Microparticle recovery method and microparticle recovery system

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] The present technology relates to a microparticle recovery method, a microchip for microparticle sorting, a microparticle recovery device, a method for producing an emulsion, and an emulsion. More specifically, the present technology relates to a microparticle recovery method for recovering microparticles in an emulsion, a microchip for microparticle sorting, a microparticle recovery device, an emulsion production method including a step performed in the recovery method, and an emulsion. [Background technology]

[0002] To perform single cell analysis, it has been proposed to use emulsions containing one cell per emulsion particle, and several techniques have been developed to form such emulsions.

[0003] For example, Non-Patent Document 1 below describes a method of randomly capturing particles in an emulsion. In this method, a solution containing cells is diluted to 100 cells / μl or less so that one emulsion contains one cell. In this method, the efficiency of generating emulsion particles containing one cell follows a Poisson distribution, so the ratio of empty emulsion particles (emulsion particles without cells) is high, making it inefficient. Therefore, Non-Patent Document 1 below proposes sorting emulsions by controlling particles or fluids, for example, by electric fields, dielectrophoresis, or local heating, in order to increase the ratio of cell-containing emulsion particles to empty emulsion particles. In addition, in order to increase the ratio of single-cell-containing emulsion particles in the generated emulsion, it has been proposed to utilize the Plateau-Rayleigh instability induced by cells, or to utilize the self-organization that occurs when a high-density suspension is quickly passed through a microchannel, as described in Non-Patent Document 1 below, for example. [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 Summary of the Invention [Problem to be solved by the invention]

[0005] To perform single-cell analysis using emulsions, it is desirable to increase the ratio of emulsion particles containing a single cell in the emulsion. The purpose of this technology is to provide a new method for more efficiently generating emulsion particles containing a single microparticle. [Means for solving the problem]

[0006] The present inventors have discovered that the above problems can be solved by a specific method for recovering microparticles. That is, the present technology provides a main flow path through which microparticles flow, a collection flow path through which collection target particles are collected from among the microparticles; 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 liquid to the connection flow path, in a microparticle sorting mechanism having a flow path structure including the following steps: a determination step of determining whether the microparticles flowing through the main flow path are particles to be collected; A recovery step of recovering the recovery target particles into the recovery flow path, and In the recovery step, the recovery target particles are recovered in a state where they are contained in the first liquid into a second liquid that is immiscible with the first liquid in the recovery flow path. A method for recovering microparticles is provided. By carrying out the microparticle recovery method, an emulsion can be formed in the recovery passageway, the emulsion including the second liquid as a dispersion medium and the first liquid as a dispersoid. By carrying out the microparticle 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 kinetic viscosity of the second liquid may be 1 / 100 to 100 times the kinetic viscosity of the first liquid. The flowing step, the determining step, and the recovering step can be performed while the second liquid is being supplied from the liquid supply flow channel to the connecting flow channel. The main flow path may branch into the connection flow path and at least one waste flow path through which microparticles other than the particles to be collected flow, The liquid supply channel may supply liquid to the connecting channel. The connecting passage may be provided with a valve that prevents the first liquid from proceeding to the recovery passage. In the flowing step, the microparticles can flow in the main flow path toward the connecting flow path in a substantially lined up state. The microparticle sorting mechanism may have a flow path structure in which a sample flow path through which a liquid containing microparticles flows and a sheath flow path through which a liquid not containing microparticles flows are connected to a main flow path at a junction, and the microparticles flow in the main flow path after the junction in a manner such that the microparticles flow in a substantially line within the main flow path, The flow channel structure can form a laminar flow containing microparticles flowing in a substantially aligned line. In the determination step, light is irradiated onto the microparticles flowing through the main channel, and it can be determined whether the microparticles are particles to be collected based on the light generated by the irradiation. In the recovery step, the recovery target particles can be recovered into the recovery flow path through the connection flow path due to pressure fluctuations in the recovery flow path. The main flow path, the connection flow path, and the recovery flow path may be aligned in a straight line. The microparticles may be cells or cell clusters, and the first liquid may be a culture medium for the microparticles. The microparticles may be cells, cell clumps, or synthetic particles, and may be disrupted after the recovery step. The microparticles collected in the collection channel may be subjected to further microparticle sorting processing. The microchip for sorting microparticles may include one or more of the flow channel structures.

[0007] In addition, the present technology provides a flow path for microparticles, a collection flow path through which collection target particles are collected from among the microparticles; 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 liquid, the main flow path has a determination region used for determining whether microparticles flowing in a state of being contained in the first liquid are particles to be collected, The microparticles determined to be particles to be collected are collected in a state where they are contained in the first liquid into a second liquid that is immiscible with the first liquid in the collection flow path. A microchip for sorting microparticles is also provided.

[0008] In addition, the present technology provides a flow path for microparticles, a collection flow path through which collection target particles are collected from among the microparticles; 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 liquid; A microchip for sorting microparticles having a flow path structure including the following: a first liquid supply unit that supplies a first liquid containing microparticles 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 channel; a determination unit that determines whether the microparticles flowing in the main flow path are particles to be collected; A microparticle collection device comprising: The microchip for sorting microparticles may be removable from the microparticle recovery device.

[0009] In addition, the present technology provides a flow path for microparticles, a collection flow path through which collection target particles are collected from among the microparticles; 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 liquid to the connection flow path, in a microparticle sorting mechanism having a flow path structure including the following steps: a determination step of determining whether the microparticles flowing through the main flow path are particles to be collected; A recovery step of recovering the recovery target particles into the recovery flow path, and In the recovery step, the recovery target particles are recovered in a state where they are contained in the first liquid, into a second liquid that is immiscible with the first liquid and is in the recovery flow path. A method for making an emulsion comprising microparticle-containing emulsion particles is also provided.

[0010] The present technology also provides an emulsion containing microparticle-containing emulsion particles, in which the ratio of the number of emulsion particles containing one microparticle to the total number of emulsion particles is 70% or more. [Brief description of the drawings]

[0011] [Figure 1] 1A to 1C are diagrams showing an example of the configuration of a microchip for sorting microparticles used in the microparticle recovery method of the present technology. [Diagram 2] 1 is a diagram showing an example of a flow of a microparticle recovery method according to the present technology. [Diagram 3] FIG. 2 is an enlarged view of an example of a particle sorting section. [Figure 4] FIG. 4 is a block diagram of an example of a control unit. [Diagram 5] FIG. 1 is a diagram showing a configuration example of a microchip for sorting microparticles to which a container is connected. [Figure 6A] FIG. [Figure 6B] FIG. [Figure 7A] FIG. [Figure 7B] FIG. [Figure 8A] 1 is a photograph showing that an emulsion particle containing one microparticle is formed in a recovery channel. [Figure 8B] 13 is a photograph showing that different driving conditions of the piezoelectric element result in emulsion particles having different sizes. [Figure 9] FIG. 1 is a schematic diagram of an example of a microchip for sorting microparticles. [Figure 10] FIG. 1 is a schematic diagram of an example of a microchip for sorting microparticles. [Figure 11] FIG. 1 is a schematic diagram of an example of a microchip for sorting microparticles. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Preferred embodiments for carrying out the present technology will be described below. Note that the embodiments described below are representative embodiments of the present technology, and the scope of the present technology is not limited to these embodiments. Note that the present technology will be described in the following order. 1. First embodiment (microparticle recovery method) (1) Description of the First Embodiment (2) 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) Microparticle sorting mechanism and microparticles (3) Other examples of flow channel structure (3-1) A flow path structure in which the main flow path and the waste flow path are aligned in a straight line (3-2) Flow path structure having multiple recovery flow paths 2. Second embodiment (microchip for sorting microparticles) 3. Third embodiment (microparticle recovery device) 4. Fourth embodiment (method of producing emulsion) 5. Fifth embodiment (emulsion)

[0013] 1. First embodiment (microparticle recovery method)

[0014] (1) Description of the First Embodiment

[0015] The microparticle collection method of the present technology is performed using a microparticle sorting mechanism having a flow path structure including a main flow path through which microparticles flow, a collection flow path through which collection target particles among the microparticles are collected, a connection flow path connecting the main flow path and the collection flow path, and a liquid supply flow path connected to the connection flow path so as to be able to supply liquid. The microparticle collection method of the present technology includes a flowing step of flowing a first liquid containing microparticles into the main flow path in the microparticle sorting mechanism, a determination step of determining whether the microparticles flowing in the main flow path are collection target particles, and a collection step of collecting the collection target particles into the collection flow path, and in the collection step, the collection target particles are collected in a state contained in the first liquid into a second liquid that is immiscible with the first liquid in the collection flow path.

[0016] According to the present technology, the particles to be collected are contained in the first liquid and collected in the second liquid that is immiscible with the first liquid in the collection flow path, thereby forming, for example, emulsion particles containing the particles to be collected in the collection flow path. In addition, in the present technology, the microparticles collected into the collection channel are those determined to be particles to be collected in the determination step, and the collection operation is performed at an appropriate timing. Further, since the collection operation is not performed when no microparticles flow in or when microparticles determined not to be particles to be collected in the determination step arrive, emulsion particles not containing microparticles or emulsion particles containing particles other than the particles to be collected are not formed in the collection channel. Therefore, the probability that one particle to be collected is contained in the emulsion particles is extremely high. For example, by the method according to the present technology, emulsion particles containing one microparticle (particularly a particle to be collected) can be generated with a success rate of, for example, 70% or more, particularly 80% or more, more particularly 90% or more, and further 95% or more. In the present technology, the particle to be collected refers to the microparticles determined to be collected in the determination step.

[0017] For single-cell analysis using an emulsion, it is important to increase the content ratio of single-cell-containing emulsion particles in the emulsion. Increasing the content ratio is particularly important when performing single-cell analysis on a sample with a small number of cells. However, for example, as described in Non-Patent Document 2 above, the probability that a certain number of cells enter one emulsion particle is considered to follow a Poisson distribution. In conventional emulsion formation techniques, it is said that the probability that one emulsion particle contains one cell is at most about 65%. In addition, in order to increase the ratio, it is conceivable to increase the number of cells in the sample. However, for example, the number of analysis target cells contained in a clinical sample is often small. For example, the number of analysis target cells in one sample can be, for example, 10 4 ~10 5 or the like. Also, for example, when analyzing rare cells such as CTCs (circulating tumor cells in the blood), the number of available cells is limited. According to the present technology, as described above, the ratio of emulsion particles containing one microparticle (for example, a cell) can be increased. Therefore, the present technology is extremely effective for performing single-cell analysis using an emulsion.

[0018] In addition, in single-cell analysis using an emulsion in which each emulsion particle contains one cell, generally, before forming the emulsion, for example, a cell sorting device such as a cell sorter is used to sort the target cell group for single-cell analysis, and the target cells are purified. Therefore, in order to perform the single-cell analysis, in addition to an emulsifying device, a cell sorter is required. An increase in the number of devices used is not desirable, for example, from the perspective of cost. In addition, in order to form the emulsion, in addition to the emulsion forming step, a cell sorting step is also required. An increase in the number of steps is not desirable, for example, from the perspectives of time and cost. There is also a technique of single-cell sorting into a well plate based on a detection signal using a cell sorter, but the upper limit of the number of wells in the well plate is 384, and the analysis scale and throughput are low. In addition, since sorting cannot be performed while the sorting nozzle moves between wells, the target cells that have flowed in during this period will be lost. In the method of the present technology, the microparticles determined to be particles to be recovered in the determination step can be recovered in the state of an emulsion. Therefore, according to the present technology, an emulsion can be formed without separately performing a cell sorting step. In addition, the recovery rate of the particles to be recovered can be increased by the method of the present technology.

[0019] In a preferred embodiment of the present technology, an emulsion is formed by implementing the microparticle recovery method, and at least a part of the droplets (hereinafter, also referred to as "emulsion particles") constituting the emulsion contains one of the particles to be recovered. More preferably, among all the droplets constituting the emulsion, preferably 70% or more, more preferably 80% or more, and even more preferably 90% or more of the number of droplets contain one particle to be recovered. The microparticle recovery method according to the present technology can thus form an emulsion in which the proportion of emulsion particles containing one particle to be recovered is high. As described above, the present technology also provides a method for producing an emulsion containing emulsion particles including one microparticle. The steps included in the production method may be the same as those of the microparticle recovery method. In addition, the present technology also provides an emulsion in which the proportion of the number of emulsion particles containing one cell is 70% or more based on 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. Thus, the present technology provides an emulsion containing one emulsion particle at an extremely high content ratio.

[0020] In the flow path structure included in the microparticle separation mechanism used in the method of the present technology, the main flow path and the recovery flow path are connected via the connection flow path, and the liquid supply flow path is connected to the connection flow path. By supplying the second liquid from the liquid supply flow path to the connection flow path, it is possible to prevent the first liquid flowing through the main flow path from entering the recovery flow path, and if necessary, to introduce the first liquid into the recovery flow path. For example, by introducing the first liquid into the recovery flow path only when the particle to be recovered reaches the vicinity of the connection flow path, the particle to be recovered can be introduced into the recovery flow path in a state contained in the first liquid.

[0021] According to a preferred embodiment of the present technology, by implementing the microparticle recovery method according to the present technology, an emulsion having the second liquid as a dispersion medium and the first liquid as a dispersed phase is formed in the recovery flow path. The emulsion formed by the microparticle recovery method according to the present technology has a high content ratio of emulsion particles containing one particle to be recovered in all emulsion particles. Therefore, the present technology is applicable 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, more preferably 1 / 100 to 100 times, even more preferably 1 / 10 to 10 times, even more preferably 1 / 5 to 5 times, and particularly preferably 1 / 2 to 2 times the kinematic viscosity of the first liquid. In the present technology, it is preferable that the kinematic viscosities of the first liquid and the second liquid are of the same degree. This makes it easier to form an emulsion. The densities of the first liquid and the second liquid at 25 °C are both, for example, 0.5 g / cm 3 ~5 g / cm 3 , preferably 0.6 g / cm 3 ~4 g / cm 3 , more preferably 0.7 g / cm 3 ~3 g / cm 3 and can be. Also, the density of the second liquid is preferably 1 / 100 to 100 times, more preferably 1 / 10 to 10 times, even more preferably 1 / 5 to 5 times, and particularly preferably 1 / 2 to 2 times the density of the first liquid. In the present technology, it is preferable that the densities of the first liquid and the second liquid are of the same degree. This makes it easier to form an emulsion. The kinematic viscosities of the first liquid and the second liquid at 25 °C can 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. When the first liquid and the second liquid have the above physical properties, an emulsion is likely to be formed in the recovery channel. Also, due to such physical properties, these liquids can easily flow in the microchannel.

[0023] In one embodiment of the present technology, the first liquid may be a hydrophilic liquid, and the second liquid may be a hydrophobic liquid. In this embodiment, an emulsion in which a hydrophobic liquid is a dispersion medium and a hydrophilic liquid is a dispersed phase can be formed in the recovery channel. For example, biological particles such as cells preferably exist in a state contained in a hydrophilic liquid such as a buffer or a culture solution. Therefore, this embodiment is suitable for recovering microparticles, particularly biological particles, more particularly cells, which preferably 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 mainly composed of one or more mixtures selected from the group consisting of water, hydrophilic alcohols, hydrophilic ethers, ketones, nitrile solvents, dimethyl sulfoxide, and N,N-dimethylformamide. In this specification, the main component refers to a component that occupies, for example, 50% by mass or more, particularly 60% by mass or more, more particularly 70% by mass or more, even more particularly 80% by mass or more, 85% by mass or more, or 90% by mass or more of the liquid. Examples of the hydrophilic alcohol include ethanol, methanol, propanol, 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, and may be, for example, water, an aqueous solution, or a water dispersion. The hydrophilic liquid may be, for example, a sheath liquid and / or a sample liquid. The hydrophilic liquid is preferably a hydrophilic liquid that does not adversely affect microparticles (for example, biological particles, particularly cells). The hydrophilic liquid may be, for example, a liquid containing a biomolecule. The biomolecule may be, for example, one or a combination of two or more 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 of the stimulated cells (such as morphology) can 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 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 using, 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) 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 / cm 3 ~3 g / cm 3 may be. 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. By having the above physical properties, the hydrophilic liquid can easily flow in the microchannel, and an emulsion is easily formed in the recovery channel.

[0028] The hydrophobic liquid may be any liquid selected from liquids immiscible with the hydrophilic liquid. The hydrophobic liquid may be, for example, a liquid mainly composed of one or more mixtures selected from the group consisting of aliphatic hydrocarbons, fluorinated oils, low-molecular or high-molecular substances containing fluorine atoms, silicone oils, aromatic hydrocarbons, aliphatic monohydric alcohols (such as n-octanol, etc.), and fluorinated polysaccharides. The aliphatic hydrocarbon is preferably an aliphatic hydrocarbon having 7 or more and 30 or less carbon atoms. By having 7 or more and 30 or less carbon atoms, the kinematic viscosity of the hydrophobic liquid is suitable for flowing in the microchannel. Examples of aliphatic hydrocarbons include mineral oil; oils derived from animals and plants 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 the present technology, from the viewpoint of good immiscibility 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 perfluorocarbon include Fluorinert (trademark) FC40 and Fluorinert FC-770 (manufactured by 3M). Examples of perfluoropolyether include Krytox (manufactured by DuPont). Examples of hydrofluoroether include HFE7500 (manufactured by 3M).

[0029] The density of the hydrophobic liquid at 25°C is, for example, 0.5 g / cm 3 ~5 g / cm 3 , preferably 0.6 g / cm 3 ~4 g / cm 3 , more preferably 0.7 g / cm 3 ~3 g / cm 3 and can be such. The kinematic viscosity of the hydrophobic liquid at 25°C can be, for example, 0.3 cSt to 5 cSt, preferably 0.4 cSt to 4 cSt, more preferably 0.5 cSt to 3 cSt. Since the hydrophobic liquid has the above physical properties, an emulsion is likely to be formed in the recovery channel. For example, if the density or kinematic viscosity is too high, the possibility that it will not flow smoothly in the connection channel increases.

[0030] In a preferred embodiment of the present technology, one or both of the first liquid and the second liquid may contain a surfactant. In particular, one or both of the hydrophobic liquid and the hydrophilic liquid contain a surfactant, and more particularly, the hydrophobic liquid contains a surfactant. The surfactant makes it easier to form emulsion particles and can also stably maintain the emulsion particles. Examples of the surfactant include nonionic surfactants and fluorosurfactants. 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 the fluorosurfactant include perfluoropolyether-based surfactants and pseudosurfactants. Examples of the former include Krytox (manufactured by DuPont), and examples of the latter include perfluorooctanol.

[0031] The surfactant may be present, for example, at a concentration equal to or higher than the critical micelle concentration of the surfactant in the hydrophobic liquid. The critical micelle concentration may be, for example, 1 μM to 1000 μM, particularly 10 μM to 100 mM. Further, the surface tension of the surfactant may be, for example, 40 mN / m or less, particularly 20 mN / m or less.

[0032] In another embodiment of the present technology, the first liquid may be a hydrophobic liquid and the second liquid may be a hydrophilic liquid. In this embodiment, an emulsion in which a hydrophilic liquid is a dispersion medium and a hydrophobic liquid is a dispersed phase may be formed in the recovery channel. The present technology may be used to recover microparticles in the emulsion. Examples of the hydrophobic liquid and the hydrophilic liquid are as described above. Further, this embodiment may be applied, for example, when only the target microparticles are recovered from an emulsion in which the dispersion medium and the dispersed phase are a hydrophobic liquid and a hydrophilic liquid, respectively, and the emulsion particles contain microparticles. In addition, as an assay system that can be used in the present technology, not only a system in which microparticles emit fluorescence but also a system in which emulsion particles emit fluorescence can be used. Therefore, in the determination step, determination may be performed on the microparticles, determination may be performed on the emulsion particles, or determination may be performed on both the microparticles and the emulsion particles in order to recover the emulsion particles containing the microparticles. Thus, in the present technology, based on the information obtained from the microparticles and / or the emulsion particles, it may be determined whether the microparticles or the emulsion particles are the objects to be recovered.

[0033] According to a preferred embodiment of the present technology, the flow-through step, the determination step, and the recovery step are performed while supplying the second liquid from the liquid supply channel to the connection channel. Thereby, the connection channel can be filled with the second liquid, and the first liquid can be prevented from unnecessarily advancing into the recovery channel.

[0034] (2) First Example of the First Embodiment

[0035] The microparticle recovery method of the present technology is performed using a microparticle separation mechanism. Hereinafter, with reference to FIG. 1 showing a microchip for microparticle separation which is a configuration example of the microparticle separation mechanism used in the microparticle recovery method of the present technology and FIG. 2 showing an example of the flow of the microparticle recovery method of the present technology, an example of an embodiment of the method of the present technology will be described.

[0036] As shown in FIG. 1, the microchip 150 for microparticle separation used in the method of the present technology includes a main flow path 155 through which microparticles flow and a recovery flow path 159 in which target particles to be recovered among the microparticles are recovered. A particle separation unit 157 is provided in the microchip 150 for microparticle separation. An enlarged view of the particle separation unit 157 is shown in FIG. 3. As shown in A of FIG. 3, the particle separation unit 157 includes a connection flow path 170 that connects the main flow path 155 and the recovery flow path 159. A liquid supply flow path 161 capable of supplying liquid to the connection flow path 170 is connected to the connection flow path 170. As described above, the microchip 150 for microparticle separation has a flow path structure including the main flow path 155, the recovery flow path 159, the connection flow path 170, and the liquid supply flow path 161.

[0037] Also, as shown in FIG. 1, the microchip 150 for microparticle separation constitutes a part of the microparticle recovery device 100 including a light irradiation unit 101, a detection unit 102, and a control unit 103 in addition to the microchip. As shown in FIG. 4, the control unit 103 may include a signal processing unit 104, a determination unit 105, and a separation control unit 106.

[0038] As shown in FIG. 2, the method of the present technology includes, in the microchip 150 for microparticle separation, a flowing step S101 of flowing a first liquid containing microparticles through the main flow path 155, a determination step S102 of determining whether the microparticles flowing through the main flow path 155 are target particles to be recovered, and a recovery step S103 of recovering the target particles to be recovered into the recovery flow path 159. Each step will be described below.

[0039] (2-1) Flowing step

[0040] In the flow-through process S101, a first liquid containing fine particles is caused to flow through the main flow path 155. The first liquid flows through the main flow path 155 from the confluence part 162 toward the particle separation part 157. The first liquid may be a laminar flow formed from a sample liquid containing fine particles and a sheath liquid, and in particular, may be 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 provided with a sample liquid inlet 151 and a sheath liquid inlet 153. A sample liquid containing fine particles and a sheath liquid not containing fine particles are introduced into a sample liquid flow path 152 and a sheath liquid flow path 154 from these inlets, respectively.

[0042] The microchip 150 for separating fine particles has a flow path structure in which the sample flow path 142 through which the sample liquid flows and the sheath liquid flow path 154 through which the sheath liquid flows merge at the confluence part 162 to form the main flow path 155. The sample liquid and the sheath liquid merge at the confluence part 162 to form, for example, a laminar flow in which the sample liquid is surrounded by the sheath liquid. Preferably, the fine particles are arranged in a substantially single row in the laminar flow. Thus, in the present technology, a laminar flow containing fine particles flowing in a substantially single row is formed by the flow path structure.

[0043] The laminar flow flows through the main flow path 155 toward the particle separation part 157. Preferably, the fine particles flow in a single row in the main flow path 155. Thereby, in the light irradiation in the detection region 156 described below, it becomes easier to distinguish the light generated by the light irradiation to one fine particle from the light generated by the light irradiation to other fine particles.

[0044] (2-2) Judgment process

[0045] In the determination step S102, it is determined whether the microparticles flowing through the main flow path 155 are particles to be recovered. This determination can be performed by the determination unit 105. The determination unit 105 can perform this determination based on the light generated by irradiating the microparticles with light from the light irradiation unit 101. 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 the microparticles flowing through the main flow path 155 (particularly the detection region 156) in the microchip 150 for microparticle separation with light (e.g., excitation light, etc.), and the detection unit 102 detects the light generated by this light irradiation. According to the characteristics of the light detected by the detection unit 102, the determination unit 105 included in the control unit 103 determines whether the microparticles are particles 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 or / and a bright field image, etc.). In the recovery step S103 described later, the control unit 103 controls the flow in the microchip 150 for microparticle separation, so that the particles to be recovered are recovered into the recovery flow path 159.

[0047] The light irradiation unit 101 irradiates the microparticles flowing in the flow path in the microchip 150 for microparticle separation with light (e.g., excitation light, etc.). The light irradiation unit 101 can include a light source that emits light and an objective lens that condenses the excitation light onto the microparticles flowing through the detection region. The light source can be appropriately selected by those skilled in the art according to the purpose of analysis, and can be, for example, a laser diode, a SHG laser, a solid-state laser, a gas laser, a high-intensity LED, or a halogen lamp, or a combination of two or more of these. The light irradiation unit may include other optical elements as necessary in addition to the light source and the objective lens.

[0048] (Discrimination of the object to be separated based on the fluorescence signal or / and the scattered light signal)

[0049] In one embodiment of the present technology, the detection unit 102 detects scattered light and / or fluorescence generated from the microparticles by light irradiation from the light irradiation unit 101. The detection unit 102 may include a condenser lens that condenses fluorescence and / or scattered light generated from the microparticles and a detector. As the detector, a PMT, a photodiode, a CCD, a CMOS, etc. may be used, but is not limited thereto. The detection unit 102 may include other optical elements as necessary in addition to the condenser lens and the detector. The detection unit 102 may further include, for example, a spectroscopic unit. Examples of the optical components constituting the spectroscopic unit include a grating, a prism, and an optical filter. By the spectroscopic unit, for example, light of a wavelength to be detected can be separated from light of other wavelengths and detected. The detection unit 102 can convert the detected light into an analog electrical signal by photoelectric conversion. The detection unit 102 can 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 and generate information (data) regarding the characteristics of the light used for the determination by the determination unit 105. As the information regarding the characteristics of the light, the signal processing unit 104 can acquire, for example, one, two, or three of the width of the waveform, the height of the waveform, and the area of the waveform from the waveform of the digital electrical signal. Further, the information regarding the characteristics of the light may include, for example, the time when the light is detected. The processing by the signal processing unit 104 as described above can be performed particularly in an embodiment in which the scattered light and / or fluorescence is detected.

[0051] The determination unit 105 included in the control unit 103 determines whether the microparticle is a particle to be collected based on the light generated by irradiating the microparticle flowing in the flow path with light. In an embodiment where the scattered light and / or fluorescence is detected, the waveform of the digital electrical signal obtained by the detection unit 102 is processed by the control unit 103, and based on the information regarding the characteristics of the light generated by the processing, the determination unit 105 determines whether the microparticle is a particle to be collected. For example, in the determination based on scattered light, the characteristics of the outer shape and / or internal structure of the microparticle may be specified, and based on the characteristics, it may be determined whether the microparticle is a particle to be collected. Further, for example, by performing pretreatment on microparticles such as cells in advance, it is also possible to determine whether the microparticle is a particle to be collected based on characteristics similar to those used in flow cytometry. Also, for example, by labeling microparticles such as cells with an antibody or a dye (particularly a fluorescent dye), it is also possible to determine whether the microparticle is a particle to be collected based on the characteristics of the surface antigen of the microparticle.

[0052] (Discrimination of the object to be sorted based on the bright-field image)

[0053] In another embodiment of the present 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 includes, for example, a halogen lamp, and the detection unit 102 may include a CCD or a CMOS. For example, light can be irradiated onto the microparticle by the halogen lamp, and the CCD or the CMOS can acquire the bright-field image of the irradiated microparticle.

[0054] In the embodiment where the bright-field image is acquired, the determination unit 105 included in the control unit 103 determines whether the microparticle is a particle to be collected based on the acquired bright-field image. For example, based on one or a combination of two or more of the morphology, size, and color of the microparticle (particularly a cell), it can be determined whether the microparticle is a particle to be collected.

[0055] (Discrimination of the object to be sorted based on the dark-field image)

[0056] In still other embodiments of the present technology, the detection unit 102 may acquire a dark-field image generated by the light irradiation of the light irradiation unit 101. In this embodiment, the light irradiation unit 101 includes, for example, a laser light source, and the detection unit 102 may include a CCD or a CMOS. For example, light can be irradiated onto microparticles by a laser, and a CCD or a CMOS can acquire a dark-field image (for example, a fluorescence image) of the irradiated microparticles.

[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 microparticle is a particle to be collected based on the acquired dark-field image. For example, based on one or a combination of two or more of the form, size, and color of the microparticle (especially a cell), it can be determined whether the microparticle is a particle to be collected.

[0058] In any of the above-described "discrimination of the sorting target based on the fluorescence signal or / and the scattered light signal", "discrimination of the sorting target based on the bright-field image", and "discrimination of the sorting target based on the dark-field image", the detection unit 102 may be an image pickup device in which a substrate incorporating a CMOS sensor and a substrate incorporating a DSP (Digital Signal Processor) are laminated. By operating the DSP of the image pickup device as a machine learning unit, the image pickup device can operate as a so-called AI sensor. The detection unit 102 including the image pickup device can determine whether the microparticle is a particle to be collected, for example, based on a learning model. Further, the learning model may be updated in real time while the method according to the present technology is being performed. For example, during the reset of the pixel array unit in the CMOS sensor, during the exposure of the pixel array unit, or during the readout of the pixel signal from each unit pixel of the pixel array unit, the DSP can perform machine learning processing. As an example of an image pickup device operating as an AI sensor, for example, the image pickup device described in International Publication No. WO2018 / 051809 can be mentioned. When an AI sensor is used as the image pickup device, the raw data acquired from the image array is directly learned, so the speed of the sorting discrimination process is high.

[0059] The determination can be made, for example, based on whether information regarding the characteristics of the light satisfies a preset criterion. The criterion can be a criterion indicating that the microparticle is a particle to be collected. The criterion may be appropriately set by those skilled in the art and can be a criterion regarding the characteristics of light, such as a criterion used in technical fields such as flow cytometry.

[0060] One light may be irradiated to one position in the detection region 156, or lights may be irradiated to each of a plurality of positions in the detection region 156. For example, the microchip 150 may be configured such that lights are irradiated to each of two different positions in the detection region 156 (that is, there are two positions in the detection region 156 where lights are irradiated). In this case, for example, based on the light (such as fluorescence and / or scattered light, etc.) generated by irradiating the microparticles at one position, it can be determined whether the microparticles are particles to be collected. Further, based on the difference between the detection time of the light generated by the light irradiation at the one position and the detection time of the light generated by the light irradiation at another position, the velocity of the microparticles in the flow path can also be calculated. For this calculation, the distance between the two irradiation positions may be determined in advance, and the velocity of the microparticles can be determined based on the difference between the two detection times and the distance. Further, based on the velocity, the arrival time at the particle separation unit 157 described below can be accurately predicted. By accurately predicting the arrival time, the timing of forming the flow into the recovery flow path 159 can be optimized. Also, when the difference between the arrival time of a certain microparticle at the particle separation unit 157 and the arrival time of the microparticle before or after the certain microparticle at the particle separation unit 157 is equal to or less than a predetermined threshold value, it can also be determined not to collect the certain microparticle. When the distance between the certain microparticle and the microparticle before or after it is narrow, the possibility that the microparticle before or after it is collected together increases when the certain microparticle is sucked. By determining not to collect the certain microparticle when the possibility of being collected together is high, it is possible to prevent the microparticle before or after it from being collected. Thereby, the purity of the target microparticles among the collected microparticles can be increased. Specific examples of the microchip in which lights are irradiated to each of two different positions in the detection region 156 and the device including the microchip are described in, for example, Japanese Patent Application Laid-Open No. 2014-202573.

[0061] Note that the control unit 103 may control the light irradiation by the light irradiation unit 101 and / or the light detection by the detection unit 102. Further, the control unit 103 can control the driving of a pump for supplying fluid into the microchip 150 for microparticle separation. The control unit 103 may be constituted by, for example, a hard disk, a CPU, and a memory in which a program and an OS for causing the microparticle recovery device to execute the microparticle recovery method according to the present technology are stored. 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, an SD memory card, or a flash memory. The drive (not shown) provided in the microparticle recovery device 100 reads the program recorded on the recording medium, and the control unit 103 may cause the microparticle recovery device 100 to execute the microparticle recovery method according to the present technology according to the read program.

[0062] (2-3) Recovery step

[0063] In the recovery step S103, the microparticles determined to be 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 being contained in the first liquid. Thereby, an emulsion having the second liquid as a dispersion medium and the first liquid as a dispersed phase can be formed in the recovery channel 159, and each emulsion particle of the emulsion contains one particle to be recovered. Therefore, the emulsion is suitable for single-cell analysis. The recovery step will be described in more detail below.

[0064] The recovery process S103 is performed in the particle separation unit 157 in the microchip 150. In the particle separation unit 157, the laminar flow that has flowed through the main flow path 155 branches off and flows into two waste flow paths 158. Although the particle separation unit 157 described in FIG. 1 has two waste flow paths 158, the number of branch flow paths is not limited to two. The particle separation unit 157 may be provided with, for example, one or a plurality (such as two, three, or four) of branch flow paths. The branch flow paths may be configured to branch in a Y shape on one plane as shown in FIG. 1, or may be configured to branch three-dimensionally.

[0065] In the particle separation unit 157, only when the particles to be recovered flow in, a flow is formed from the main flow path 155 through the connection flow path 170 into the recovery flow path 159, and the particles to be recovered are recovered into the recovery flow path 159. An enlarged view of the particle separation unit 157 is shown in FIG. 3. As shown in FIG. 3A, the main flow path 155 and the recovery flow path 159 are communicated via a connection flow path 170 that is coaxial with the main flow path 155. The particles to be recovered flow into the recovery flow path 159 through the connection flow path 170 as shown in FIG. 3B. Micro-particles that are not the particles to be recovered flow into the waste flow path 158 as shown in FIG. 3C.

[0066] Enlarged views of the vicinity of the connection flow path 170 are shown in FIGS. 6A and 6B. FIG. 6A is a schematic perspective view of the vicinity of the connection flow path 170. FIG. 7B is a schematic cross-sectional view in a plane passing through the center line of the liquid supply flow path 161 and the center line of the connection flow path 170. The connection flow path 170 includes a flow path 170a on the detection region 156 side (hereinafter also referred to as the upstream connection flow path 170a), a flow path 170b on the recovery flow path 159 side (hereinafter also referred to as the downstream connection flow path 170b), and a connection portion 170c between the connection flow path 170 and the liquid supply flow path 161. The liquid supply flow path 161 is provided so as to be substantially perpendicular to the axis of the flow path of the connection flow path 170. In FIGS. 6A and 6B, two liquid supply flow paths 161 are provided so as to face each other at a substantially central position of the connection flow path 170, but only one liquid supply flow path may be provided.

[0067] The shape and dimensions of the cross-section of the upstream connection channel 170a may be the same as those of the downstream connection channel 170b. For example, as shown in FIGS. 6A and 6B, both the cross-section of the upstream connection channel 120a and the cross-section of the downstream connection channel 120b may be substantially circular with the same dimensions. Alternatively, both of these two cross-sections may be rectangular (e.g., square or rectangular, etc.) with the same dimensions.

[0068] From the two liquid supply channels 161, the second liquid is supplied to the connection channel 170 as indicated by the arrows in FIG. 6B. The second liquid flows from the connection portion 170c into both the upstream connection channel 170a and the downstream connection channel 170b.

[0069] When the recovery process is not performed, the second liquid flows as follows. The second liquid that has flowed into the upstream connection channel 170a exits from the connection surface with the main channel 155 of the connection channel 170 and then flows separately into the two waste channels 158. By the second liquid exiting from the connection surface in this way, it is possible to prevent the first liquid and fine particles that do not need to be recovered into the recovery channel 159 from entering the recovery channel 159 through the connection channel 170. The second liquid that has flowed into the downstream connection channel 170b flows into the recovery channel 159. As a result, the inside of the recovery channel 159 is filled with the second liquid, and the second liquid becomes, for example, a dispersion medium for emulsion formation.

[0070] Even when the recovery process is performed, the second liquid can be supplied from the two liquid supply channels 161 to the connection channel 170. However, due to the pressure fluctuations in the recovery channel 159, particularly by generating a negative pressure in the recovery channel 159, a flow is formed from the main channel 155 through the connection channel 170 to the recovery channel 159. That is, a flow is formed from the main channel 155 through the upstream connection channel 170a, the connection portion 170c, and the downstream connection channel 170b in this 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 being wrapped in the first liquid. By performing the recovery process, an emulsion can be formed, for example, in the recovery channel 159 or in a container connected to the end 163 of the recovery channel via a channel, for example.

[0071] The shape and / or dimensions of the cross-section of the upstream connection channel 120a may be different from the shape and / or dimensions of the downstream connection channel 120b. Examples of the different dimensions of these two channels are shown in FIGS. 7A and 7B. As shown in FIGS. 7A and 7B, the connection channel 180 includes a channel 180a on the detection region 156 side (hereinafter also referred to as the upstream connection channel 180a), a channel 180b on the recovery channel 159 side (hereinafter also referred to as the downstream connection channel 180b), and a connection portion 180c between the connection channel 180 and the liquid supply channel 161. The cross-sections of both the upstream connection channel 180a and the downstream connection channel 180b have a substantially circular shape, but the diameter of the cross-section of the latter is larger than the diameter of the cross-section of the former. By making the diameter of the cross-section of the latter larger than that of the former, compared with the case where the diameters of both are the same, the recovered particles to be collected that have already been separated into the recovery channel 159 immediately after the microparticle separation operation by the negative pressure described above can be more effectively prevented from being released from the connection channel 180 to the main channel 155. For example, when the cross-sections of both the upstream connection channel 180a and the downstream connection channel 180b are rectangular, by making the area of the cross-section of the latter larger than the area of the cross-section of the former, as described above, the already recovered microparticles can be more effectively prevented from being released from the connection channel 180 to the main channel 155.

[0072] In the recovery step S103, due to the pressure fluctuation in the recovery channel 159, the particles to be recovered are recovered into the recovery channel through the connection channel. This recovery may be performed, for example, by generating a negative pressure in the recovery channel 159 as described above. The negative pressure may be generated, for example, by deforming the wall defining the recovery channel 159 by an actuator 107 (particularly a piezo actuator) attached outside the microchip 150. Due to the negative pressure, the flow entering the recovery channel 159 can be formed. In order to generate the negative pressure, for example, the actuator 107 can be attached outside the microchip 150 so that the wall of the recovery channel 159 can be deformed. By the deformation of the wall, the internal space of the recovery channel 159 can be changed and a negative pressure can be generated. The actuator 107 can be, for example, a piezo actuator. When the particles to be recovered are sucked into the recovery channel 159, the sample liquid constituting the laminar flow or the sample liquid and the sheath liquid constituting the laminar flow can 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 recovered in a second liquid that is immiscible with the first liquid in the recovery channel 159 while being wrapped in the first liquid. Thereby, as described above, an emulsion having the second liquid as a dispersion medium and the first liquid as a dispersed phase is formed in the recovery channel 159.

[0074] In order to prevent fine particles other than the particles to be recovered from entering the recovery channel 159 through the connection channel 170, the connection channel 170 is provided with a liquid supply channel 161. A second liquid that is immiscible with the liquid (sample liquid and sheath liquid) flowing in the main channel 155 is introduced into the connection channel 170 from the liquid supply channel 161. A part of the second liquid introduced into the connection channel 170 forms a flow from the connection channel 170 toward the main channel 155, thereby preventing fine particles other than the particles to be recovered from entering the recovery channel 159. The second liquid forming the flow from the connection channel 170 toward the main channel 155 flows through the waste channel 158 in the same manner as the first liquid without flowing through the inside of the main channel 155 due to the flow of the first liquid flowing through the main channel 155 into the waste channel 158. Note that the remainder of the second liquid introduced into the connection channel 170 flows into the recovery channel 159. Thereby, the inside of 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 inside of the recovery channel 159 with the second liquid, the second liquid may be supplied from the liquid supply channel 161 to the connection channel 170. By this supply, the second liquid flows from the connection channel 170 into the recovery channel 159, and thereby the inside of the recovery channel 159 can be filled with the second liquid.

[0076] The laminar flow that has flowed into the waste channel 158 can be discharged to the outside of the microchip at the waste channel end 160. Also, the particles to be recovered that have been recovered into the recovery channel 159 can be discharged to the outside of the microchip at the recovery channel end 161.

[0077] A container 171 can be connected to the recovery channel end 163 via a channel such as a tube 172 as shown in FIG. 5, for example. As shown in the figure, an emulsion in which the first liquid containing the particles to be recovered is a disperse phase and the second liquid is a dispersion medium is recovered into the container 171. Thus, according to one embodiment of the present technology, the fine particle recovery device 100 may include a channel for recovering an emulsion containing the particles to be recovered into a container. Also, when the recovery operation is performed with the recovery channel end 163 closed, a plurality of emulsion particles can be retained in the recovery channel 159. After the completion of the recovery operation, an assay such as single cell analysis can be continuously performed in the recovery channel 159.

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

[0079] Also, as shown in FIGS. 1 and 2, in the microparticle separation microchip used in the method of the present technology, the main flow path, the connection flow path, and the recovery flow path may be arranged linearly. When these three flow paths are arranged linearly (particularly coaxially), for example, compared with the case where the connection flow path and the recovery flow path are arranged at an angle with respect to the main flow path, the recovery process can be performed more efficiently. For example, the suction amount required to guide the particles to be recovered into the connection flow path can be reduced. Also, as shown in FIGS. 1 and 2, in the microparticle separation microchip used in the method of the present technology, the fine particles are arranged in a substantially single row in the main flow path and flow toward the connection flow path. Therefore, the suction amount in the recovery process can also be reduced.

[0080] Also, in the method of the present technology, the liquid supply flow path supplies liquid (particularly, a second liquid) to the connection flow path. As a result, a flow is formed in the connection flow path that flows from the connection position between the liquid supply flow path and the connection flow path toward the main flow path, preventing the liquid flowing through the main flow path from entering the connection flow path, and also preventing fine particles other than the particles to be recovered from flowing through the connection flow path into the recovery flow path. When performing the recovery process, as described above, for example, due to the negative pressure generated in the recovery flow path, the first liquid containing one particle to be recovered passes through the connection flow path and is recovered into the second liquid in the recovery flow path. Thereby, emulsion particles containing one particle to be recovered are formed in the second liquid.

[0081] In the present technology, when the microparticles determined to be particles to be collected in the determination step reach, for example, a piezo actuator at an appropriate timing (for example, when they reach the particle separation unit 157), the hydrophilic solution containing the particles to be collected is collected into the collection channel 159 to form emulsion particles. In the determination step, for example, by using a peak signal and an area signal to determine whether the particles are those to be collected, it is also possible to determine whether the particles are a single microparticle (singlet), a combination of two microparticles (doublet), or a combination of three microparticles (triplet). Therefore, it is possible to avoid the formation of emulsion particles containing two or more microparticles in one emulsion particle. Therefore, 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 a combination of two or more microparticles in this way, for example, an operation of removing a combination of two or more microparticles before the emulsion formation operation by a cell sorter or the like can be omitted. Conversely, it is also possible to determine the characteristics of each of two or more microparticles that are close enough to be drawn into the connection channel simultaneously in one collection operation. For example, two microparticles having the same characteristics can be confined in one emulsion particle, or a combination of microparticles having specified different characteristics can also be confined in one emulsion particle.

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

[0083] The microchip 150 for separating microparticles is provided with a piezo element (piezo actuator) attached to the outer surface of the microchip (particularly, the outer surface corresponding to the bulging portion near the connection portion of the recovery channel 159 with the connection channel 170) so as to be able to vary the volume in the recovery channel 159. 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 the hydrophilic sheath solution merged at the merging portion 162, and a laminar flow in which the hydrophilic sample solution was surrounded by the hydrophilic sheath solution was formed. The laminar flow contained beads arranged in a substantially single row, and the laminar flow flowed in the main channel 155 toward the connection channel 170. In parallel with the introduction of the hydrophilic sample solution and the hydrophilic sheath solution, a hydrophobic liquid was supplied from the liquid supply channel 161 to the connection channel 170. By supplying the hydrophobic liquid from the liquid supply channel 161 to the connection channel 170, the laminar flow was prevented from entering the recovery channel 159 through the connection channel 170, and the recovery channel 159 was filled with the hydrophobic liquid. When the beads passed through the irradiation position of the laser light irradiated to the detection region 156 in the main channel 155, the beads were irradiated with the laser light and light was generated. The generated light was detected, and based on the characteristics of the detected light, it was determined whether each bead was to be recovered.

[0084] At the timing when the beads determined to be recovered reach the vicinity of the connection channel 170, the piezoelectric element is driven, and the inner cavity of the recovery channel 159 is deformed. As a result, the beads are recovered into the recovery channel 159 through the connection channel 170. The operations for the recovery were as follows: (i) deforming the recovery channel 159 over 10 μs to apply a negative pressure into the recovery channel 159, (ii) maintaining the deformed state for 10 μs, and (iii) releasing the negative pressure over 10 μs to restore the deformation. By repeating the operations (i) to (iii) above, an emulsion containing emulsion particles including one bead in the hydrophobic liquid was formed in the recovery channel 159. The emulsion had the hydrophobic liquid as the dispersion medium and emulsion particles containing a hydrophilic liquid as the dispersed phase.

[0085] By performing the operations (i) to (iii) above, it is shown in FIG. 8A that emulsion particles containing one bead are formed in the recovery channel 159. FIG. 8A will be described below. (a) of FIG. 8A is a photograph showing a state where beads (indicated by white arrows) are flowing toward the connection channel 170. At the time point of FIG. 8A, the hydrophilic liquid is flowing from the main channel 155 toward the particle separation section 157 and, without flowing into the connection channel 170, is flowing into two waste channels 158 that branch off from the main channel 155. The hydrophobic liquid is supplied from the liquid supply channel 161 to the connection channel 170 and flows into both the main channel 155 and the recovery channel 159. The hydrophobic liquid that has flowed into the main channel 155 immediately after exiting the connection channel 170 is separated and flows into the two waste channels 158 by the flow of the hydrophilic liquid (the vicinity indicated by a in FIG. 8A flows along the wall of the waste channel 158). (b) of FIG. 8A is a photograph at the time when the beads are closer to the connection channel. At this time point, the operation for bead recovery is started. That is, by driving the piezoelectric element, the deformation of the inner cavity of the recovery channel 159 is started. Figure 8A (c) is a photograph showing the state in which the hydrophilic liquid advances into the recovery channel 159. From this photograph, it can be seen that at the time of Figure 8C, the beads are enveloped in the hydrophilic liquid, which is in turn surrounded by the hydrophobic liquid. In other words, an emulsion particle P containing one bead is formed. FIG. 8A(d) is a photograph of the emulsion particles P immediately before they enter the recovery channel 159 from the connection channel 170. FIG. 8A(e) is a photograph of the emulsion particle P immediately after it enters the recovery channel 159. It can be seen that the emulsion particle P contains one bead. FIG. 8A(f) is a photograph showing that the emulsion particles P have flowed further downstream within the ocean current flow path 159. As described above, emulsion particles containing one bead were formed by the method according to the present technology.

[0086] The size of the emulsion particles can be adjusted by controlling the time and amount of deformation of each of the above operations (i) to (iii). In other words, the size of the emulsion particles in the recovery channel can be controlled by the volume of the recovery channel that deforms and expands. Therefore, it can be easily controlled by the amount of deformation of the piezoelectric element, i.e., the electrical drive waveform of the piezoelectric element. For example, by increasing the amount of deformation of the recovery channel lumen by the piezoelectric element, the amount of hydrophilic liquid drawn into recovery channel 159 increases, thereby making it possible to increase the size of the emulsion particles. Conversely, by decreasing the amount of deformation, the amount of hydrophilic liquid drawn into recovery channel 159 decreases, thereby making it possible to reduce the size of the emulsion particles. Also, by making the retention time in (ii) longer or shorter, the size of the emulsion particles can be made larger or smaller. This is shown in FIG. 8B. FIG. 8B is a photograph showing the sizes of the emulsion particles when the retention time in (ii) 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). Also, by adjusting the volume of the connection flow path 170 (particularly each of the upstream connection flow path 170a, the connection portion 170c, and the downstream connection flow path 170b), the drawing of the emulsion particles can be adjusted.

[0087] In the example described above, by supplying the second liquid from the liquid supply flow path to the connection flow path, the first liquid flowing in the main flow path is prevented from entering the connection flow path. In the present technology, in order to prevent the first liquid flowing in the main flow path from entering the connection flow path, a valve for preventing the first liquid from proceeding to the recovery flow path may be provided in the connection flow path. Only when the recovery step is performed, the valve is released, and the first liquid can flow through the connection flow path to the recovery flow path.

[0088] (2-4) Other steps

[0089] The method for recovering fine particles of the present technology may further include other steps in addition to the flow-through step, the determination step, and the recovery step described above. Examples of the other steps will be described below.

[0090] (2-4-1) Culturing step

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

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

[0093] According to one preferred embodiment of the present technology, the microparticles are cells, cell aggregates, or synthetic particles, and the microparticles can 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 in the emulsion particles constituting the emulsion can be destroyed. As described above, the microparticle recovery method of the present technology may include a destruction step of destroying the recovered microparticles after the recovery step. According to another preferred embodiment of the present technology, particles other than cells, cell aggregates, and synthetic particles may be destroyed. That is, the microparticle recovery method of the present technology may include a destruction step of destroying the particles to be recovered recovered after the recovery step.

[0094] The destruction can preferably be carried out while maintaining the emulsion particles. Thereby, for example, components of cell components (such as intracellular components, cell membrane components, and cell wall components) or synthetic particles are released into the emulsion particles, and the cell components or the components can be processed separately from the components of other microparticles. The processing can be, for example, analysis, separation, or amplification of the cell components or the components. Examples of the cell components or the components include, but are not limited to, DNA, RNA, proteins, peptides, amino acids, lipids, saccharides, or cell organelles. For example, when the cell component or the component is DNA, a sample for DNA barcoding can be prepared by the destruction step or by further processing the emulsion particles obtained by the destruction step.

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

[0096] According to one preferred embodiment of the present technology, after the recovery step, detection or analysis of the components of the particles to be recovered, or a reaction between the components of the particles to be recovered and other components can be performed. In order to perform the detection or the reaction, for example, emulsion particles formed according to the present technology can be merged with other emulsion particles. After the merging, detection or analysis of the components of the particles to be recovered, or a reaction between the components and other components can be performed within the merged emulsion particles. By the merging, for example, Cellular Indexing of Transcriptomes and Epitopes by Sequencing (also referred to as CITE-seq) becomes possible. For example, assume that the particles to be recovered are cells to which an antibody bound with an oligo barcode having a poly-A sequence is bound. In the recovery step, emulsion particles containing the cells are formed. Then, the emulsion particles are merged with emulsion particles containing beads or gels having barcode sequences. By the merging, cell surface proteins or intracellular mRNAs of the cells can be detected. Furthermore, the emulsified cells or cell aggregates (such as spheroids or organoids, etc.) may be reacted with a drug. Thereby, the response of the cells or cell aggregates to the drug can be detected or analyzed. Also, in the detection, biomolecules may be detected. Detection of biomolecules can be performed, for example, by a wash free assay. In a wash free assay, for example, LOCI, FRET, BRET, or FlimPIA methods can be used.

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

[0098] According to one preferred embodiment of the present technology, after the recovery step, synthesis of a chemical substance by the particles to be recovered can be performed. For example, the synthesis may be performed within the emulsion particles formed in the recovery step. For example, by encapsulating reagents for cell-free expression within the emulsion particles, it becomes possible to produce antibodies in vitro. Examples of the reagents for cell-free expression include linear DNA and E.coli S30 Extract system for linear DNA (promega). Also, for example, protein synthesis by a cell-free protein synthesis system may be performed within the emulsion particles.

[0099] (2-5) Microparticle separation mechanism and microparticles

[0100] The microparticle separation mechanism used in the microparticle separation method of the present technology may be a structure or device having the flow path structure described above, for example, a chip having a microchannel, particularly a microchip for microparticle separation. In the present technology, "micro" means that at least a part of the flow path included in the microchip for microparticle separation has dimensions on the order of μm, particularly a cross-sectional dimension on the order of μm. That is, in the present technology, a "microchip" refers to a chip including a flow path on the order of μm, particularly a chip including a flow path having a cross-sectional dimension on the order of μm. For example, a chip including a particle separation section composed of a flow path having a cross-sectional dimension on the order of μm can be called a microchip according to the present technology. For example, among the particle separation section 157, the cross-section of the main flow path 155 is, for example, rectangular, and the width of the main flow path 155 is, for example, 100 μm to 500 μm within the particle separation section 157, and particularly can be 100 μm to 300 μm. The width of the branch flow path branching from the main flow path 155 may be smaller than the width of the main flow path 155. The cross-section of the connection flow path 170 is, for example, circular, and the diameter of the connection flow path 170 at the connection portion between the connection flow path 170 and the main flow path 155 can be, for example, 10 μm to 60 μm, particularly 20 μm to 50 μm. These dimensions regarding the flow path may be appropriately changed according to the size of the microparticles, particularly the size of the particles to be collected.

[0101] The microchip 150 for microparticle separation can be manufactured by a method known in the art. For example, the microchip 150 for biological particle separation can be manufactured by bonding two or more substrates on which predetermined flow paths are formed. The flow paths may be formed in all of the two or more substrates (particularly two substrates), or may be formed only in a part of the two or more substrates (particularly one of the two substrates). In order to make it easier to adjust the position when bonding the substrates, it is preferable that the flow paths are formed only in one substrate.

[0102] As the material for forming the microchip 150 for separating microparticles, materials known in the art can be used. For example, polycarbonate, cycloolefin polymer, polypropylene, PDMS (polydimethylsiloxane), polymethyl methacrylate (PMMA), polyethylene, polystyrene, glass, and silicon can be mentioned, but are not limited thereto. In particular, polymer materials such as polycarbonate, cycloolefin polymer, and polypropylene are particularly preferred because they have excellent processability and can be used to manufacture microchips inexpensively using a molding device.

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

[0104] In the above description, the embodiment in which the above-described flow path group is formed in the disposable microchip 150 for separating microparticles has been described. However, in the present technology, the above-described flow path group may not be formed in the microchip 150. For example, the above-described flow path group may be formed in a substrate such as plastic or glass. Further, the above-described flow path group may have a two-dimensional or three-dimensional structure.

[0105] In the present technology, the microparticles may be particles having a size that can flow in the flow path in the microparticle separation mechanism (for example, the microchip for separating microparticles). In the present technology, the microparticles may be appropriately selected by those skilled in the art. In the present technology, the microparticles may include biological microparticles such as cells, cell aggregates, microorganisms, and liposomes, and synthetic microparticles such as gel particles, beads, latex particles, polymer particles, and industrial particles. Biological microparticles (also referred to as biological particles) may include chromosomes, liposomes, mitochondria, and organelles (cell organelles) that make up various cells. Cells may include animal cells (such as blood cells) and plant cells. Cells may be, in particular, blood cells or tissue cells. The blood cells may be, for example, floating cells such as T cells and B cells. The tissue cells may be, for example, adherent cultured cells or adherent cells separated from tissues. Cell aggregates may include, for example, spheroids and organoids. 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, for example, those extracted from cells or those contained in blood samples or other liquid samples. Synthetic microparticles may be, for example, microparticles composed of 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 colloids 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 or substantially spherical, or may be non - spherical. The size and mass of the microparticles can be appropriately selected by those skilled in the art according to the size of the flow path of the microchip. On the other hand, the size of the flow path of the microchip can also be appropriately selected according to the size and mass of the microparticles. In the present technology, chemical or biological labels, such as fluorescent dyes or fluorescent proteins, may be attached to the microparticles as needed. Such labels can make the detection of the microparticles easier. The labels to be attached can be appropriately selected by those skilled in the art. Molecules that specifically react with the microparticles (such as antibodies, aptamers, DNA, or RNA) can bind to the labels. According to one embodiment of the present technology, the microparticles may be biological particles, particularly cells.

[0106] In the present technology, the microparticles collected in the recovery channel may be further subjected to microparticle fractionation processing. The further microparticle fractionation processing may be performed, for example, using the microparticle fractionation microchip 150 described above, or may be performed using other microparticle fractionation microchips. For example, in the further microparticle fractionation processing, any one or a combination of two or more of the steps described in (2-1) to (2-4) above may be performed. For example, when the determination step described in (2-2) above is performed in the further microparticle fractionation processing, in order to recover emulsion particles containing microparticles, in the determination step, determination may be performed on the microparticles, determination may be performed on the emulsion particles, or determination may be performed on both the microparticles and the emulsion particles. Thus, also in the further microparticle fractionation processing, based on the information obtained from the microparticles and / or the emulsion particles, it may be determined whether the microparticles or the emulsion particles are the objects to be recovered. Further, in the further microparticle fractionation processing, an emulsion containing emulsion particles containing microparticle-containing emulsion particles may be subjected to the fractionation processing.

[0107] (3) Another example of the flow path structure

[0108] (3-1) Flow path structure in which the main flow path and the waste flow path are arranged linearly side by side

[0109] In one embodiment of the present technology, the connection channel and the recovery channel may not be arranged linearly with the main flow path. An example of the microparticle fractionation microchip in this embodiment will be described below with reference to FIG. 9.

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

[0111] In addition to the main flow path 355, the microchip 350 for microparticle separation includes a recovery flow path 359 where particles to be recovered are recovered, a connection flow path 370 connecting the main flow path 355 and the recovery flow path 359, and a liquid supply flow path 361 connected to the connection flow path 370 so as to be able to supply liquid.

[0112] The main flow path 355 further includes a waste flow path 358 through which microparticles that are not particles to be recovered flow. In the microchip 150 for microparticle separation described with reference to FIG. 1, two waste flow paths 158 branch off from the main flow path 155, and the connection flow path 170, the recovery flow path 159, and the main flow path 155 are arranged linearly. On the other hand, the microchip 350 for microparticle separation in FIG. 9 has a flow path structure in which the main flow path 355 and the waste flow path 358 are arranged linearly, and the connection flow path 370 and the recovery flow path 359 branch off from the main flow path 355. Thus, in the microchip for microparticle separation used in the microparticle recovery method of the present technology, the connection flow path and the recovery flow path do not have to be arranged linearly with the main flow path. For example, the main flow path and the waste flow path may be arranged linearly, and the connection flow path and the recovery flow path may have a flow path structure that branches off from the main flow path.

[0113] (3-2) Flow path structure having a plurality of recovery flow paths

[0114] In other embodiments of the present technology, the microchip for microparticle separation may include one or more channel structures including a main channel through which microparticles flow, a recovery channel for recovering particles to be recovered among the microparticles, a connection channel connecting the main channel and the recovery channel, and a liquid supply channel connected to the connection channel so as to be able to supply a liquid. The microchip for microparticle separation described above with reference to FIG. 1 has one such channel structure. In contrast, as described below with reference to FIGS. 10 and 11, the microchip for microparticle separation used in the microparticle recovery method of the present technology may have two or more such channel structures.

[0115] FIG. 10 shows a schematic diagram of a region including a detection region and a particle separation section of a microchip for microparticle separation according to the present technology. The microchip 450 for microparticle separation shown in FIG. 10 has a channel structure in which a sample liquid channel 452 and a sheath liquid channel 454 merge at a merging section 462 to form a main channel 455, and a detection region 456 is provided in the main channel 455, similar to the microchip 150 for microparticle separation described with reference to FIG. 1. A laminar flow in which a sample liquid containing microparticles P flows in the main channel 455 surrounded by the sheath liquid.

[0116] In addition to the main channel 455, the microchip 450 for microparticle separation includes a recovery channel 459-1 for recovering particles to be recovered, a connection channel 470-1 connecting the main channel 455 and the recovery channel 459-1, and a liquid supply channel 461-1 connected to the connection channel 470-1 so as to be able to supply a liquid. The microchip 450 for microparticle separation further includes a recovery channel 459-2 for recovering particles to be recovered, a connection channel 470-2 connecting the main channel 455 and the recovery channel 459-2, and a liquid supply channel 461-2 connected to the connection channel 470-2 so as to be able to supply a liquid. That is, the microchip 450 for microparticle separation has two such channel structures. In the microchip 450 for microparticle separation, the main channel 455 is shared by these two channel structures. Also, the connection channel 470-1 is downstream of the connection channel 470-2 and is connected to the main channel 455.

[0117] For example, two emulsions containing different particles to be recovered can be formed by the microparticle separation microchip 450. In this case, in the determination step, it is determined whether the microparticle is, for example, either 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 belonging to the particle A or B to be recovered may be appropriately selected by the user. In the recovery step, when the microparticle is the particle A to be recovered, the microparticle is recovered through the connection channel 470-1 to the recovery channel 459-1. The microparticle, which is the particle A to be recovered, is recovered into the second liquid in the recovery channel 459-1 while being contained in the first liquid. In the recovery step, when the microparticle is the particle B to be recovered, the microparticle is recovered through the connection channel 470-2 to the recovery channel 459-2. The microparticle, which is the particle B to be recovered, is recovered into the second liquid in the recovery channel 459-2 while being contained in the first liquid. In the recovery step, when the microparticle is neither the particle A nor the particle B to be recovered, the microparticle flows into the waste channel 458. In the above manner, an emulsion containing the particle A to be recovered and an emulsion containing the particle B to be recovered are formed.

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

[0119] Even with the microparticle separation microchip 550, two emulsions containing different particles to be recovered can be formed. Also in this case, in the determination step, it is determined whether the microparticle is, for example, either 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 belonging to the particle A or B to be recovered may be appropriately selected by the user. In the recovery step, when the microparticle is the particle A to be recovered, the microparticle is recovered through the connection channel 570-1 to the recovery channel 559-1. The microparticle, which is the particle A to be recovered, is recovered in the second liquid in the recovery channel 559-1 in a state of being contained in the first liquid. In the recovery step, when the microparticle is the particle B to be recovered, the microparticle is recovered through the connection channel 570-2 to the recovery channel 559-2. The microparticle, which is the particle B to be recovered, is recovered in the second liquid in the recovery channel 559-2 in a state of being contained in the first liquid. In the recovery step, when the microparticle is neither the particle A nor the particle B to be recovered, the microparticle flows into the waste channel 558. In the above manner, an emulsion containing the particle A to be recovered and an emulsion containing the particle B to be recovered are formed.

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

[0121] The present technology also provides a microchip for microparticle separation having a flow path structure including a main flow path through which microparticles flow, a recovery flow path in which target 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. The main flow path in the microchip has a determination region used for determining whether the microparticles flowing in the first liquid are target particles to be recovered. The microparticles determined to be target 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. The microchip for microparticle separation is the same as the microchip for microparticle separation described in 1. above, and the description thereof also applies to the present embodiment.

[0122] 3. Third Embodiment (Microparticle Recovery Device)

[0123] The present technology also provides a microparticle recovery device including a microchip for microparticle separation having a flow path structure including a main flow path through which microparticles flow, a recovery flow path in which target 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, a first liquid supply unit that supplies a first liquid containing microparticles 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 microparticles flowing in the main flow path are target particles to be recovered. The microparticle recovery device is the same as the microparticle recovery device described in 1. above, and the description thereof also applies to the present embodiment.

[0124] The microchip for microparticle separation is also the same as the microchip for microparticle separation described in 1. above, and the description thereof also applies to the present embodiment. The first liquid supply unit may be, for example, at least one component for forming the laminar flow described in (2) of 1. above, and may include, for example, a sample liquid flow path 152 and a sheath liquid flow path 154. Further, the first liquid supply unit may also include flow paths (such as tubes) respectively connected to these inlets for introducing the sample liquid and the sheath liquid to the sample liquid inlet 151 and the sheath liquid inlet 153, and a sample liquid-containing container and a sheath liquid-containing container connected to the flow paths. The second liquid supply unit may be, for example, at least one component for supplying a second liquid to the liquid supply flow path 161 described in (2) of 1. above, and may include, for example, a container for storing the second liquid supplied to the liquid supply flow path 161, and a flow path (such as a tube connecting the chip and the container) connecting the container and the liquid supply flow path. The determination unit is the same as the determination unit described in 1. above, and the description thereof also applies to this embodiment.

[0125] Further, the microchip for microparticle separation may be removable from the microparticle recovery device. By the microchip for microparticle separation being removable from the device, a new microchip for microparticle separation 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 producing an emulsion, which includes the through-flow step, the determination step, and the recovery step described in the above "1. First Embodiment (Method for Recovering Microparticles)". The production method may include other steps described in the above "(2-4) Other Steps". The description given in the above "1. First Embodiment (Method for Recovering Microparticles)" also applies to the method for producing an emulsion of this technology.

[0128] By the emulsion production method of the present technology, an emulsion containing emulsion particles containing one microparticle (particularly a particle to be recovered) at a high content ratio can be produced. For example, by the production method of the present technology, an emulsion in which the ratio of the number of emulsion particles containing one microparticle is, for example, 70% or more, preferably 80% or more, more preferably 90% or more, and even more preferably 95% or more based on the total number of emulsion particles can be produced. In single-cell analysis using an emulsion, it is important to increase the content ratio of the emulsion containing one cell, and when the number of cells to be analyzed is small, it is particularly important to increase the content ratio. Since the production method of the present technology can produce an emulsion with a high ratio of the number of emulsion particles containing one microparticle, it is extremely useful for the single-cell analysis.

[0129] 5. Fifth Embodiment (Emulsion)

[0130] The present technology also provides an emulsion containing microparticle-containing emulsion particles in which the ratio of the number of emulsion particles containing one microparticle is 70% or more based on the total number of emulsion particles. The ratio is preferably 80% or more, more preferably 90% or more, and even more preferably 95% or more. Since the emulsion of the present technology contains emulsion particles containing one microparticle at such a high content ratio, it is suitable for, for example, single-cell analysis. The emulsion particles containing the one microparticle can be formed as described in the above "1. First Embodiment (Microparticle Recovery Method)". Further, the emulsion can be produced as described in, for example, the above "1. First Embodiment (Microparticle Recovery Method)" or "4. Fourth Embodiment (Emulsion Production Method)".

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

[0132] Incidentally, the present technology can also have the following configuration. 〔1〕A main flow path through which fine particles flow, A recovery flow path in which target particles to be recovered among the fine particles are recovered, A connection flow path connecting the main flow path and the recovery flow path, In a fine particle separation mechanism having a flow path structure including a liquid supply flow path connected to the connection flow path so as to be able to supply a liquid, a flowing step of flowing a first liquid containing fine particles into the main flow path, A determination step of determining whether the fine particles flowing through the main flow path are target particles to be recovered, A recovery step of recovering the target particles to be recovered into the recovery flow path, and In the recovery step, the target particles to be recovered are recovered into a second liquid that is immiscible with the first liquid in the recovery flow path while being contained in the first liquid, Fine particle recovery method. 〔2〕The fine particle recovery method according to 〔1〕, wherein an emulsion in which the second liquid is a dispersion medium and the first liquid is a dispersed phase is formed in the recovery flow path by implementing the fine particle recovery method. 〔3〕The fine particle recovery method according to 〔1〕 or 〔2〕, wherein an emulsion is formed by implementing the fine particle recovery method, and at least a part of the droplets constituting the emulsion contains one of the target particles to be recovered. 〔4〕The fine particle recovery method according to any one of 〔1〕 to 〔3〕, wherein the first liquid is hydrophilic and the second liquid is hydrophobic. 〔5〕The method for recovering microparticles according to any one of 〔1〕to 〔4〕, wherein the kinematic viscosity of the second liquid is 1 / 100 to 100 times the kinematic viscosity of the first liquid. 〔6〕The method for recovering microparticles according to any one of 〔1〕to 〔5〕, wherein the flowing step, the determination step, and the recovery step are performed while supplying the second liquid from the liquid supply channel to the connection channel. 〔7〕The main channel branches into at least one waste channel through which microparticles other than the connection channel and the particles to be recovered flow, and the liquid supply channel supplies liquid to the connection channel. The method for recovering microparticles according to any one of 〔1〕to 〔6〕. 〔8〕The method for recovering microparticles according to any one of 〔1〕to 〔7〕, wherein a valve is provided in the connection channel to prevent the first liquid from advancing to the recovery channel. 〔9〕The method for recovering microparticles according to any one of 〔1〕to 〔8〕, wherein in the flowing step, the microparticles flow in a substantially single row in the main channel toward the connection channel. 〔10〕The microparticle separation mechanism has a channel structure in which a sample channel through which a liquid containing microparticles flows and a sheath channel through which a liquid not containing microparticles flow are connected to the main channel of the confluence part, and the microparticles in the main channel after the confluence part flow in a substantially single row in the main channel, and a laminar flow containing microparticles flowing in a single row is formed by the channel structure. The method for recovering microparticles according to any one of 〔1〕to 〔9〕. 〔11〕The method for recovering microparticles according to any one of 〔1〕to 〔10〕, wherein in the determination step, light is irradiated onto the microparticles flowing in the main channel, and based on the light generated by the irradiation, it is determined whether the microparticles are particles to be recovered. 〔12〕The method for recovering microparticles according to any one of 〔1〕to 〔11〕, wherein in the recovery step, due to the pressure fluctuation in the recovery channel, the particles to be recovered are recovered into the recovery channel through the connection channel. 〔13〕The method for recovering microparticles according to any one of 〔1〕to 〔12〕, wherein the main flow path, the connection flow path, and the recovery flow path are arranged linearly. 〔14〕The method for recovering microparticles according to any one of 〔1〕to 〔13〕, wherein the microparticles are cells or cell aggregates, and the first liquid is a culture solution of the microparticles. 〔15〕The method for recovering microparticles according to any one of 〔1〕to 〔14〕, wherein the microparticles are cells, cell aggregates, or synthetic particles, and the microparticles are destroyed after the recovery step. 〔16〕The method for recovering microparticles according to any one of 〔1〕to 〔13〕, wherein the recovered microparticles in the recovery flow path are subjected to further microparticle separation processing. 〔17〕The method for recovering microparticles according to any one of 〔1〕to 〔6〕, wherein the microparticle separation microchip includes one or more of the flow path structures. 〔18〕A main flow path through which microparticles flow, A recovery flow path for recovering target particles to be recovered among the microparticles, 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, and having 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 target particles to be recovered, The microparticles determined to be target particles to be recovered are recovered in the second liquid immiscible with the first liquid in the recovery flow path in a state of being contained in the first liquid. A microchip for microparticle separation. 〔19〕A main flow path through which microparticles flow, A recovery flow path for recovering target particles to be recovered among the microparticles, 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 for supplying a first liquid containing microparticles 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; A determination unit that determines whether the microparticles flowing in the main flow path are particles to be recovered; A microparticle recovery device comprising the same. 〔20〕The microparticle separation microchip is detachable from the microparticle recovery device, the microparticle recovery device according to 〔19〕. 〔21〕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 that connects the main flow path and the recovery flow path, In a microparticle separation mechanism having a flow path structure including a liquid supply flow path connected to be able to supply liquid to the connection flow path, a flowing step of flowing a first liquid containing microparticles through the main flow path, A determination step of determining whether the microparticles flowing through the main flow path are particles to be recovered, A recovery step of recovering the particles to be recovered into the recovery flow path, and In the recovery step, the particles to be recovered are recovered into the second liquid that is immiscible with the first liquid in the recovery flow path while being contained in the first liquid. A method for producing an emulsion containing emulsion particles containing microparticle-containing emulsion particles. 〔22〕An emulsion containing emulsion particles containing microparticle-containing emulsion particles, wherein the ratio of the number of emulsion particles containing one microparticle is 70% or more with respect to the total number of emulsion particles.

Explanation of Signs

[0133] 100 Microparticle recovery device 150 Microparticle separation microchip 155 Main flow path 159 Recovery flow path 161 Liquid supply flow path 170 Connection flow path

Claims

1. A main flow path through which microparticles flow; a collection flow path through which collection target particles are collected from among the microparticles; 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 liquid to the connection flow path, in a microparticle sorting mechanism having a flow path structure including the following steps: a determination step of determining whether the microparticles flowing through the main flow path are particles to be collected; A recovery step of recovering the recovery target particles into the recovery flow path, and In the recovery step, the recovery target particles are recovered in a state where they are contained in the first liquid, into a second liquid that is immiscible with the first liquid and is in the recovery flow path. Microparticle recovery method.

2. 2. The method for recovering fine particles according to claim 1, wherein, by carrying out the method for recovering fine particles, an emulsion is formed in the recovery flow path, the emulsion including the second liquid as a dispersion medium and the first liquid as a dispersoid.

3. 2. The method for recovering fine particles according to claim 1, wherein an emulsion is formed by carrying out the method for recovering fine particles, and at least a part of droplets constituting the emulsion includes one of the particles to be recovered.

4. The method for recovering fine particles according to claim 1 , wherein the first liquid is hydrophilic and the second liquid is hydrophobic.

5. 2. The method for recovering fine particles according to claim 1, wherein the kinetic viscosity of the second liquid is 1 / 100 to 100 times the kinetic viscosity of the first liquid.

6. The method for recovering fine particles according to claim 1 , wherein the flowing step, the determining step, and the recovering step are performed while the second liquid is being supplied from the liquid supply flow path to the connecting flow path.

7. The main flow path branches into the connection flow path and at least one waste flow path through which fine particles other than the particles to be collected flow, The liquid supply flow path supplies liquid to the connection flow path. The method for recovering microparticles according to claim 1 .

8. The method for recovering fine particles according to claim 1 , wherein the connecting flow passage is provided with a valve that prevents the first liquid from proceeding to the recovery flow passage.

9. The method for recovering fine particles according to claim 1 , wherein in the flowing step, the fine particles flow in the main flow path toward the connecting flow path in a substantially lined up state.

10. the microparticle sorting mechanism has a flow path structure in which a sample flow path through which a liquid containing microparticles flows and a sheath flow path through which a liquid not containing microparticles flows are connected to a main flow path at a junction, and the microparticles flow in the main flow path after the junction in a substantially lined-up manner; The flow path structure forms a laminar flow including microparticles flowing in a substantially aligned line. The method for recovering microparticles according to claim 1 .

11. 2. The method for collecting fine particles according to claim 1, wherein in the determining step, light is irradiated onto the fine particles flowing through the main flow path, and it is determined whether the fine particles are particles to be collected based on the light generated by the irradiation.

12. 2. The method for recovering fine particles according to claim 1, wherein in the recovering step, the particles to be recovered are recovered into the recovery flow path through the connection flow path by a pressure fluctuation in the recovery flow path.

13. The method for recovering fine particles according to claim 1 , wherein the main flow path, the connection flow path, and the recovery flow path are aligned in a straight line.

14. 2. The method for recovering fine particles according to claim 1, wherein the fine particles are cells or cell clumps, and the first liquid is a culture medium for the fine particles.

15. The method for recovering microparticles according to claim 1 , wherein the microparticles are cells, cell clumps, or synthetic particles, and the microparticles are destroyed after the recovery step.

16. The method for recovering fine particles according to claim 1 , wherein the recovered fine particles in the recovery passageway are subjected to a further fine particle sorting process.

17. The method for recovering microparticles according to claim 1 , wherein the microparticle sorting mechanism includes one or more of the flow path structures.

18. A main flow path through which microparticles flow; a collection flow path through which collection target particles are collected from among the microparticles; 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 liquid, the main flow path has a determination region used for determining whether microparticles flowing in a state of being contained in the first liquid are particles to be collected, The microparticles determined to be particles to be collected are collected in a state where they are contained in the first liquid into a second liquid that is immiscible with the first liquid in the collection flow path. A microchip for sorting microparticles.

19. A main flow path through which microparticles flow; a collection flow path through which collection target particles are collected from among the microparticles; 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 liquid; A microchip for sorting microparticles having a flow path structure including the following: a first liquid supply unit that supplies a first liquid containing microparticles 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 channel; a determination unit that determines whether the microparticles flowing in the main flow path are particles to be collected; A microparticle collection device comprising:

20. The microparticle recovery device according to claim 19, wherein the microparticle sorting microchip is removable from the microparticle recovery device.

21. A main flow path through which microparticles flow; a collection flow path through which collection target particles are collected from among the microparticles; 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 liquid to the connection flow path, in a microparticle sorting mechanism having a flow path structure including the following steps: a determination step of determining whether the microparticles flowing through the main flow path are particles to be collected; A recovery step of recovering the recovery target particles into the recovery flow path, and In the recovery step, the recovery target particles are recovered in a state where they are contained in the first liquid, into a second liquid that is immiscible with the first liquid and is in the recovery flow path. A method for producing an emulsion comprising microparticle-containing emulsion particles.

22. An emulsion comprising microparticle-containing emulsion particles, the ratio of the number of emulsion particles each containing one microparticle being 70% or more relative to the total number of emulsion particles.

Images