path, A second liquid supply unit supplies a second liquid, which is immiscible with the first liquid, to the liquid supply channel, A determination unit that determines whether the fine particles flowing in the main channel are particles to be collected, We also provide a microparticle recovery device equipped with this feature. The microchip for separating fine particles may be removable from the fine particle collection device.

[0009] Furthermore, this technology involves a main channel through which fine particles are passed, A collection channel from which the particles to be collected are collected, A connecting channel that connects the main channel and the recovery channel, A fine particle sorting mechanism having a flow channel structure including a liquid supply channel connected to the aforementioned connecting channel so as to be able to supply liquid, comprising a flow step of flowing a first liquid containing fine particles into the main channel, A determination step of determining whether the fine particles flowing through the main channel are particles to be collected, The process includes a recovery step of recovering the particles to be recovered into the recovery channel, and In the recovery process, the particles to be recovered are recovered in the first liquid while contained within the second liquid, which is immiscible with the first liquid, within the recovery channel. A method for producing an emulsion containing fine particle-containing emulsion particles is also provided.

[0010] Furthermore, this technology also provides emulsions containing microparticle-containing emulsion particles, where the proportion of emulsion particles containing a single microparticle is 70% or more of the total number of emulsion particles. [Brief explanation of the drawing]

[0011] [Figure 1] This figure shows an example configuration of a microchip used for separating fine particles in the fine particle collection method of this technology. [Figure 2] This figure shows an example of the flow chart for the microparticle recovery method using this technology. [Figure 3] This is a magnified view of an example of a particle sorting section. [Figure 4] This is a block diagram of an example of a control unit. [Figure 5] This figure shows an example configuration of a microchip for separating fine particles with a container attached. [Figure 6A] This is a magnified view of the connecting channel section. [Figure 6B] This is a magnified view of the connecting channel section. [Figure 7A] This is a magnified view of the connecting channel section. [Figure 7B] This is a magnified view of the connecting channel section. [Figure 8A] This photograph shows the formation of emulsion particles containing a single microparticle within the recovery channel. [Figure 8B] This photograph shows that emulsion particles of different sizes are formed under different driving conditions for a piezoelectric element. [Figure 9] This is a schematic diagram of an example of a microchip for separating fine particles. [Figure 10] This is a schematic diagram of an example of a microchip for separating fine particles. [Figure 11] This is a schematic diagram of an example of a microchip for separating fine particles. [Modes for carrying out the invention]

[0012] The following describes preferred embodiments for implementing this technology. The embodiments described below are representative examples of this technology, and the scope of this technology is not limited to these embodiments. The description of this technology will proceed in the following order. 1. First Embodiment (Method for Collecting Fine Particles) (1) Description of the first embodiment (2) A first example of the first embodiment (2-1) Flow process (2-2) Judgment process (2-3) Recovery process (2-4) Other processes (2-4-1) Culture process (2-4-2) Destruction Process (2-4-3) Detection process (2-4-4) Synthesis process (2-5) Mechanism for separating fine particles and fine particles (3) Other examples of flow channel structures (3-1) A flow channel structure in which the main flow channel and the waste flow channel are aligned in a straight line. (3-2) Flow channel structure having multiple recovery channels 2. Second Embodiment (Microchip for Microparticle Separation) 3. Third Embodiment (Fine Particle Recovery Device) 4. Fourth Embodiment (Method for Producing Emulsion) 5. Fifth Embodiment (Emulsion)

[0013] 1. First Embodiment (Method for Collecting Fine Particles)

[0014] (1) Description of the first embodiment

[0015] The fine particle recovery method of this technology is carried out using a fine particle sorting mechanism having a flow channel structure that includes a main flow channel through which fine particles are passed, a recovery flow channel for which particles to be recovered are recovered 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. The fine particle recovery method of this technology includes a flow step of passing a first liquid containing fine particles through the main flow channel in the fine particle sorting mechanism, a determination step of determining whether the fine particles flowing through the main flow channel are particles to be recovered, and a recovery step of recovering the particles to be recovered into the recovery flow channel, wherein in the recovery step, the particles to be recovered are recovered in the first liquid and into a second liquid in the recovery flow channel that is immiscible with the first liquid.

[0016] This technology allows the particles to be recovered to be collected in a second liquid that is immiscible with the first liquid in the recovery channel, while still contained within the first liquid. This makes it possible to form, for example, emulsion particles containing the particles to be recovered within the recovery channel. Furthermore, in this technology, the fine particles recovered into the recovery channel are those determined to be recovery targets in the determination step, and the recovery operation is performed at an appropriate timing. Moreover, since no recovery operation is performed when no fine particles flow in or when fine particles that have been determined not to be recovery targets in the determination step arrive, emulsion particles that do not contain fine particles or emulsion particles that contain particles other than recovery targets are not formed in the recovery channel. Therefore, the probability that one recovery target particle is included in the emulsion particles is extremely high. For example, by following the method of this technology, emulsion particles containing one fine particle (particularly a recovery target particle) can be produced with a success rate of, for example, 70% or more, particularly 80% or more, even more particularly 90% or more, and even 95% or more. In this technology, recovery target particles refer to fine particles that have been determined to be recovered in the determination step.

[0017] For single-cell analysis using emulsions, it is important to increase the proportion of single-cell emulsion particles in the emulsion. This is especially important when performing single-cell analysis on samples with a small number of cells. However, as described in Non-Patent Document 2 above, for example, the probability of a certain number of cells being present in a single emulsion particle is thought to follow a Poisson distribution. In conventional emulsion formation techniques, the probability of one cell being present in a single emulsion particle is said to be at most about 65%. Furthermore, to increase this ratio, one could consider increasing the number of cells in the sample. However, the number of cells to be analyzed in a clinical sample is often small; for example, the number of cells to be analyzed in one sample is, for example, 10 4 ~10 5 This is possible. Furthermore, when analyzing rare cells such as CTCs (circulating tumor cells), the number of available cells is limited. As described above, this technology makes it possible to increase the proportion of emulsion particles containing a single microparticle (e.g., a cell). Therefore, this technology is extremely effective for single-cell analysis using emulsions.

[0018] Furthermore, in single-cell analysis using an emulsion containing one cell per emulsion particle, the target cell population for single-cell analysis is generally sorted and purified using a cell sorting device, such as a cell sorter, before the emulsion is formed. Therefore, in order to perform this single-cell analysis, a cell sorter is required in addition to the emulsion formation device. Increasing the number of devices used is undesirable, for example, from a cost perspective. Also, in order to form the emulsion, a cell sorting step is required in addition to the emulsion formation step. Increasing the number of steps is undesirable, for example, from a time and cost perspective. While there is a technique that uses a cell sorter to sort cells into single cells in a well plate based on the detection signal, the number of wells in such a well plate is limited to 384, resulting in low analytical scale and throughput. Furthermore, sorting cannot occur while the sorting nozzle is moving between wells, so target cells that pass through during this time are lost. In this technology, the fine particles determined to be the particles to be recovered in the determination step can be recovered in the form of an emulsion. Therefore, this technology allows for the formation of an emulsion without the need for a separate cell sorting step. Furthermore, this technology can increase the recovery rate of the particles to be recovered.

[0019] In a preferred embodiment of this technology, an emulsion is formed by carrying out the fine particle recovery method, and at least a portion of the droplets constituting the emulsion (hereinafter also referred to as "emulsion particles") contain one of the particles to be recovered. More preferably, of the total droplets constituting the emulsion, preferably 70% or more, more preferably 80% or more, and even more preferably 90% or more contain one of the particles to be recovered. The fine particle recovery method according to this technology can form an emulsion in which the proportion of emulsion particles containing one of the particles to be recovered is high in this way. As described above, this technology also provides a method for producing an emulsion containing an emulsion particle containing a single fine particle. The steps included in this production method may be the same as those in the fine particle recovery method. Furthermore, this technology also provides an emulsion in which the proportion of emulsion particles containing a single cell is 70% or more of the total number of emulsion particles. The proportion may preferably be 75% or more, more preferably 80% or more, 85% or more, or 90% or more. In this way, this technology provides an emulsion containing a single emulsion particle at an extremely high concentration.

[0020] In the flow channel structure included in the fine particle sorting mechanism used in the method of this technology, the main flow channel and the recovery flow channel are connected via the connecting flow channel, and the liquid supply flow channel is connected to the connecting flow channel. By supplying the second liquid from the liquid supply flow channel to the connecting flow channel, it is possible to prevent the first liquid flowing through the main flow channel from entering the recovery flow channel, and to guide the first liquid into the recovery flow channel as needed. For example, by guiding the first liquid into the recovery flow channel only when the particles to be recovered reach the vicinity of the connecting flow channel, the particles to be recovered can be guided into the recovery flow channel while contained in the first liquid.

[0021] In accordance with a preferred embodiment of this technology, by implementing the microparticle recovery method according to this technology, an emulsion is formed in the recovery channel in which the second liquid is the dispersion medium and the first liquid is the dispersed phase. The emulsion formed by the microparticle recovery method according to this technology has a high proportion of emulsion particles containing one target particle to the total number of emulsion particles. Therefore, this technology can be applied even when the number of cells to be analyzed is small.

[0022] The kinematic viscosity of the second liquid is preferably 1 / 1000 to 1000 times the kinematic viscosity of the first liquid, 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. 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. 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 It 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 approximately the same. This facilitates the formation of an emulsion. 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. The first and second liquids having the above-described physical properties facilitate the formation of emulsions within the recovery channel. Furthermore, these physical properties facilitate the flow of these liquids within the microchannel.

[0023] In one embodiment of this technology, the first liquid may be a hydrophilic liquid and the second liquid may be a hydrophobic liquid. In this embodiment, an emulsion can be formed in the recovery channel in which the hydrophobic liquid is the dispersion medium and the hydrophilic liquid is the dispersed phase. For example, it is desirable for biological particles such as cells to exist in a state contained in a hydrophilic liquid such as a buffer or culture medium. Therefore, this embodiment is suitable for recovering fine particles, especially biological particles, and more particularly cells, which are desirable to exist in a hydrophilic liquid.

[0024] The hydrophilic liquid includes, for example, water and liquids miscible with water. 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, the main component refers to a component that accounts for, for example, 50% or more by mass, more particularly 60% or more by mass, more particularly 70% or more by mass, even more particularly 80% or more by mass, 85% or more by mass, or 90% or more by mass of the liquid. Examples of the hydrophilic alcohol include ethanol, methanol, propanol, and glycerin. Examples of the hydrophilic ether include tetrahydrofuran, polyethylene oxide, and 1,4-dioxane. Examples of the ketone include acetone and methyl ethyl ketone. Examples of the nitrile solvent include acetonitrile.

[0025] The hydrophilic liquid may preferably be a liquid mainly composed of water, such as water, an aqueous solution, or a water dispersion. The hydrophilic liquid may, for example, be a sheath liquid and / or a sample liquid. The hydrophilic liquid is preferably a hydrophilic liquid that does not adversely affect fine particles (e.g., biological particles, especially cells). The hydrophilic liquid may be, for example, a liquid containing biomolecules. These biomolecules may be, for example, one or more combinations selected from amino acids, peptides, and proteins. In addition, 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, which are also called poloxamers or pluronic surfactants. A more specific example of a pluronic surfactant is Pluronic (trademark) F68.

[0026] Examples of the hydrophilic liquid include, but are not limited to, culture media and buffers. The buffer is preferably a Good buffer. By using a culture medium as the hydrophilic liquid, the cells recovered as the particles to be recovered can be cultured while being retained in the emulsion particles. In addition, when the hydrophilic liquid (particularly the sheath liquid) contains a cell-stimulating component, the cells recovered as the particles to be recovered can be stimulated while being retained in the emulsion particles. Furthermore, characteristics (such as morphology) of the stimulated cells can also be observed using a microscope or the like. In addition, the hydrophilic liquid (for example, the sheath liquid or the sample liquid) may contain an assay system that enables observation of the response to cell stimulation. By means of the assay system, the response from the cells recovered as the particles to be recovered can be optically detected, for example, while being retained in the emulsion particles. The assay system is preferably a wash-free assay system, and a system utilizing, for example, fluorescence resonance energy transfer (FRET) or bioluminescence resonance energy transfer (BRET) is preferred. As described above, in the present technology, when the microparticles are biological particles (particularly cells), various analyses of single biological particles (particularly single cell analysis, such as single cell imaging, etc.) can be performed.

[0027] The density of the hydrophilic liquid at

[25] °C is, for example, 0.5 g / cm 3 ~5 g / cm 3 , preferably 0.6 g / cm 3 ~4 g / cm3 , more preferably 0.7 g / cm³ 3 ~3g / cm 3 It is possible. The kinematic viscosity of the hydrophilic 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. Because the hydrophilic liquid has the above-mentioned physical properties, it becomes easier for it to flow through the microchannel and for an emulsion to form in the recovery channel.

[0028] The hydrophobic liquid may be any liquid selected from liquids that are miscible with the hydrophilic liquid. The hydrophobic liquid may be a liquid whose main component is one or more mixtures selected from the group consisting of aliphatic hydrocarbons, fluorinated oils, low-molecular-weight or high-molecular-weight substances containing fluorine atoms, silicone oils, aromatic hydrocarbons, aliphatic monohydric alcohols (e.g., n-octanol), and fluorinated polysaccharides. The aliphatic hydrocarbon is preferably an aliphatic hydrocarbon having 7 to 30 carbon atoms. Having 7 to 30 carbon atoms makes the kinematic viscosity of the hydrophobic liquid suitable for flowing through a microchannel. Examples of aliphatic hydrocarbons include mineral oil; plant and animal-derived oils such as squalane oil and olive oil; paraffinic hydrocarbons having 10 to 20 carbon atoms such as decane and hexadecane; and olefinic hydrocarbons having 10 to 20 carbon atoms. In this technology, from the viewpoint of good miscibility with the hydrophilic liquid, the hydrophobic liquid is preferably a fluorinated oil. Examples of the fluorinated oil include perfluorocarbon (PFC), perfluoropolyether (PFPE), and hydrofluoroether (HFE). Examples of perfluorocarbons include Fluorinert® FC40 and Fluorinert FC-770 (manufactured by 3M). Examples of perfluoropolyethers include Krytox (manufactured by DuPont). Examples of hydrofluoroethers include HFE7500 (manufactured by 3M).

[0029] The density of the hydrophobic liquid at 25°C is, 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 It is possible. The kinematic viscosity of the hydrophobic 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. Because the hydrophobic liquid has the above-mentioned physical properties, an emulsion is more likely to form in the recovery channel. For example, if the density or kinematic viscosity is too high, there is a higher possibility that it will not flow smoothly in the connecting channel.

[0030] In a preferred embodiment of this technology, one or both of the first liquid and the second liquid may contain a surfactant. More particularly, one or both of the hydrophobic liquid and the hydrophilic liquid may contain a surfactant, and more particularly, the hydrophobic liquid may contain a surfactant. The surfactant facilitates the formation of emulsion particles and also helps to maintain the stability of the emulsion particles. Examples of surfactants include nonionic surfactants and fluorinated surfactants. Examples of nonionic surfactants include, but are not limited to, Span80 and Abil EM. The type of surfactant may be appropriately selected by those skilled in the art. 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.

[0031] 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 may be, for example, 1 μM to 1000 μM, and more particularly 10 μM to 100 mM. Furthermore, the interfacial tension of the surfactant may be, for example, 40 mN / m or less, and more particularly 20 mN / m or less.

[0032] In another embodiment 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 recovery channel, with a hydrophilic liquid as the dispersion medium and a hydrophobic liquid as the dispersed phase. This technique may be used to recover fine particles in the emulsion. Examples of the hydrophobic and hydrophilic liquids are as described above. Furthermore, this embodiment can be applied, for example, when the dispersion medium and dispersed phase are a hydrophobic liquid and a hydrophilic liquid, respectively, and the emulsion particles contain fine particles, and only the desired fine particles are recovered from the emulsion. Furthermore, the assay system that can be used in this technology may include not only a system in which microparticles emit fluorescence, but also a system in which emulsion particles emit fluorescence. Therefore, in order to recover emulsion particles containing microparticles, the determination step may involve determining the microparticles, determining the emulsion particles, or determining both the microparticles and the emulsion particles. Thus, in this technology, it may be determined whether microparticles or emulsion particles are to be recovered based on information obtained from microparticles and / or emulsion particles.

[0033] In a preferred embodiment of this technology, the flow step, the determination step, and the recovery step are performed while supplying the second liquid from the liquid supply channel to the connecting channel. This fills the connecting channel with the second liquid, preventing the first liquid from unnecessarily flowing into the recovery channel.

[0034] (2) A first example of the first embodiment

[0035] The microparticle collection method of this technology is performed using a microparticle sorting mechanism. Below, an example of one embodiment of the method of this technology will be described with reference to Figure 1, which shows a microparticle sorting microchip that is an example of the configuration of the microparticle sorting mechanism used in the microparticle collection method of this technology, and Figure 2, which shows an example of the flow of the microparticle collection method of this technology.

[0036] As shown in Figure 1, the microchip 150 for separating fine particles used in the method of this technology includes a main channel 155 through which fine particles flow and a recovery channel 159 through which particles to be recovered are recovered. The microchip 150 for separating fine particles is provided with a particle separation section 157. 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 recovery channel 159. A liquid supply channel 161 capable of supplying liquid to the connecting channel 170 is connected to the connecting channel 170. As described above, the microchip 150 for separating fine particles has a flow channel structure including the main channel 155, the recovery channel 159, the connecting channel 170, and the liquid supply channel 161.

[0037] Furthermore, as shown in Figure 1, the microchip 150 for separating fine particles constitutes part of the fine particle collection device 100, which includes, in addition to the microchip itself, a light irradiation unit 101, a detection unit 102, and a control unit 103. The control unit 103 may include a signal processing unit 104, a determination unit 105, and a separation control unit 106, as shown in Figure 4.

[0038] As shown in Figure 2, the method of this technology includes a flow step S101 in which a first liquid containing fine particles is flowed into a main channel 155 in a microchip 150 for separating fine particles, a determination step S102 in which it is determined whether the fine particles flowing in the main channel 155 are particles to be recovered, and a recovery step S103 in which the particles to be recovered are recovered into a recovery channel 159. The following explains each step.

[0039] (2-1) Flow process

[0040] In the flow passage process S101, a first liquid containing fine particles is passed through the main flow passage 155. The first liquid flows through the main flow passage 155 from the confluence section 162 toward the particle separation section 157. The first liquid may be a laminar flow formed from a sample liquid containing fine particles and a sheath liquid, and in particular, a laminar flow in which the sample liquid is surrounded by the sheath liquid. The flow path structure for forming the laminar flow will be described below.

[0041] The microchip 150 for separating fine particles is equipped with a sample liquid inlet 151 and a sheath liquid inlet 153. A sample liquid containing fine particles and a sheath liquid that does not contain fine particles are introduced from these inlets into the sample liquid channel 152 and the sheath liquid channel 154, respectively.

[0042] The microchip 150 for separating fine particles has a flow channel structure in which the sample channel 142 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 fine particles are arranged in a substantially straight line in the laminar flow. Thus, in this technology, the flow channel structure forms a laminar flow containing fine particles flowing in a substantially straight line.

[0043] The laminar flow flows through the main channel 155 toward the particle sorting section 157. Preferably, the fine particles flow in a single line within the main channel 155. This makes it easier to distinguish between the light generated by the irradiation of one fine particle and the light generated by the irradiation of other fine particles during the light irradiation in the detection region 156 described below.

[0044] (2-2) Judgment process

[0045] In the determination step S102, it is determined whether the fine particles flowing through the main channel 155 are particles to be recovered. This determination may be performed by the determination unit 105. The determination unit 105 may perform this determination based on the light generated by the light irradiation unit 101 irradiating the fine particles with light. An example of the determination step S102 will be described in more detail below.

[0046] In the determination step S102, the light irradiation unit 101 irradiates light (e.g., excitation light) onto the fine particles flowing through the main channel 155 (particularly the detection region 156) in the microparticle sorting microchip 150, and the detection unit 102 detects the light generated by the light irradiation. Based on the characteristics of the light detected by the detection unit 102, the determination unit 105 included in the control unit 103 determines whether the fine particles are to be recovered. For example, the determination unit 105 can perform a determination based on scattered light, a determination based on fluorescence, or a determination based on an image (e.g., a dark-field image and / or a bright-field image). In the recovery step S103 described later, the control unit 103 controls the flow in the microparticle sorting microchip 150 so that the particles to be recovered are recovered into the recovery channel 159.

[0047] The light irradiation unit 101 irradiates the microparticles flowing in the channel within the microchip 150 for microparticle sorting with light (e.g., excitation light). The light irradiation unit 101 may include a light source that emits light and an objective lens that focuses the excitation light onto the microparticles flowing in the detection area. The light source may be appropriately selected by those skilled in the art depending on the purpose of the analysis, and may be, for example, a laser diode, SHG laser, solid-state laser, gas laser, high-brightness LED, or halogen lamp, or a combination of two or more of these. In addition to the light source and objective lens, the light irradiation unit may include other optical elements as needed.

[0048] (Discrimination of objects to be sorted based on fluorescence signals and / or scattered light signals)

[0049] In one embodiment of this technology, the detection unit 102 detects scattered light and / or fluorescence generated from the minute particles by light irradiation by the light irradiation unit 101. The detection unit 102 may include a focusing lens and a detector for focusing the fluorescence and / or scattered light generated from the minute particles. The detector may be, but is not limited to, a PMT, a photodiode, a CCD, and a CMOS. In addition to the focusing lens and the detector, 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, a prism, and an optical filter. 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.

[0050] The signal processing unit 104 included in the control 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 determination by the determination unit 105. As information regarding the characteristics of the light, the signal processing unit 104 can 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 signal processing unit 104 described above can be performed in an embodiment in which scattered light and / or fluorescence are detected.

[0051] The determination unit 105 included in the control unit 103 determines whether the fine particles flowing in the channel are particles to be recovered, based on the light generated by the irradiation of the fine particles with light. In the embodiment in which scattered light and / or fluorescence are detected, the waveform of the digital electrical signal obtained by the detection unit 102 is processed by the control unit 103, and the determination unit 105 determines whether the fine particle is a particle to be recovered based on the information regarding the characteristics of the light generated by the processing. For example, in the determination based on scattered light, the external shape and / or internal structure characteristics of the fine particle may be identified, and it may be determined whether the fine particle is a particle to be recovered based on these characteristics. Furthermore, by pre-treating the fine particle, such as a cell, it is also possible to determine whether the fine particle is a particle to be recovered based on characteristics similar to those used in flow cytometry. In addition, by labeling the fine particle, such as a cell, with an antibody or dye (particularly a fluorescent dye), it is also possible to determine whether the fine particle is a particle to be recovered based on the characteristics of the surface antigen of the fine particle.

[0052] (Discrimination of target objects based on bright-field images)

[0053] In another embodiment of this technology, the detection unit 102 may acquire a bright-field image generated by light irradiation by the light irradiation unit 101. In this embodiment, the light 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 minute particles by a halogen lamp, and a bright-field image of the irradiated minute particles may be acquired by the CCD or CMOS.

[0054] In the embodiment in which the bright-field image is acquired, the determination unit 105 included in the control unit 103 determines whether the microparticles are to be recovered based on the acquired bright-field image. For example, whether the microparticles (especially cells) are to be recovered can be determined based on one or more combinations of the morphology, size, and color of the microparticles.

[0055] (Target sorting based on dark-field imaging)

[0056] In yet another embodiment of this technology, the detection unit 102 may acquire a dark-field image generated by light irradiation by the light irradiation unit 101. In this embodiment, the light 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 minute particles by a laser, and the CCD or CMOS may acquire a dark-field image (e.g., a fluorescence image) of the irradiated minute particles.

[0057] In the embodiment in which the dark-field image is acquired, the determination unit 105 included in the control unit 103 determines whether the microparticles are to be collected based on the acquired dark-field image. For example, whether the microparticles (especially cells) are to be collected can be determined based on one or more combinations of the morphology, size, and color of the microparticles.

[0058] In any of the above-mentioned methods of "discrimination of target to be collected based on fluorescence signals and / or scattered light signals," "discrimination of target to be collected based on bright-field images," and "discrimination of target to be collected based on dark-field images," the detection unit 102 may be 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 operate as a so-called AI sensor. The detection unit 102 including the image sensor can determine, for example, whether a minute particle is a target particle to be collected based on a learning model. Furthermore, the learning model may be updated in real time while the method according to this technology 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. As an example of an image sensor operating as an AI sensor, for example, the imaging device described in International Publication No. 2018 / 051809 can be cited. When using an AI sensor as an image sensor, the raw data acquired from the image array is used directly for learning, resulting in faster sorting and discrimination processing.

[0059] The determination may be made, for example, by 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.

[0060] One beam of light may be shone at one location within the detection area 156, or light may be shone at each of multiple locations within the detection area 156. For example, the microchip 150 may be configured such that light is shone at each of two different locations within the detection area 156 (i.e., there are two locations within the detection area 156 where light is shone). In this case, for example, it may be determined whether a microparticle is a particle to be collected based on the light (e.g., fluorescence and / or scattered light) produced by the light shone at one location. Furthermore, the velocity of the microparticle in the flow path can be calculated based on the difference between the detection time of the light produced by the light shone at the one location and the detection time of the light produced by the light shone at the other location. For this calculation, the distance between the two shone locations may be determined in advance, and the velocity of the microparticle can be determined based on the difference between the two detection times and the distance. Furthermore, based on this velocity, the arrival time at the particle sorting unit 157 described below can be accurately predicted. By accurately predicting the arrival time, the timing of the flow formation entering the recovery channel 159 can be optimized. Furthermore, if the difference between the arrival time of a certain microparticle at the particle sorting unit 157 and the arrival time of a microparticle preceding or succeeding that microparticle is below a predetermined threshold, it can be determined that the microparticle should not be recovered. When the distance between the microparticle and the microparticles preceding or succeeding it is small, the likelihood of the preceding or succeeding microparticles being recovered together with the microparticle increases during the aspiration of the microparticle. By determining that the microparticle should not be recovered when there is a high probability of them being recovered together, the recovery of the preceding or succeeding microparticles can be prevented. This makes it possible to increase the purity of the target microparticles among the recovered microparticles. Specific examples of a microchip in which light is irradiated at two different positions in the detection region 156 and an apparatus including the microchip are described, for example, in Japanese Patent Application Publication No. 2014-202573.

[0061] The control unit 103 may also control light irradiation by the light irradiation unit 101 and / or light detection by the detection unit 102. Furthermore, the control unit 103 may control the drive of a pump for supplying fluid into the microchip 150 for fine particle sorting. The control unit 103 may consist of, for example, a hard disk, CPU, and memory containing a program and OS for causing the fine particle recovery device to execute the fine particle recovery method according to this technology. For example, the functions of the control 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 (not shown) provided in the fine particle recovery device 100 may read the program recorded on the recording medium, and the control unit 103 may cause the fine particle recovery device 100 to execute the fine particle recovery method according to this technology in accordance with the read program.

[0062] (2-3) Recovery process

[0063] In the recovery step S103, the fine particles determined to be the particles to be recovered in the determination step S102 are recovered into the recovery channel 159. In the recovery step S103, the particles to be recovered are recovered into a second liquid that is immiscible with the first liquid in the recovery channel while still contained in the first liquid. This allows for the formation of an emulsion in the recovery channel 159 with the second liquid as the dispersion medium and the first liquid as the dispersed phase, and each emulsion particle in the emulsion contains one particle to be recovered. Therefore, this emulsion is suitable for single-cell analysis. The recovery process will be explained in more detail below.

[0064] The recovery process S103 is performed in the particle sorting section 157 of the microchip 150. In the particle sorting section 157, the laminar flow that has flowed through the main channel 155 splits and flows into two waste channels 158. The particle sorting section 157 shown in Figure 1 has two waste channels 158, but the number of branch channels is not limited to two. The particle sorting section 157 may be provided with, for example, 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.

[0065] In the particle sorting section 157, a flow is formed from the main channel 155 through the connecting channel 170 to the recovery channel 159 only when particles to be recovered are flowing in, and the particles to be recovered are recovered into the recovery channel 159. An enlarged view of the particle sorting section 157 is shown in Figure 3. As shown in Figure 3A, the main channel 155 and the recovery channel 159 are connected via the connecting channel 170, which is coaxial with the main channel 155. As shown in Figure 3B, the particles to be recovered flow through the connecting channel 170 to the recovery channel 159. Fine particles that are not to be recovered flow to the waste channel 158, as shown in Figure 3C.

[0066] Enlarged views of the vicinity of the connecting channel 170 are shown in Figures 6A and 6B. Figure 6A is a schematic perspective view of the vicinity of the connecting channel 170. Figure 7B 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 area 156 side (hereinafter also referred to as the upstream connecting channel 170a), a channel 170b on the recovery 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 6A and 6B, 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 may be provided.

[0067] 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 6A and 6B, 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 or rectangle) with the same dimensions.

[0068] From the two liquid supply channels 161, a second liquid is supplied to the connecting channel 170 as shown by the arrows in Figure 6B. This second liquid flows from the connection part 170c to both the upstream connecting channel 170a and the downstream connecting channel 170b.

[0069] If the recovery process is not performed, the second liquid will flow as follows. The second liquid that flows into the upstream connecting channel 170a exits from 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 fine particles, which do not need to be recovered into the recovery channel 159, from entering the recovery channel 159 through the connecting channel 170. The second liquid that flows into the downstream connecting channel 170b flows into the recovery channel 159. As a result, the recovery channel 159 is filled with the second liquid, which then acts as a dispersion medium for emulsion formation, for example.

[0070] Even when the recovery process 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 recovery channel 159, particularly by generating negative pressure within the recovery channel 159, create a flow from the main channel 155 through the connecting channel 170 to the recovery channel 159. That is, a flow is formed 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 recovery channel 159. As a result, the particles to be recovered are recovered into the second liquid in the recovery channel 159 while encased in the first liquid. By performing this recovery process, an emulsion, for example, may be formed within the recovery channel 159 or in a container connected to the end of the recovery channel 163, for example, via a channel.

[0071] The cross-sectional shape and / or dimensions of the upstream connecting channel 120a may differ from those of the downstream connecting channel 120b. Examples of these two channels with different dimensions are shown in Figures 7A and 7B. As shown in Figures 7A and 7B, 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 recovery 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 recovered particles already separated into the recovery channel 159 from being released into the main channel 155 through the connecting channel 180 immediately after the negative pressure-induced particle separation operation 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 already collected fine particles from being released into the main channel 155 through the connecting channel 180, as described above.

[0072] In the recovery process S103, the particles to be recovered are recovered into the recovery channel through the connecting channel due to pressure fluctuations within the recovery channel 159. This recovery may be performed, for example, by generating negative pressure within the recovery channel 159, as described above. This negative pressure can be generated by, for example, an actuator 107 (particularly a piezo actuator) attached to the outside of the microchip 150, which deforms the wall defining the recovery channel 159. This negative pressure can form the flow entering the recovery channel 159. To generate this negative pressure, the actuator 107 can be attached to the outside of the microchip 150 so as to be able to deform the wall of the recovery channel 159. This deformation of the wall changes the internal space of the recovery channel 159, which can generate negative pressure. The actuator 107 may be, for example, a piezo actuator. When the particles to be recovered are drawn into the recovery 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 recovery channel 159. In this way, the particles to be recovered are separated in the particle separation unit 157 and recovered into the recovery channel 159.

[0073] The particles to be recovered are encapsulated in the first liquid and then recovered into a second liquid, which is immiscible with the first liquid, within the recovery channel 159. As a result, as described above, an emulsion is formed within the recovery channel 159, with the second liquid as the dispersion medium and the first liquid as the dispersed phase.

[0074] To prevent fine particles that are not to be recovered from entering the recovery 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, preventing fine particles other than those to be recovered from entering the recovery 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, just like the first liquid, without flowing through the main channel 155, due to the flow of the first liquid flowing through the main channel 155 toward the waste channel 158. Furthermore, any remaining second liquid introduced into the connecting channel 170 flows into the recovery channel 159. As a result, the recovery channel 159 can be filled with the second liquid.

[0075] The recovery channel 159 may be filled with a second liquid that is immiscible with the first liquid. In order to fill the recovery 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 recovery channel 159, thereby filling the recovery channel 159 with the second liquid.

[0076] The laminar flow that flows into the waste channel 158 can be discharged outside the microchip at the end of the waste channel 160. Furthermore, the particles to be recovered that are collected into the recovery channel 159 can be discharged outside the microchip at the end of the recovery channel 161.

[0077] A container 171 may be connected to the end 163 of the recovery channel via a channel such as a tube 172, as shown in Figure 5. As shown in the same figure, an emulsion in which the first liquid containing the particles to be recovered is used as the dispersion phase and the second liquid as the dispersion medium is recovered into the container 171. Thus, according to one embodiment of this technology, the fine particle recovery device 100 may be equipped with a channel for recovering the emulsion containing the particles to be recovered into a container. Furthermore, by closing the end 163 of the recovery channel and performing the recovery operation, multiple emulsion particles can be retained within the recovery channel 159. After the completion of the recovery operation, assays such as single-cell analysis can be performed continuously within the recovery channel 159.

[0078] As described above, in this technology, the main channel may branch into the connecting channel and the at least one waste channel. The at least one waste channel is a channel through which fine particles other than the particles to be recovered flow.

[0079] Furthermore, as shown in Figures 1 and 2, in the microchip for separating fine particles used in the method of this technology, the main channel, the connecting channel, and the recovery channel may be arranged in a straight line. When these three channels are arranged in a straight line (particularly coaxially), the recovery process can be performed more efficiently compared to, for example, when the connecting channel and the recovery channel are arranged at an angle to the main channel. For example, the amount of suction required to guide the particles to be recovered into the connecting channel can be reduced. Furthermore, as shown in Figures 1 and 2, in the microchip for separating fine particles used in this technology, the fine particles are arranged in approximately a single line within the main channel and flow toward the connecting channel. Therefore, the amount of material collected during the recovery process can be reduced.

[0080] Furthermore, in the method of this technology, the liquid supply channel supplies liquid (particularly a second liquid) to the connecting channel. This creates a flow within the connecting channel that flows from the connection point between the liquid supply channel and the connecting channel toward the main channel, preventing the liquid flowing in the main channel from entering the connecting channel and preventing fine particles other than the target particles from flowing through the connecting channel into the recovery channel. When performing the recovery step, as described above, for example, the negative pressure generated in the recovery channel causes the first liquid containing one target particle to be recovered through the connecting channel and into the second liquid in the recovery channel. As a result, emulsion particles containing one target particle are formed in the second liquid.

[0081] Furthermore, in this technology, when a microparticle determined to be a particle to be recovered in the determination step is driven, for example, by driving a piezo actuator at an appropriate timing (for example, when it reaches the particle sorting unit 157), the hydrophilic solution containing the particle to be recovered is recovered into the recovery channel 159 and emulsion particles are formed. In the determination step, for example, by using peak signals and area signals to determine whether a particle is a particle to be recovered, it is also possible to determine whether it is a single microparticle (singlet), a doublet of two microparticles combined, or a triplet of three microparticles combined. Therefore, it is possible to avoid the formation of emulsion particles containing two or more microparticles in a single emulsion particle. As a result, emulsion particles containing one microparticle can be formed with high probability and high efficiency. In addition, since it is possible to avoid the formation of emulsion particles containing two or more combined microparticles, it is possible to omit the operation of removing combined microparticles of two or more particles before the emulsion formation operation, for example, by using a cell sorter. Conversely, it is also possible to determine the characteristics of two or more microparticles that are close enough to be simultaneously drawn into the connecting channel in a single retrieval operation. For example, two microparticles with identical characteristics can be confined within a single emulsion particle, or a combination of microparticles with specified different characteristics can be confined within a single emulsion particle.

[0082] (Examples) Using a microchip for microparticle sorting having the same channel structure as the microchip 150 for microparticle sorting shown in Figure 1, emulsion particles containing one microparticle were formed as follows. The procedure for forming these emulsion particles will be described below with reference to Figure 1.

[0083] The microchip 150 for separating fine particles was equipped with a piezoelectric element (piezo actuator) on its outer surface (particularly on the outer surface corresponding to the bulging portion of the collection channel 159 that is close to the connection point with the connecting channel 170) so that the volume within the collection channel 159 could be varied. A hydrophilic sample solution containing beads with a diameter of 10 μm was introduced from the sample solution inlet 151 into the sample solution channel 152, and a hydrophilic sheath solution was introduced from the sheath solution inlet 153 into the sheath solution channel 154. The introduced hydrophilic sample solution and hydrophilic sheath solution merged at the confluence 162, and a laminar flow was formed in which the hydrophilic sample solution was surrounded by the hydrophilic sheath solution. This laminar flow contained beads arranged in approximately a single line, and the laminar flow flowed through the main channel 155 toward the connecting channel 170. In parallel with the introduction of the hydrophilic sample liquid and the hydrophilic sheath liquid, a hydrophobic liquid was supplied from the liquid supply channel 161 to the connecting channel 170. The supply of the hydrophobic liquid from the liquid supply channel 161 to the connecting channel 170 prevented the laminar flow from entering the recovery channel 159 through the connecting channel 170, and the recovery channel 159 was filled with the hydrophobic liquid. When a bead passes through the irradiation position of the laser beam that is irradiated into the detection region 156 of the main channel 155, the laser beam irradiates the bead and generates light. This generated light is detected, and based on the characteristics of the detected light, it is determined whether to collect each bead.

[0084] When a bead determined to be recovered reaches the vicinity of the connecting channel 170, the piezoelectric element is activated, deforming the lumen of the recovery channel 159, thereby recovering the bead through the connecting channel 170 into the recovery channel 159. The recovery operation was as follows: (i) deform the recovery channel 159 over 10 microseconds to create negative pressure inside the recovery channel 159, (ii) maintain the deformed state for 10 microseconds, and (iii) release the negative pressure over 10 microseconds to return the deformation to its original state. By repeating operations (i) to (iii) above, an emulsion containing emulsion particles with one bead in the hydrophobic liquid was formed inside the recovery channel 159. This emulsion used the hydrophobic liquid as the dispersion medium and the emulsion particles containing the hydrophilic liquid as the dispersed phase.

[0085] As shown in Figure 8A, by performing the operations (i) to (iii) above, an emulsion particle containing one bead is formed in the recovery channel 159. Figure 8A will be explained below. Figure 8A(a) is a photograph showing beads (indicated by white arrows) flowing toward the connecting channel 170. At the point shown in Figure 8A, the hydrophilic liquid flows through the main channel 155 toward the particle sorting section 157, and then flows into the two waste channels 158 that branch off from the main channel 155, without flowing into the connecting channel 170. The hydrophobic liquid is supplied from the liquid supply channel 161 to the connecting channel 170 and flows into both the main channel 155 and the recovery channel 159. The hydrophobic liquid that flowed into the main channel 155 is then separated and flows into the two waste channels 158 immediately after leaving the connecting channel 170, due to the flow of the hydrophilic liquid (flowing along the wall of the waste channel 158 in the vicinity indicated by a in Figure 8A). Figure 8A(b) is a photograph taken when the beads are closer to the connecting channel. At this point, the operation for bead retrieval is initiated. That is, the deformation of the lumen of the retrieval channel 159 is initiated by driving the piezoelectric element. Figure 8A(c) is a photograph showing the state in which the hydrophilic liquid is moving into the recovery channel 159. From this photograph, it can be seen that at the time of Figure 8C, the bead is surrounded by the hydrophilic liquid, and that hydrophilic liquid is further surrounded by the hydrophobic liquid. In other words, an emulsion particle P containing one bead has been formed. Figure 8A(d) is a photograph taken just before the emulsion particles P enter the recovery channel 159 from the connecting channel 170. Figure 8A(e) is a photograph taken immediately after the emulsion particle P entered the recovery channel 159. It can be seen that the emulsion particle P contains one bead. Figure 8A(f) is a photograph showing that the emulsion particles P continued to flow downstream within the ocean current channel 159. As described above, emulsion particles containing one bead were formed by the method according to this technology.

[0086] The size of the emulsion particles can be adjusted by controlling the time and deformation amount of each of the operations (i) to (iii) described above. In other words, the size of the emulsion particles in the recovery channel can be controlled by the channel volume as the recovery channel deforms and expands. Therefore, it can be easily controlled by the deformation amount of the piezoelectric element, i.e., by the electrical drive waveform of the piezoelectric element. For example, by increasing the amount of deformation of the lumen of the recovery channel by the piezoelectric element, the amount of hydrophilic liquid drawn into the recovery channel 159 increases, thereby increasing the size of the emulsion particles. Conversely, by decreasing the amount of deformation, the amount of hydrophilic liquid drawn into the recovery channel 159 decreases, thereby decreasing the size of the emulsion particles. Furthermore, the size of the emulsion particles can be made larger or smaller by making the retention time in (ii) above longer or shorter. This is shown in Figure 8B. Figure 8B is a photograph showing the size of the emulsion particles when the retention time in (ii) above is 10 μs, 15 μs, 25 μs, and 35 μs. From this photograph, it can be seen that the size of the emulsion particles can be adjusted by changing the retention time in (ii) above. Furthermore, the drawing of emulsion particles can also be adjusted by adjusting the volume of the connecting channel 170 (particularly the upstream connecting channel 170a, the connection section 170c, and the downstream connecting channel 170b).

[0087] In the example described above, supplying a second liquid from the liquid supply channel to the connecting channel prevents the first liquid flowing through the main channel from entering the connecting channel. In this technology, in order to prevent the first liquid flowing through the main channel from entering the connecting channel, a valve may be provided in the connecting channel to prevent the first liquid from proceeding to the recovery channel. Only when the recovery process is performed, the valve is opened, allowing the first liquid to flow through the connecting channel to the recovery channel.

[0088] (2-4) Other processes

[0089] The fine particle recovery method of this technology may include other steps in addition to the flow path, determination step, and recovery step described above. Examples of such other steps are described below.

[0090] (2-4-1) Culture process

[0091] In one preferred embodiment of this technology, the microparticles are cells or cell aggregates, and the first liquid is a culture medium for the microparticles. In this embodiment, an emulsion can be formed in which the culture medium is the dispersed phase and the second liquid, which is immiscible with the culture medium, is the dispersion medium. Each emulsion particle formed by the dispersed phase may contain one cell or cell aggregate. This allows one cell or cell aggregate to be cultured within each emulsion particle. As described above, in this embodiment, the microparticle recovery method may further include a culture step in which the recovered particles (i.e., cells or cell aggregates) recovered in the recovery step are cultured in emulsion particles formed from the culture medium. The type of culture medium can be appropriately selected by those skilled in the art depending on the cells to be cultured. This embodiment is suitable, for example, when it is required to culture each cell or cell aggregate separately in single-cell analysis.

[0092] (2-4-2) Destruction Process

[0093] In one preferred embodiment of this technology, the fine particles are cells, cell aggregates, or synthetic particles, and the fine particles may be destroyed after the recovery step. In this embodiment, an emulsion is formed by the recovery step, and each cell, cell aggregate, or synthetic particle within the emulsion particles constituting the emulsion may be destroyed. As described above, the fine particle recovery method of this technology may include a destruction step after the recovery step in which the recovered fine particles are destroyed. In other preferred embodiments of this technology, particles other than cells, cell aggregates, and synthetic particles may be destroyed. That is, the microparticle recovery method of this technology may include a destruction step after the recovery step in which the recovered particles are destroyed.

[0094] The disruption can preferably be carried out while maintaining the emulsion particles. This releases, for example, cellular components (e.g., intracellular components, cell membrane components, and cell wall components) or components of the synthetic particles into the emulsion particles, allowing these cellular components or components to be processed separately from the components of other microparticles. Such processing may include, for example, analysis, separation, or amplification of the cellular components or components. Examples of such cellular components or components include, but are not limited to, DNA, RNA, proteins, peptides, amino acids, lipids, sugars, or intracellular organelles. For example, if the cellular component or component is DNA, a sample for DNA barcoding can be prepared by the disruption step, or by further processing of the emulsion particles obtained by the disruption step.

[0095] (2-4-3) Detection process

[0096] According to one preferred embodiment of this technology, after the recovery step, the components of the recovered particles may be detected or analyzed, or the components of the recovered particles may react with other components. For the purpose of performing the aforementioned detection or reaction, emulsion particles formed according to this technology may be merged with other emulsion particles. After such merging, the detection or analysis of components contained in the recovered particles, or the reaction between those components and other components, may be carried out within the merged emulsion particles. This merging process enables, for example, Cellular Indexing of Transcriptomes and Epitopes by Sequencing (also known as CITE-seq). For example, consider a case where the particles to be recovered are cells to which an antibody conjugated with an oligo barcode having a poly-A sequence is attached. In the recovery step, emulsion particles containing these cells are formed. Subsequently, these emulsion particles are merged with emulsion particles containing beads or gel having a barcode sequence. This merging process allows for the detection of cell surface proteins or intracellular mRNA from these cells. Furthermore, the emulsion of cells or cell aggregates (e.g., spheroids or organoids) may be reacted with the drug. This allows for the detection or analysis of the response of the cells or cell aggregates to the drug. Furthermore, biomolecules may be detected in the detection process. Biomolecules can be detected, for example, by a wash-free assay. Examples of wash-free assays include LOCI, FRET, BRET, or FlimPIA.

[0097] (2-4-4) Synthesis process

[0098] In one preferred embodiment of this technology, after the recovery step, chemical synthesis may be performed using the recovered particles. For example, this synthesis may be carried out within the emulsion particles formed in the recovery step. For example, by encapsulating a cell-free expression reagent within the emulsion particles, in vitro antibody production becomes possible. Examples of the cell-free expression reagent include linear DNA and the E. coli S30 Extract system for linear DNA (promega). Alternatively, for example, protein synthesis may be performed within the emulsion particles using a cell-free protein synthesis system.

[0099] (2-5) Mechanism for separating fine particles and fine particles

[0100] The microparticle separation mechanism used in the microparticle separation method of this technology may be a structure or device having the channel structure described above, for example, a chip having a microchannel, and in particular, a microchip for microparticle separation. In this technology, "micro" means that at least a portion of the channels included in the microchip for separating fine particles has dimensions on the order of μm, and more particularly, has cross-sectional dimensions on the order of μm. That is, in this technology, "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 particle separation section composed of channels having cross-sectional dimensions on the order of μm may be called a microchip according to this technology. For example, in the particle separation section 157, the cross-section of the main channel 155 is, for example, rectangular, and the width of the main channel 155 within the particle separation section 157 may be, 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 particularly 20 μm to 50 μm. These dimensions of the channel may be appropriately changed depending on the size of the fine particles, in particular the size of the particles to be recovered.

[0101] The microchip 150 for separating fine particles can be manufactured by methods known in the art. For example, the microchip 150 for separating biological particles 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). To facilitate adjustment of the position when bonding the substrates, it is preferable that the channels be formed on only one substrate.

[0102] Materials known in the art can be used as the material for forming the microchip 150 for separating fine particles. Examples include, but are not limited to, polycarbonate, cycloolefin polymer, polypropylene, PDMS (polydimethylsiloxane), polymethyl methacrylate (PMMA), polyethylene, polystyrene, glass, and silicon. Polymeric materials such as polycarbonate, cycloolefin polymer, and polypropylene are particularly preferred because they have excellent processability and allow for inexpensive manufacturing of microchips using molding equipment.

[0103] The microchip 150 for separating fine particles is preferably transparent. For example, the microchip 150 for separating fine particles is transparent in at least the portion through which light (laser light and scattered light) passes, and for example, the detection area may be transparent. The entire microchip 150 for separating fine particles may also be transparent.

[0104] In the above description, an embodiment has been described in which the above-mentioned channel group is formed on a disposable microchip 150 for sorting fine particles. However, in this technology, the channel group does not have to be formed on the microchip 150. For example, the channel group may be formed in a substrate such as plastic or glass. Furthermore, the channel group may have a two-dimensional or three-dimensional structure.

[0105] In this technology, the microparticles may be particles having dimensions that allow them to flow through the channels in the microparticle sorting mechanism (e.g., a microchip for microparticle sorting). In this technology, the microparticles may be appropriately selected by those skilled in the art. In this technology, the microparticles may include biological microparticles such as cells, cell aggregates, microorganisms, and liposomes, as well as synthetic microparticles such as gel particles, beads, latex particles, polymer particles, and industrial particles. Biological microparticles (also called bioparticles) may include chromosomes, liposomes, mitochondria, organelles, and other components that make up various cells. Cells may include animal cells (such as hematopoietic cells) and plant cells. Cells may be hematopoietic cells or tissue cells in particular. Hematopoietic cells may be suspension cells such as T cells and B cells. Tissue cells may be adherent cultured cells or adherent cells separated from tissue. Cell aggregates may include spheroids and organoids, for example. Microorganisms may include bacteria such as Escherichia coli, viruses such as tobacco mosaic virus, and fungi such as yeast. Furthermore, biological microparticles may also include biological macromolecules such as nucleic acids, proteins, and complexes thereof. These biological macromolecules may be extracted from cells, for example, or contained in blood samples or other liquid samples. Synthetic microparticles may be microparticles made of, for example, organic or inorganic polymer materials or metals. Organic polymer materials may include polystyrene, styrene-divinylbenzene, and polymethyl methacrylate. Inorganic polymer materials may include glass, silica, and magnetic materials. Metals may include gold colloid and aluminum. The synthetic microparticles may be, for example, gel particles or beads, and more particularly, gel particles or beads to which one or more combinations selected from oligonucleotides, peptides, proteins, and enzymes are bound. The shape of the microparticles may be spherical, nearly spherical, or non-spherical. The size and mass of the microparticles may be appropriately selected by those skilled in the art depending on the size of the microchip's channel. On the other hand, the size of the microchip's channel may also be appropriately selected depending on the size and mass of the microparticles. In this technology, chemical or biological labels, such as fluorescent dyes or fluorescent proteins, may be attached to the microparticles as needed. Such labels may make the detection of the microparticles easier. The labels to be attached may be appropriately selected by those skilled in the art. Molecules that react specifically with the microparticles (e.g., antibodies, aptamers, DNA, or RNA) may be bound to the labels. According to one embodiment of this technology, the microparticles are biological particles, and in particular, may be cells.

[0106] In this technology, the fine particles collected in the collection channel may be subjected to further fine particle sorting. This further fine particle sorting may be performed, for example, using the fine particle sorting microchip 150 described above, or using other fine particle sorting microchips. For example, in the further fine particle separation process, one or more combinations of the steps described in (2-1) to (2-4) above may be performed. For example, if the determination step described in (2-2) above is performed in the further fine particle separation process, in order to recover emulsion particles containing fine particles, the determination step may be performed on fine particles, on emulsion particles, or on both fine particles and emulsion particles. In this way, even in the further fine particle separation process, it may be determined whether fine particles or emulsion particles are to be recovered based on information obtained from the fine particles and / or emulsion particles. Furthermore, in the aforementioned further fine particle separation process, an emulsion containing fine particle-containing emulsion particles may be subjected to the separation process.

[0107] (3) Other examples of flow channel structures

[0108] (3-1) A flow channel structure in which the main flow channel and the waste flow channel are aligned in a straight line.

[0109] In one embodiment of this technology, the connecting channel and the collection channel do not have to be aligned in a straight line with the main channel. An example of a microchip for separating fine particles in this embodiment will be described below with reference to Figure 9.

[0110] Figure 9 shows a schematic diagram of the detection region and the region including the particle sorting section of a microchip for fine particle sorting according to this technology. The microchip 350 for fine particle sorting shown in Figure 9 has a flow path structure similar to the microchip 150 for fine particle sorting described with reference to Figure 1, in which the sample liquid flow path 352 and the sheath liquid flow path 354 merge at a confluence 362 to form a main flow path 355, and a detection region 356 is provided in the main flow path 355. A laminar flow of the sample liquid containing fine particles P, surrounded by the sheath liquid, flows through the main flow path 355.

[0111] The microchip 350 for separating fine particles includes, in addition to the main channel 355, a recovery channel 359 into which the particles to be recovered are collected, a connecting channel 370 connecting the main channel 355 and the recovery channel 359, and a liquid supply channel 361 connected to the connecting channel 370 so as to be able to supply liquid.

[0112] The main channel 355 further includes a waste channel 358 through which fine particles that are not the target of collection flow. The microchip 150 for fine particle sorting, as described with reference to Figure 1, has two waste channels 158 branching off from the main channel 155, and the connecting channel 170, collection channel 159, and main channel 155 are arranged in a straight line. In contrast, the microchip 350 for fine particle sorting in Figure 9 has a channel structure in which the main channel 355 and the waste channel 358 are arranged in a straight line, and the connecting channel 370 and collection channel 359 branch off from the main channel 355. Thus, in the microparticle sorting microchip used in the microparticle collection method of this technology, the connecting channel and the collection channel do not necessarily have to be aligned linearly with the main channel. For example, the main channel and the waste channel may be aligned linearly, and the connecting channel and the collection channel may have a channel structure in which they branch off from the main channel.

[0113] (3-2) Flow channel structure having multiple recovery channels

[0114] In other embodiments of this technology, the microchip for separating fine particles may include one or more flow path structures, each comprising: a main flow path through which fine particles flow; a recovery flow path for which particles to be recovered are recovered; a connecting flow path connecting the main flow path and the recovery flow path; and a liquid supply flow path connected to the connecting flow path so as to be able to supply liquid. The microchip for separating fine particles described above with reference to Figure 1 has one such flow path structure. In contrast, as will be described below with reference to Figures 10 and 11, the microchip for separating fine particles used in the fine particle recovery method of this technology may have two or more such flow path structures.

[0115] Figure 10 shows a schematic diagram of the detection region and the region including the particle sorting section of a microchip for sorting fine particles according to this technology. The microchip for sorting fine particles 450 shown in Figure 10 has a flow path structure similar to the microchip for sorting fine particles 150 described with reference to Figure 1, in which the sample liquid flow path 452 and the sheath liquid flow path 454 merge at a confluence 462 to form a main flow path 455, and a detection region 456 is provided in the main flow path 455. A laminar flow of the sample liquid containing fine particles P, surrounded by the sheath liquid, flows through the main flow path 455.

[0116] The microchip 450 for fine particle sorting includes, in addition to the main channel 455, a recovery channel 459-1 through which target particles are collected, a connecting channel 470-1 connecting the main channel 455 and the recovery channel 459-1, and a liquid supply channel 461-1 connected to the connecting channel 470-1 so as to be able to supply liquid. The microchip 450 for fine particle sorting further includes a recovery channel 459-2 through which target particles are collected, a connecting channel 470-2 connecting the main channel 455 and the recovery channel 459-2, and a liquid supply channel 461-2 connected to the connecting channel 470-2 so as to be able to supply liquid. In other words, the microchip 450 for fine particle sorting has two of these channel structures. In the microchip 450 for fine particle sorting, the main channel 455 is shared by these two channel structures. The connecting channel 470-1 is downstream of the connecting channel 470-2 and is connected to the main channel 455.

[0117] The microchip 450 for separating fine particles can, for example, form two emulsions containing different target particles. In this case, the determination step determines whether the fine particles are, for example, one of two types of target particles A and B, or neither of the two types of target particles. The criteria for determining whether a particle belongs to target particle A or B may be selected as appropriate by the user. In the recovery process described above, if the fine particles are particle A to be recovered, the fine particles are recovered through the connecting channel 470-1 into the recovery channel 459-1. The fine particles, which are particle A to be recovered, are recovered into the second liquid in the recovery channel 459-1 while still contained in the first liquid. In the recovery process described above, if the fine particles are the target particle B, the fine particles are recovered through the connecting channel 470-2 into the recovery channel 459-2. The fine particles, which are the target particle B, are recovered into the second liquid in the recovery channel 459-2 while still contained in the first liquid. In the recovery process described above, if the fine particles are neither of the target particles A nor B, the fine particles flow into the waste channel 458. As described above, an emulsion containing particle A to be recovered and an emulsion containing particle B to be recovered are formed.

[0118] Figure 11 shows a schematic diagram of the detection region and the region including the particle sorting section of a microchip for sorting fine particles according to this technology. The microchip 550 for sorting fine particles shown in Figure 11 is the same as the microchip 450 for sorting fine particles described with reference to Figure 10, except that two connecting channels 570-1 and 570-2 are connected to the main channel 555 at the same position. That is, the microchip 550 for sorting fine particles has two of the channel structures, and the main channel 555 is shared by these two channel structures.

[0119] The microchip 550 for separating fine particles can also be used to form two emulsions containing different target particles. In this case as well, the determination step determines whether the fine particles are, for example, one of two types of particles A and B to be recovered, or neither of the two types of particles to be recovered. The criteria for determining whether a particle belongs to particle A or B may be selected as appropriate by the user. In the recovery process described above, if the fine particles are particle A to be recovered, the fine particles are recovered through the connecting channel 570-1 into the recovery channel 559-1. The fine particles, which are particle A to be recovered, are recovered into the second liquid in the recovery channel 559-1 while still contained in the first liquid. In the recovery process described above, if the fine particles are the target particle B, the fine particles are recovered through the connecting channel 570-2 into the recovery channel 559-2. The fine particles, which are the target particle B, are recovered into the second liquid in the recovery channel 559-2 while still contained in the first liquid. In the recovery process described above, if the fine particles are neither of the target particles A nor B, the fine particles flow into the waste channel 558. As described above, an emulsion containing particle A to be recovered and an emulsion containing particle B to be recovered are formed.

[0120] 2. Second Embodiment (Microchip for Microparticle Separation)

[0121] This technology also provides a microchip for separating fine particles having a flow channel structure that includes a main flow channel through which fine particles flow, a recovery flow channel for which particles to be recovered are recovered 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. The main flow channel in the microchip has a determination region used to determine whether fine particles flowing while contained in a first liquid are particles to be recovered. Fine particles determined to be particles to be recovered are recovered while contained in the first liquid into a second liquid in the recovery flow channel that is immiscible with the first liquid. This microchip for separating fine particles is the same as the microchip for separating fine particles described in 1. above, and that description also applies to this embodiment.

[0122] 3. Third Embodiment (Fine Particle Recovery Device)

[0123] This technology also provides a microchip for separating fine particles having a flow path structure including a main flow path through which fine particles flow, a recovery flow path for which particles to be recovered are recovered from the fine particles, a connecting flow path connecting the main flow path and the recovery flow path, and a liquid supply flow path connected to the connecting flow path so as to be able to supply liquid; a fine particle recovery device comprising a first liquid supply unit that supplies a first liquid containing fine particles to the main flow path, a second liquid supply unit that supplies a second liquid that is immiscible with the first liquid to the liquid supply flow path, and a determination unit that determines whether the fine particles flowing in the main flow path are particles to be recovered. This fine particle recovery device is the same as the fine particle recovery device described in 1. above, and the description therein also applies to this embodiment.

[0124] The microchip for separating fine particles is the same as the microchip for separating fine particles described in 1. above, and that description also applies to this embodiment. The first liquid supply unit may be, for example, at least one component for forming the laminar flow described in 1.(2) above, and may include, for example, a sample liquid channel 152 and a sheath liquid channel 154. The first liquid supply unit may also include channels (e.g., tubes, etc.) connected to the sample liquid inlet 151 and the sheath liquid inlet 153, respectively, for introducing the sample liquid and sheath liquid into these inlets, and a sample liquid-containing container and a sheath liquid-containing container connected to these channels. The second liquid supply unit may be, for example, at least one component for supplying the second liquid to the liquid supply channel 161 described in 1.(2) above, and may include, for example, a container for containing the second liquid supplied to the liquid supply channel 161, and a channel connecting the container and the liquid supply channel (for example, a tube connecting the tip and the container). The determination unit is the same as the determination unit described in 1. above, and the same description applies to this embodiment as well.

[0125] Furthermore, the microchip for separating fine particles may be removable from the microparticle collection device. By making the microchip for separating fine particles removable from the device, a new microchip can be used for each sample, thereby preventing contamination between samples.

[0126] 4. Fourth Embodiment (Method for Producing Emulsion)

[0127] This technology also provides a method for manufacturing an emulsion, which includes the flow step, determination step, and recovery step described in "1. First Embodiment (Fine Particle Recovery Method)" above. This manufacturing method may include other steps described in "(2-4) Other Steps" above. The description given in "1. First Embodiment (Fine Particle Recovery Method)" above also applies to the emulsion manufacturing method of this technology.

[0128] The emulsion manufacturing method of this technology makes it possible to produce emulsions containing a high proportion of emulsion particles containing a single microparticle (particularly the particle to be recovered). For example, the manufacturing method of this technology can produce emulsions in which the proportion of emulsion particles containing a single microparticle is, for example, 70% or more, preferably 80% or more, more preferably 90% or more, and even more preferably 95% or more, relative to the total number of emulsion particles. In single-cell analysis using emulsions, it is important to increase the proportion of emulsions containing a single cell, and this is especially important when the number of cells to be analyzed is small. Since the manufacturing method of this technology can produce emulsions with a high proportion of emulsion particles containing a single microparticle, it is extremely useful for the aforementioned single-cell analysis.

[0129] 5. Fifth Embodiment (Emulsion)

[0130] This technology also provides an emulsion containing microparticle-containing emulsion particles, wherein the proportion of emulsion particles containing a single microparticle is 70% or more of the total number of emulsion particles. This proportion is preferably 80% or more, more preferably 90% or more, and even more preferably 95% or more. Because the emulsion of this technology contains emulsion particles containing a single microparticle at such a high proportion, it is suitable, for example, for single-cell analysis. The emulsion particles containing the single fine particle can be formed as described in "1. First Embodiment (Method for Recovering Fine Particles)" above. Furthermore, the emulsion can be manufactured, for example, as described in "1. First Embodiment (Method for Recovering Fine Particles)" or "4. Fourth Embodiment (Method for Manufacturing Emulsion)" above.

[0131] The emulsion may be, for example, an emulsion in which the second liquid is the dispersion medium and the first liquid is the dispersed phase. The description in "1. First Embodiment (Method for Recovering Fine Particles)" above applies to these liquids. The description in "1. First Embodiment (Method for Recovering Fine Particles)" above also applies to the fine particles contained in the emulsion particles, and such fine particles may be, for example, cells, cell aggregates, or synthetic particles.

[0132] Furthermore, this technology can also be configured as follows: [1] The main channel through which fine particles are passed, A collection channel from which the particles to be collected are collected, A connecting channel that connects the main channel and the recovery channel, A fine particle sorting mechanism having a flow channel structure including a liquid supply channel connected to the aforementioned connecting channel so as to be able to supply liquid, comprising a flow step of flowing a first liquid containing fine particles into the main channel, A determination step of determining whether the fine particles flowing through the main channel are particles to be collected, The process includes a recovery step of recovering the particles to be recovered into the recovery channel, and In the recovery process, the particles to be recovered are recovered in the first liquid while contained within the second liquid, which is immiscible with the first liquid, within the recovery channel. Method for collecting fine particles. [2] The fine particle recovery method according to [1], wherein by carrying out the fine particle recovery method, an emulsion is formed in the recovery channel in which the second liquid is the dispersion medium and the first liquid is the dispersed phase. [3] The fine particle recovery method according to [1] or [2], wherein an emulsion is formed by carrying out the fine particle recovery method, and at least a portion of the droplets constituting the emulsion contains one of the particles to be recovered. [4] A method for recovering fine particles according to any one of [1] to [3], wherein the first liquid is hydrophilic and the second liquid is hydrophobic. [5] A method for recovering fine particles according to any one of [1] to [4], wherein the kinematic viscosity of the second liquid is 1 / 100 to 100 times that of the first liquid. [6] The fine particle recovery method according to any one of [1] to [5], wherein the flow step, the determination step, and the recovery step are performed while the second liquid is supplied from the liquid supply channel to the connecting channel. [7] The main channel branches off to the connecting channel and at least one waste channel through which fine particles other than the particles to be recovered flow, The liquid supply channel supplies liquid to the connecting channel. A method for recovering fine particles as described in any one of [1] to [6]. [8] The fine particle recovery method according to any one of [1] to [7], wherein the connecting channel is provided with a valve to prevent the first liquid from proceeding to the recovery channel. [9] A method for recovering fine particles according to any one of [1] to [8], wherein in the flow step, the fine particles flow in a substantially straight line through the main flow channel toward the connecting flow channel.

[10] The fine particle sorting mechanism has a flow path structure such that a sample flow path through which a liquid containing fine particles flows and a sheath flow path through which a liquid not containing fine particles flows are connected to the main flow path of the confluence, and fine particles flow in the main flow path after the confluence in a substantially straight line within the main flow path. This channel structure creates a laminar flow containing fine particles flowing in a line. A method for recovering fine particles as described in any one of [1] to [9].

[11] A method for recovering fine particles according to any one of [1] to

[10] , wherein in the determination step, light is irradiated onto fine particles flowing in the main channel, and it is determined whether the fine particles are particles to be recovered based on the light generated by the irradiation.

[12] A method for recovering fine particles according to any one of [1] to

[11] , wherein, in the recovery step, the particles to be recovered are recovered into the recovery channel through the connecting channel due to pressure fluctuations in the recovery channel.

[13] A method for recovering fine particles according to any one of [1] to

[12] , wherein the main channel, the connecting channel, and the recovery channel are arranged in a straight line.

[14] A method for recovering fine particles according to any one of [1] to

[13] , wherein the fine particles are cells or cell aggregates, and the first liquid is a culture medium for the fine particles.

[15] A method for recovering fine particles according to any one of [1] to

[14] , wherein the fine particles are cells, cell aggregates, or synthetic particles, and the fine particles are destroyed after the recovery step.

[16] A method for recovering fine particles according to any one of [1] to

[13] , wherein the recovered fine particles in the recovery channel are subjected to further fine particle sorting treatment.

[17] The method for collecting fine particles according to any one of [1] to [6], wherein the microchip for separating fine particles includes one or more of the channel structures.

[18] The main channel through which fine particles are passed, A collection channel from which the particles to be collected are collected, A connecting channel that connects the main channel and the recovery channel, The flow path structure includes a liquid supply channel connected to the aforementioned connecting channel so as to be able to supply liquid, The main flow channel has a determination region used to determine whether the fine particles flowing while contained in the first liquid are particles to be recovered. Fine particles determined to be particles to be recovered are recovered in the first liquid while contained within the recovery channel into a second liquid that is immiscible with the first liquid. Microchip for separating fine particles.

[19] The main channel through which fine particles are passed, A collection channel from which the particles to be collected are collected, A connecting channel that connects the main channel and the recovery channel, A liquid supply channel connected to the aforementioned connecting channel so as to be able to supply liquid, A microchip for separating fine particles having a flow channel structure including, The main channel is supplied with a first liquid supply unit containing fine particles, A second liquid supply unit supplies a second liquid, which is immiscible with the first liquid, to the liquid supply channel, A determination unit that determines whether the fine particles flowing in the main channel are particles to be collected, A microparticle recovery device equipped with the following features.

[20] The microparticle collection device according to

[19] , wherein the microchip for separating fine particles is removable from the microparticle collection device.

[21] The main channel through which fine particles are passed, A collection channel from which the particles to be collected are collected, A connecting channel that connects the main channel and the recovery channel, A fine particle sorting mechanism having a flow channel structure including a liquid supply channel connected to the aforementioned connecting channel so as to be able to supply liquid, comprising a flow step of flowing a first liquid containing fine particles into the main channel, A determination step of determining whether the fine particles flowing through the main channel are particles to be collected, The process includes a recovery step of recovering the particles to be recovered into the recovery channel, and In the recovery process, the particles to be recovered are recovered in the first liquid while contained within the second liquid, which is immiscible with the first liquid, within the recovery channel. A method for producing an emulsion containing fine particle-containing emulsion particles.

[22] An emulsion containing fine particle-containing emulsion particles, wherein the proportion of emulsion particles containing one fine particle is 70% or more of the total number of emulsion particles. [Explanation of Symbols]

[0133] 100 Fine Particle Recovery Device 150 Microchips for Separating Fine Particles 155 Main channel 159 Recovery channel 161 Liquid supply channel 170 Connection channel

Claims

1. A flow step of flowing a first liquid containing fine particles into a main channel, A determination step of determining whether the fine particles flowing in the main channel are particles to be collected, A recovery step in which, among the aforementioned fine particles, the particles to be recovered are recovered into the recovery channel by pressure fluctuations within the recovery channel, Includes, The particles to be recovered are recovered into the second liquid within the recovery channel while contained in the first liquid. The second liquid is immiscible with the first liquid, and A method for recovering fine particles, wherein the second liquid acts as a dispersion medium and the first liquid acts as a dispersed phase within the recovery channel.

2. The method for recovering fine particles according to claim 1, wherein the particles to be recovered are recovered into an emulsion within the recovery channel.

3. The method for recovering fine particles according to claim 1, wherein the particles to be recovered are one or more selected from the group consisting of cells, cell aggregates, and synthetic particles.

4. The method for recovering fine particles according to claim 1, wherein the shape of the fine particles is spherical, substantially spherical, or non-spherical.

5. The fine particles are synthetic fine particles, The method for recovering fine particles according to claim 1, wherein the synthetic fine particles are one or more selected from the group consisting of synthetic gel particles, beads, latex particles, polymer particles, and industrial particles.

6. The method for recovering fine particles according to claim 5, wherein a fluorescent dye or fluorescent protein is attached to the synthetic fine particles.

7. The method for recovering fine particles according to claim 1, wherein the determination in the determination step is determination based on scattered light, determination based on fluorescence, or determination based on an image.

8. The method for recovering fine particles according to claim 1, wherein the determination in the determination step is performed by a determination unit in the fine particle recovery system.

9. The method for recovering fine particles according to claim 1, wherein in the recovery step, two or more adjacent fine particles are recovered into the recovery channel.

10. The method for recovering fine particles according to claim 9, wherein the two or more adjacent fine particles have the same characteristics.

11. The method for recovering fine particles according to claim 9, wherein the two or more adjacent fine particles are a combination of fine particles having different characteristics.

12. The method for collecting fine particles according to claim 1, wherein the pressure fluctuation is controlled by a piezoelectric drive.

13. The dispersion medium is a hydrophobic liquid, The method for recovering fine particles according to claim 1, wherein the dispersed phase is a hydrophilic liquid.

14. The method for recovering fine particles according to claim 2, wherein the emulsion from which the particles to be recovered have been recovered is merged with other emulsions.

15. The particles to be recovered are cells or cell aggregates, The first liquid is the culture medium for the particles to be recovered. The method for recovering fine particles according to claim 2, further comprising a culture step of culturing the cells or cell aggregates in the emulsion from which the particles to be recovered have been recovered.

16. The particles to be recovered are cells, cell aggregates, or synthetic particles, and The method for recovering fine particles according to claim 1, further comprising a destruction step in which the particles to be recovered are destroyed after the recovery step.

17. A main channel through which a first liquid containing fine particles flows, A recovery channel that recovers the target particles into the recovery channel due to pressure fluctuations within the recovery channel where the target particles are recovered, A microchip for separating fine particles having a flow channel structure including, A determination unit that determines whether the fine particles flowing in the main channel are particles to be collected, Equipped with, The particles to be recovered are recovered into the second liquid within the recovery channel while contained in the first liquid. The second liquid is immiscible with the first liquid, and A fine particle recovery system in which the second liquid acts as a dispersion medium and the first liquid acts as a dispersed phase within the recovery channel.

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