Microparticle recovery method, microchip for separating microparticles, microparticle recovery device, emulsion production method, and emulsion

Abstract

To provide a new method for more efficiently generating emulsion particles each containing one fine particle.SOLUTION: In a fine particle sorting mechanism having a channel structure including a main channel through which fine particles flow, a collection channel into which particles to be collected are collected from among the fine particles, a connection channel that connects the main channel with the collection channel, and a liquid supply channel connected to the connection channel so as to supply a liquid, a fine particle collection method includes: a flow step of causing a first liquid containing the fine particles to flow through the main channel; a determination step of determining whether or not the fine particles flowing through the main channel are the particles to be collected; and a collection step of collecting the particles to be collected into the collection channel. In the collection step, the particles to be collected are collected into a second liquid that is immiscible with the first liquid in the collection channel while being contained 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 proportion of emulsion particles containing a single cell. 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. In addition, this technology: a flowing step of flowing a first liquid containing microparticles through a main flow path; 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 particles to be recovered into a recovery flow path; and In the recovery step, droplets containing a plurality of recovery target particles are recovered. death, the plurality of recovery target particles are recovered in the recovery flow path in a second liquid while being contained in the first liquid, the second liquid is immiscible with the first liquid; and In the recovery flow path, the second liquid becomes a dispersion medium and the first liquid becomes a dispersoid. A method for recovering microparticles is also provided. 。 before In the recovery step, the plurality of recovery target particles can be recovered into the recovery flow path through a connection flow path that connects the main flow path and the recovery flow path. In the microparticle recovery method, the second liquid can be supplied into the connecting flow path from a liquid supply flow path that is connected to the connecting flow path so as to be able to supply liquid. By supplying the second liquid from the liquid supply flow channel into the connecting flow channel, a flow that flows from a connection position between the liquid supply flow channel and the connecting flow channel toward the main flow channel can be formed. 。 before By carrying out the microparticle recovery method, a plurality of droplets are formed, and at least a portion of the plurality of droplets may contain the plurality of particles to be recovered. In the flowing step, the microparticles can flow in the main flow path toward the connecting flow path in a substantially lined up state. In the flowing step, the microparticles can flow in a laminar flow state within the main flow path toward the connecting flow path. 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. In addition, this technology: a flowing step of flowing a first liquid containing microparticles through a main flow path; 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 particles to be recovered into a recovery flow path; and In the recovery step, droplets containing a plurality of recovery target particles are recovered. death, the plurality of recovery target particles are recovered in the recovery flow path in a second liquid while being contained in the first liquid, the second liquid is immiscible with the first liquid; and In the recovery flow path, the second liquid becomes a dispersion medium and the first liquid becomes a dispersoid. A method for producing droplets is also provided. In addition, this technology: a first liquid supply unit that supplies a first liquid containing microparticles to a main flow path in a microchip for sorting microparticles, the main flow path having a flow path structure including a main flow path through which microparticles flow and a recovery flow path through which recovery target particles among the microparticles are recovered; a determination unit that determines whether the microparticles flowing in the main flow path are particles to be collected; Equipped with The particles to be collected are collected into the collection flow path. death, the plurality of recovery target particles are recovered in the recovery flow path in a second liquid while being contained in the first liquid, the second liquid is immiscible with the first liquid; and In the recovery flow path, the second liquid becomes a dispersion medium and the first liquid becomes a dispersoid. A droplet collection device is also provided. In addition, this technology: A microchip for sorting microparticles having a flow path structure including a main flow path through which microparticles flow and a recovery flow path through which target particles among the microparticles are recovered, The microchip includes a flowing step of flowing a first liquid containing microparticles through the main flow path, a determining step of determining whether the microparticles flowing through the main flow path are particles to be collected, and a collecting step of collecting the particles to be collected into the collection flow path, and in the collecting step, droplets containing a plurality of particles to be collected are collected. the plurality of recovery target particles are recovered in a second liquid in the recovery flow path while being contained in the first liquid, the second liquid is immiscible with the first liquid, and in the recovery flow path, the second liquid serves as a dispersion medium and the first liquid serves as a dispersoid; Used to carry out the microparticle recovery method, The microchip is also provided. [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 a 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) Cultivation 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 flow path are those determined to be collection target particles in the determination step, and the collection operation is performed at an appropriate timing. Furthermore, when no microparticles flow or when a microparticle determined not to be a collection target particle in the determination step arrives, the collection operation is not performed, so emulsion particles not containing microparticles or emulsion particles containing particles other than the collection target particles are not formed in the collection flow path. Therefore, the probability that one collection target particle is included in the emulsion particles is extremely high. For example, by the method according to the present technology, emulsion particles containing one microparticle (particularly a collection target particle) can be generated with a success rate of, for example, 70% or more, particularly 80% or more, more particularly 90% or more, and even 95% or more. In the present technology, the collection target particle refers to the microparticle determined to be collected in the determination step.

[0017] For single-cell analysis using emulsions, it is important to increase the content of single-cell-containing emulsion particles in the emulsion, which is particularly important when single-cell analysis is performed on samples with a small number of cells. However, as described in the above Non-Patent Document 2, for example, the probability that a certain number of cells are contained in one emulsion particle is considered to follow a Poisson distribution. With conventional emulsion formation techniques, it is said that the probability that one cell is contained in one emulsion particle is at most about 65%. In order to increase the ratio, it is possible to increase the number of cells in the sample. However, the number of cells to be analyzed contained in a clinical sample is often small, for example, the number of cells to be analyzed in one sample is, for example, 10 4 ~10 5 In addition, when analyzing rare cells such as circulating tumor cells (CTCs), the number of available cells is limited. As described above, this technique can increase the ratio of emulsion particles that contain a single microparticle (e.g., a cell), making it extremely useful for single-cell analysis using emulsions.

[0018] In addition, in single cell analysis using an emulsion containing one cell in each emulsion particle, generally, before forming the emulsion, a cell sorter or other cell sorting device is used to sort the target cell group for single cell analysis to purify the target cells. Therefore, in order to perform the single cell analysis, a cell sorter is required in addition to the emulsification device. Increasing the number of devices used is undesirable, for example, from the viewpoint of cost. In addition, in order to form the emulsion, a cell sorting step is also required in addition to the emulsion formation step. Increasing the number of steps is undesirable, for example, from the viewpoint of time and cost. Although there is a technique for single-cell sorting into a well plate based on a detection signal using a cell sorter, the number of wells in the well plate is limited to 384, and the analysis scale and throughput are low. In addition, sorting cannot be performed while the sort nozzle is moving between wells, so the target cells that flow in during this time are lost. In the method of the present technology, the microparticles determined to be particles to be collected in the determination step can be collected in an emulsion state. Therefore, the present technology can form an emulsion without a separate cell sorting step. In addition, the method of the present technology can increase the collection rate of particles to be collected.

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

[0020] In the flow path structure included in the microparticle sorting 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 it is possible to guide the first liquid into the recovery flow path as necessary. For example, by guiding the first liquid into the recovery flow path only when the particles to be recovered reach the vicinity of the connection flow path, it is possible to guide the particles to be recovered contained in the first liquid into the recovery flow path.

[0021] According to a preferred embodiment of the present technology, an emulsion is formed in the recovery flow path, with the second liquid as a dispersion medium and the first liquid as a dispersoid, by carrying out the microparticle recovery method according to the present technology. The emulsion formed by the microparticle recovery method according to the present technology has a high content of emulsion particles including one recovery target particle among all emulsion particles. Therefore, the present technology is applicable even when the number of cells to be analyzed is small.

[0022] The kinetic 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, still more preferably 1 / 5 to 5 times, and particularly preferably 1 / 2 to 2 times, of the kinetic viscosity of the first liquid. In the present technology, it is preferable that the kinetic viscosities of the first liquid and the second liquid are approximately the same. This makes it easier to form an emulsion. The density of each of the first liquid and the second liquid at 25° C. is, for example, 0.5 g / cm 3 ~5g / cm 3 , preferably 0.6 g / cm 3 ~4g / cm 3 , more preferably 0.7 g / cm 3 ~3g / cm 3 It is possible. 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, of 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 approximately the same. This makes it easier to form an emulsion. The kinematic viscosity at 25° C. of the first liquid and the second liquid 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. The first liquid and the second liquid having the above physical properties facilitate the formation of an emulsion in the recovery channel, and also facilitate the flow of these liquids through 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 may be formed in the recovery flow path, with the hydrophobic liquid as a dispersion medium and the hydrophilic liquid as a dispersoid. It is desirable for biological particles, such as cells, to exist in a state contained in a hydrophilic liquid, such as a buffer or culture medium. Therefore, this embodiment is suitable for recovering microparticles, particularly biological particles, more particularly cells, that are desirable to exist in a hydrophilic liquid.

[0024] The hydrophilic liquid includes, for example, water and liquids miscible with water. For example, the hydrophilic liquid may be a liquid whose main component is one or a mixture of two or more selected from the group consisting of water, hydrophilic alcohol, hydrophilic ether, ketone, nitrile solvent, dimethyl sulfoxide, and N,N-dimethylformamide. In this specification, the main component refers to a component that occupies, for example, 50% by mass or more of the liquid, particularly 60% by mass or more, more particularly 70% by mass or more, and even more particularly 80% by mass or more, 85% by mass or more, or 90% by mass or more. 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 whose main component is water, such as water, an aqueous solution, or an aqueous 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 (e.g., 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. The hydrophilic liquid may also contain, for example, a surfactant, particularly a non-ionic surfactant. An example of a non-ionic surfactant is a triblock copolymer of polyethylene oxide and polypropylene oxide, which is also called a poloxamer or pluronic surfactant. A more specific example of a pluronic surfactant is Pluronic (trademark) F68.

[0026] The hydrophilic liquid may include, but is not limited to, a culture medium and a buffer, preferably a Good's buffer. By using a culture medium as the hydrophilic liquid, the cells collected as the target particles can be cultured while being held within the emulsion particles. In addition, by including a cell stimulating component in the hydrophilic liquid (particularly the sheath liquid), the cells collected as the collection target particles can be stimulated while being held in the emulsion particles. Furthermore, the characteristics (e.g., morphology, etc.) of the stimulated cells can be observed by a microscope or the like. The hydrophilic liquid (e.g., the sheath liquid or the sample liquid) may also contain an assay system that allows for observing a response to cell stimulation. The assay system allows for, for example, optical detection of the response from the cells collected as the target particles while they are retained in the emulsion particles. The assay system is preferably a wash-free assay system, and is preferably a system that utilizes, for example, fluorescence resonance energy transfer (FRET) or bioluminescence resonance energy transfer (BRET). As described above, in the present technology, when the microparticles are bioparticles (particularly cells), various analyses of single bioparticles (particularly single-cell analyses, 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 ~5g / cm 3 , preferably 0.6 g / cm 3 ~4g / cm3 , more preferably 0.7 g / cm 3 ~3g / cm 3 It is possible. The hydrophilic liquid may have a kinetic viscosity at 25° C. of, 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 hydrophilic liquid has the above physical properties, it flows easily through 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, and may be a liquid mainly composed of one or a mixture of two or more selected from the group consisting of aliphatic hydrocarbons, fluorine-based oils, low or high molecules containing fluorine atoms, silicone oils, aromatic hydrocarbons, aliphatic monohydric alcohols (e.g., n-octanol, etc.), and fluorinated polysaccharides. The aliphatic hydrocarbon is preferably an aliphatic hydrocarbon having 7 to 30 carbon atoms. By having 7 to 30 carbon atoms, the kinetic viscosity of the hydrophobic liquid is suitable for flowing through the microchannel. Examples of aliphatic hydrocarbons include mineral oil; oils derived from animals and plants, such as squalane oil and olive oil; paraffin-based hydrocarbons having 10 to 20 carbon atoms, such as decane and hexadecane; and olefin-based 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 fluorine-based oil. The fluorine-based oil may, for example, be perfluorocarbon (PFC), perfluoropolyether (PFPE), or hydrofluoroether (HFE). The perfluorocarbon may, for example, be Fluorinert (trademark) FC40 or Fluorinert FC-770 (manufactured by 3M). The perfluoropolyether may, for example, be Krytox (manufactured by DuPont). The hydrofluoroether may, for example, be HFE7500 (manufactured by 3M).

[0029] The density of the hydrophobic liquid at 25° C. is, for example, 0.5 g / cm 3 ~5g / cm 3 , preferably 0.6 g / cm 3 ~4g / cm 3 , more preferably 0.7 g / cm 3 ~3g / cm 3 It is possible. The hydrophobic liquid may have a kinematic viscosity at 25° C. of, 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 hydrophobic liquid has the above physical properties, it is more likely to form an emulsion in the recovery flow path. For example, if the density or kinetic viscosity is too high, it is more likely that the liquid will not flow smoothly through the connecting flow path.

[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, more particularly, the hydrophobic liquid contains a surfactant. The surfactant makes it easier to form emulsion particles, and also allows the emulsion particles to be stably maintained. Examples of the surfactant include nonionic surfactants and fluorosurfactants. Examples of the nonionic surfactant 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. The former may include, for example, Krytox (manufactured by DuPont), and the latter may include, for example, perfluorooctanol.

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

[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 having a hydrophilic liquid as a dispersion medium and a hydrophobic liquid as a dispersoid 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. This embodiment can also be applied to the case where only the target microparticles are recovered from an emulsion in which the dispersion medium and dispersoid 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 order to collect emulsion particles containing microparticles, in the determination step, a determination may be made on the microparticles, a determination may be made on the emulsion particles, or a determination may be made on both the microparticles and the emulsion particles. In this way, in the present technology, based on information obtained from the microparticles and / or emulsion particles, it may be determined whether the microparticles or emulsion particles are to be collected.

[0033] According to a preferred embodiment of the present disclosure, the flowing step, the determining step, and the recovering step are performed while the second liquid is supplied from the liquid supply flow path to the connecting flow path, whereby the connecting flow path is filled with the second liquid, and it is possible to prevent the first liquid from unnecessarily proceeding to the recovery flow path.

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

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

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

[0037] 1, the microchip 150 for sorting microparticles constitutes a part of a microparticle recovery device 100 that includes, in addition to the microchip, a light irradiation unit 101, a detection unit 102, and a control unit 103. The control unit 103 can include a signal processing unit 104, a determination unit 105, and a sorting control unit 106, as shown in FIG.

[0038] As shown in Figure 2, the method of the present technology includes a flow process S101 in which a first liquid containing microparticles is flowed through a main flow path 155 in a microchip 150 for sorting microparticles, a determination process S102 in which it is determined whether the microparticles flowing through the main flow path 155 are particles to be recovered, and a recovery process S103 in which the particles to be recovered are recovered into a recovery flow path 159. Each step will be explained below.

[0039] (2-1) Flow process

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

[0041] The microchip 150 for sorting microparticles is provided with a sample liquid inlet 151 and a sheath liquid inlet 153. From these inlets, sample liquid containing microparticles and sheath liquid not containing microparticles are introduced into a sample liquid flow path 152 and a sheath liquid flow path 154, respectively.

[0042] The microchip 150 for sorting microparticles has a flow path structure in which a sample flow path 142 through which the sample liquid flows and a sheath liquid flow path 154 through which the sheath liquid flows join at a joining section 162 to form a main flow path 155. The sample liquid and the sheath liquid join at the joining section 162 to form, for example, a laminar flow in which the sample liquid is surrounded by the sheath liquid. Preferably, the microparticles are aligned in a substantially straight line in the laminar flow. In this way, in the present technology, a laminar flow containing microparticles flowing aligned in a substantially straight line is formed by the flow path structure.

[0043] The laminar flow flows through main channel 155 toward particle sorting section 157. Preferably, the microparticles flow in a line within main channel 155. This makes it easier to distinguish between light generated by irradiating one microparticle with light and light generated by irradiating another microparticle with light in light irradiation in detection region 156 described below.

[0044] (2-2) Judgment process

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

[0046] In a determination step S102, the light irradiating unit 101 irradiates light (e.g., excitation light, etc.) to the microparticles flowing in the main channel 155 (particularly the detection region 156) in the microchip 150 for sorting microparticles, and the detection unit 102 detects the light generated by the 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 microparticle is a particle to be collected. For example, the determination unit 105 can make a determination based on scattered light, a determination based on fluorescence, or a determination based on an image (e.g., a dark-field image and / or a bright-field image, etc.). In a recovery step S103 described later, the control unit 103 controls the flow in the microchip 150 for sorting microparticles, so that the particles to be collected are collected into the collection channel 159.

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

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

[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 by the light irradiation unit 101. The detection unit 102 may include a condensing lens that condenses the 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 are not limited to these. In addition to the condensing lens and the detector, the detection unit 102 may include other optical elements as necessary. The detection unit 102 may further include, for example, a spectroscopic unit. Examples of optical components constituting the spectroscopic unit include a grating, a prism, and an optical filter. The spectroscopic unit can detect, for example, light of a wavelength to be detected by separating it from light of other wavelengths. The detection unit 102 may convert the detected light into an analog electrical signal by photoelectric conversion. The detection unit 102 may further convert the analog electrical signal into a digital electrical signal by AD conversion.

[0050] The signal processing unit 104 included in the control unit 103 may process the waveform of the digital electric signal obtained by the detection unit 102 to generate information (data) on the characteristics of the light to be used for the judgment by the judgment unit 105. As the information on the characteristics of the light, the signal processing unit 104 may acquire, for example, one, two or three of the width, height and area of ​​the waveform from the waveform of the digital electric signal. Furthermore, the information on the characteristics of the light may include, for example, the time when the light was detected. The above processing by the signal processing unit 104 may be performed particularly in an embodiment in which the scattered light and / or fluorescence is detected.

[0051] A determination unit 105 included in the control unit 103 determines whether or not a microparticle flowing through the flow channel is a particle to be collected, based on light generated by irradiating the microparticle with light. In an embodiment in which the scattered light and / or fluorescence is detected, the waveform of the digital electric signal obtained by the detection unit 102 is processed by the control unit 103, and the determination unit 105 determines whether the microparticle is a particle to be collected based on information about the characteristics of the light generated by the process. For example, in the determination based on the scattered light, the characteristics of the external 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. Furthermore, by subjecting the microparticles, such as cells, to a pretreatment in advance, it is also possible to determine whether the microparticle is a particle to be collected based on the same characteristics as those used in flow cytometry. In addition, by labeling the 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 antigens of the microparticles.

[0052] (Identification of the target to be sorted based on bright-field images)

[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 may include, for example, a halogen lamp, and the detection unit 102 may include a CCD or a CMOS. For example, light may be irradiated onto a microparticle by the halogen lamp, and a bright-field image of the irradiated microparticle may be acquired by the CCD or the CMOS.

[0054] In an embodiment in which 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, whether the microparticle is a particle to be collected can be determined based on one or a combination of two or more of the shape, size, and color of the microparticle (particularly a cell).

[0055] (Discrimination of objects to be sorted based on dark-field images)

[0056] In yet another embodiment of the present technology, the detection unit 102 may acquire a dark-field image generated by light irradiation by the light irradiation unit 101. In this embodiment, the light irradiation unit 101 may include, for example, a laser light source, and the detection unit 102 may include a CCD or a CMOS. For example, light may be irradiated onto a microparticle by a laser, and a dark-field image (for example, a fluorescent image) of the irradiated microparticle may be acquired by the CCD or the CMOS.

[0057] In an 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, whether the microparticle is a particle to be collected can be determined based on one or a combination of two or more of the shape, size, and color of the microparticle (particularly a cell).

[0058] In any of the above-mentioned "discrimination of a target to be collected based on a fluorescent signal and / or a scattered light signal", "discrimination of a target to be collected based on a bright-field image", and "discrimination of a target to be collected based on a dark-field image", the detection unit 102 may be, for example, an imaging element in which a substrate incorporating a CMOS sensor and a substrate incorporating a DSP (Digital Signal Processor) are stacked. By operating the DSP of the imaging element as a machine learning unit, the imaging element can operate as a so-called AI sensor. The detection unit 102 including the imaging element can determine whether a microparticle is a particle to be collected based on, for example, a learning model. In addition, the learning model may be updated in real time while the method according to the present technology is being performed. For example, the DSP can perform machine learning processing during resetting of a pixel array unit in a CMOS sensor, during exposure of the pixel array unit, or during readout of pixel signals from each unit pixel of the pixel array unit. An example of an imaging element operating as an AI sensor is the imaging device described in International Publication No. WO 2018 / 051809. When an AI sensor is used as an imaging element, the raw data obtained from the image array is directly learned, making the speed of sorting and discrimination processing fast.

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

[0060] A single light may be irradiated to one position in the detection region 156, or light may be irradiated to each of a plurality of positions in the detection region 156. For example, the microchip 150 may be configured so that light is irradiated to each of two different positions in the detection region 156 (i.e., there are two positions in the detection region 156 where light is irradiated). In this case, for example, it may be determined whether a microparticle is a particle to be collected based on light (e.g., fluorescent light and / or scattered light, etc.) generated by irradiating a microparticle with light at one position. Furthermore, the speed of the microparticle in the flow channel may be calculated based on the difference between the detection time of light generated by the light irradiation at the one position and the detection time of light generated by the light irradiation at another position. For this calculation, the distance between the two irradiation positions may be determined in advance, and the speed of the microparticle may be determined based on the difference between the two detection times and the distance. Furthermore, based on the speed, the arrival time at the particle sorting section 157 described below can be accurately predicted. By accurately predicting the arrival time, the timing of the formation of the flow entering the recovery flow passage 159 can be optimized. In addition, when the difference between the arrival time of a certain microparticle at the particle sorting section 157 and the arrival time of a microparticle before or after the certain microparticle at the particle sorting section 157 is equal to or less than a predetermined threshold, it can be determined that the certain microparticle is not to be collected. When the distance between the certain microparticle and the microparticle before or after the certain microparticle is narrow, the possibility that the microparticle before or after the certain microparticle is collected together when the certain microparticle is sucked increases. When the possibility of the microparticles being collected together is high, it is possible to prevent the microparticle before or after the certain microparticle from being collected by determining not to collect the certain microparticle. This can increase the purity of the target microparticle among the collected microparticles. A specific example of a microchip in which light is irradiated to each of two different positions in the detection region 156 and an apparatus including the microchip are described in, for example, JP 2014-202573 A.

[0061] 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. The control unit 103 may also control the driving of a pump for supplying a fluid into the microchip 150 for microparticle sorting. The control unit 103 may be configured with, 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 function of the control unit 103 may be realized in a general-purpose computer. The program may be recorded in a recording medium such as a microSD memory card, an SD memory card, or a flash memory. The program recorded in the recording medium may be read by a drive (not shown) provided in the microparticle recovery device 100, and the control unit 103 may cause the microparticle recovery device 100 to execute the microparticle recovery method according to the present technology in accordance with the read program.

[0062] (2-3) Recovery process

[0063] In the recovery step S103, the microparticles determined to be recovery target particles in the determination step S102 are recovered into the recovery flow path 159. In the recovery step S103, the recovery target particles are recovered in a state where they are contained in the first liquid into a second liquid in the recovery flow path that is immiscible with the first liquid. This makes it possible to form an emulsion in the recovery flow path 159, in which the second liquid is a dispersion medium and the first liquid is a dispersoid, and each emulsion particle in the emulsion contains one recovery target particle. Therefore, the emulsion is suitable for single cell analysis. The recovery process is described in more detail below.

[0064] The recovery step S103 is performed in the particle sorting section 157 in the microchip 150. In the particle sorting section 157, the laminar flow that has flowed through the main channel 155 separates and flows into two waste channels 158. The particle sorting section 157 shown in FIG. 1 has two waste channels 158, but the number of branch channels is not limited to two. The particle sorting section 157 may be provided with, for example, one or more (e.g., two, three, or four) branch channels. The branch channels may be configured to branch in a Y-shape on one plane as in FIG. 1, or may be configured to branch three-dimensionally.

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

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

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

[0068] 6B, the second liquid is supplied from the two liquid supply channels 161 to the connecting channel 170. The second liquid flows from the connecting portion 170c to both the upstream connecting channel 170a and the downstream connecting channel 170b.

[0069] If no recovery step is performed, the second liquid flows as follows: The second liquid that has flowed into the upstream connecting flow channel 170a exits from the connecting surface of the connecting flow channel 170 with the main flow channel 155, and then separates and flows into the two waste flow channels 158. By the second liquid exiting from the connecting surface in this manner, it is possible to prevent the first liquid and microparticles that do not need to be recovered into the recovery flow channel 159 from passing through the connecting flow channel 170 and entering the recovery flow channel 159. The second liquid that has flowed into the downstream connection flow channel 170b flows into the recovery flow channel 159. As a result, the recovery flow channel 159 is filled with the second liquid, and the second liquid becomes, for example, a dispersion medium for forming an emulsion.

[0070] Even when the recovery step is performed, the second liquid can be supplied from the two liquid supply channels 161 to the connection channel 170. However, due to pressure fluctuations in the recovery channel 159, particularly by generating a negative pressure in the recovery channel 159, a flow is formed that flows from the main channel 155 through the connection channel 170 to the recovery channel 159. That is, a flow is formed that flows 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 in the second liquid in the recovery channel 159 while being surrounded by the first liquid. By performing the recovery step, for example, an emulsion can be formed in the recovery channel 159 or in a container connected to the recovery channel end 163 via, for example, a channel.

[0071] The shape and / or dimensions of the cross section of the upstream connecting flow channel 120a may be different from the shape and / or dimensions of the downstream connecting flow channel 120b. An example in which the dimensions of these two flow channels are different is shown in Figures 7A and 7B. As shown in Figures 7A and 7B, the connecting flow channel 180 includes a flow channel 180a on the detection region 156 side (hereinafter also referred to as the upstream connecting flow channel 180a), a flow channel 180b on the recovery flow channel 159 side (hereinafter also referred to as the downstream connecting flow channel 180b), and a connection part 180c between the connecting flow channel 180 and the liquid supply flow channel 161. The cross sections of the upstream connecting flow channel 180a and the downstream connecting flow channel 180b are both approximately circular, but the diameter of the latter cross section is larger than the diameter of the former cross section. By making the cross-sectional diameter of the latter larger than that of the former, it is possible to more effectively prevent the particles to be recovered that have already been sorted into the recovery flow path 159 from being released into the main flow path 155 through the connecting flow path 180 immediately after the microparticle sorting operation by negative pressure described above, compared to when the diameters of the two are the same. For example, if the cross-sections of the upstream connecting flow path 180a and the downstream connecting flow path 180b are both rectangular, the area of ​​the latter cross-section can be made larger than the area of ​​the former cross-section, thereby more effectively preventing already collected microparticles from being released through the connecting flow path 180 into the main flow path 155, as described above.

[0072] In the recovery step S103, the recovery target particles are recovered into the recovery channel through the connection channel due to pressure fluctuations in the recovery channel 159. The 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 by, for example, an actuator 107 (particularly a piezoelectric actuator) attached to the outside of the microchip 150, which deforms a wall defining the recovery channel 159. The flow entering the recovery channel 159 may be formed by the negative pressure. In order to generate the negative pressure, for example, the actuator 107 may be attached to the outside of the microchip 150 so as to be able to deform the wall of the recovery channel 159. The deformation of the wall may change the inner space of the recovery channel 159, generating a negative pressure. The actuator 107 may be, for example, a piezoelectric actuator. When the recovery target particles are sucked into the recovery channel 159, the sample liquid constituting the laminar flow or the sample liquid and sheath liquid constituting the laminar flow may also flow into the recovery channel 159. In this manner, the particles to be collected are sorted in particle sorting section 157 and collected into collection channel 159.

[0073] The particles to be collected are surrounded by a first liquid and collected in a second liquid that is immiscible with the first liquid in collection flow path 159. As a result, as described above, an emulsion is formed in collection flow path 159, with the second liquid as a dispersion medium and the first liquid as a dispersoid.

[0074] In order to prevent microparticles that are not target particles for recovery 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 liquids (sample liquid and sheath liquid) flowing through the main channel 155 is introduced from the liquid supply channel 161 into the connection channel 170. A flow from the connection flow channel 170 toward the main flow channel 155 is formed by a portion of the second liquid introduced into the connection flow channel 170, thereby preventing fine particles other than the particles to be recovered from entering the recovery flow channel 159. The second liquid formed by the flow from the connection flow channel 170 toward the main flow channel 155 flows through the waste flow channel 158 in the same manner as the first liquid, without flowing inside the main flow channel 155, due to the flow of the first liquid flowing through the main flow channel 155 to the waste flow channel 158. The remainder of the second liquid introduced into the connection channel 170 flows into the recovery channel 159. This allows the recovery channel 159 to be filled with the second liquid.

[0075] A second liquid that is immiscible with the first liquid may be filled inside the recovery channel 159. 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 to the recovery channel 159, and thus the inside of the recovery channel 159 may be filled with the second liquid.

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

[0077] 5, a container 171 can be connected to the recovery channel end 163 via a channel such as a tube 172. As shown in the figure, an emulsion containing the first liquid as a dispersoid and the second liquid as a dispersion medium, which contains particles to be recovered, is recovered in the container 171. Thus, according to one embodiment of the present technology, the microparticle recovery device 100 may be provided with a channel for recovering the emulsion containing particles to be recovered in the container. Furthermore, when the recovery operation is performed with the recovery channel end 163 closed, a plurality of emulsion particles can be held in the recovery channel 159. After the recovery operation is completed, 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 be branched 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 collected flow.

[0079] 1 and 2, in the microchip for sorting microparticles used in the method of the present technology, the main flow path, the connection flow path, and the recovery flow path may be arranged in a straight line. When these three flow paths are arranged in a straight line (particularly on a coaxial line), the recovery step can be performed more efficiently than when, for example, the connection flow path and the recovery flow path are arranged at an angle to the main flow path. For example, the amount of suction required to guide the particles to be recovered to the connection flow path can be reduced. 1 and 2, in the microchip for sorting microparticles used in the method of the present technology, the microparticles are aligned in a line in the main channel and flow toward the connecting channel, so that the amount of the microparticles to be aspirated in the recovery process can be reduced.

[0080] In the method of the present technology, the liquid supply flow path supplies liquid (particularly the second liquid) to the connection flow path. This forms a flow 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 microparticles other than the particles to be recovered from flowing through the connection flow path to the recovery flow path. When the recovery step is performed, as described above, for example, the negative pressure generated in the recovery flow path causes the first liquid containing one particle to be recovered to pass through the connection flow path and be recovered into the second liquid in the recovery flow path. This forms an emulsion particle containing one particle to be recovered in the second liquid.

[0081] In addition, in the present technology, when a microparticle determined to be a particle to be collected in the determination step is driven, for example, by a piezo actuator at an appropriate timing (for example, when the particle reaches the particle sorting section 157), the hydrophilic solution containing the particle to be collected is collected in the collection flow channel 159, and an emulsion particle is formed. In the determination step, for example, by using a peak signal and an area signal to determine whether the particle is a particle to be collected, it is also possible to determine whether the particle is 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, it is possible to form emulsion particles containing one microparticle with high probability and high efficiency. Also, since it is possible to avoid the formation of emulsion particles containing two or more microparticles combined in this way, it is possible to omit the operation of removing the combination of two or more microparticles before the emulsion formation operation, for example, by a cell sorter or the like. Conversely, the characteristics of two or more microparticles that are close enough together that they can be drawn into a connecting flow path in a single collection operation can also be determined. For example, two microparticles with the same characteristics can be trapped within a single emulsion particle, or a combination of microparticles with different designated characteristics can be trapped within a single emulsion particle.

[0082] (Example) Emulsion particles containing one microparticle were formed as follows, using a microchip for sorting microparticles having the same flow path structure as the microchip for sorting microparticles 150 shown in Fig. 1. The operation for forming the emulsion particles will be described below with reference to Fig. 1.

[0083] In the microchip 150 for sorting microparticles, a piezoelectric element (piezo actuator) was attached to the outer surface of the microchip (in particular, the outer surface corresponding to the bulging portion of the recovery channel 159 near the connection portion with the connecting channel 170) so as to vary the volume within the recovery channel 159. A hydrophilic sample liquid containing beads with a diameter of 10 μm was introduced from a sample liquid inlet 151 into a sample liquid flow path 152, and a hydrophilic sheath liquid was introduced from a sheath liquid inlet 153 into a sheath liquid flow path 154. The introduced hydrophilic sample liquid and the hydrophilic sheath liquid joined at a joining portion 162, and a laminar flow was formed in which the hydrophilic sample liquid was surrounded by the hydrophilic sheath liquid. The laminar flow contained beads arranged in a substantially straight line, and flowed through the main flow path 155 toward the connecting flow path 170. In parallel with the introduction of the hydrophilic sample liquid and the hydrophilic sheath liquid, a hydrophobic liquid was supplied from the liquid supply flow channel 161 to the connection flow channel 170. By supplying the hydrophobic liquid from the liquid supply flow channel 161 to the connection flow channel 170, the laminar flow was prevented from passing through the connection flow channel 170 and entering the recovery flow channel 159, and the recovery flow channel 159 was filled with the hydrophobic liquid. When a bead passes through the irradiation position of the laser light irradiated to the detection region 156 in the main flow channel 155, the bead is irradiated with the laser light and light is generated. The generated light is detected, and based on the characteristics of the detected light, it is determined whether or not to collect each bead.

[0084] When a bead determined to be collected reaches the vicinity of the connecting channel 170, the piezoelectric element is driven to deform the inner cavity of the recovery channel 159, and the bead is collected into the recovery channel 159 through the connecting channel 170. The operation for the collection was as follows: (i) the recovery channel 159 is deformed over 10 μsec to apply negative pressure inside the recovery channel 159, (ii) the state after the deformation is maintained for 10 μsec, and (iii) the negative pressure is released over 10 μsec to return the deformation to the original state. By repeating the above operations (i) to (iii), an emulsion containing emulsion particles containing one bead in the hydrophobic liquid was formed in the recovery channel 159. The emulsion had the hydrophobic liquid as a dispersion medium and emulsion particles containing a hydrophilic liquid as a dispersoid.

[0085] 8A shows that by carrying out the above operations (i) to (iii), an emulsion particle containing one bead is formed in recovery channel 159. FIG. 8A will be described below. FIG. 8A (a) is a photograph showing a state in which beads (indicated by white arrows) are flowing toward the connecting flow channel 170. At the time of FIG. 8A, the hydrophilic liquid flows through the main flow channel 155 toward the particle sorting section 157, and then flows into two waste flow channels 158 branching off from the main flow channel 155 without flowing into the connecting flow channel 170. The hydrophobic liquid is supplied from the liquid supply flow channel 161 to the connecting flow channel 170, and flows into both the main flow channel 155 and the recovery flow channel 159. The hydrophobic liquid that has flowed into the main flow channel 155 is separated into two waste flow channels 158 by the flow of the hydrophilic liquid immediately after leaving the connecting flow channel 170 (it flows along the wall of the waste flow channel 158 near the area indicated by a in FIG. 8A). 8A(b) is a photograph taken when the beads have come even closer to the connecting channel. At this point, the operation for recovering the beads is started. That is, the piezoelectric element is driven to start deforming the lumen of recovery channel 159. 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 holding time of (ii) above 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 size of the emulsion particles when the holding time of (ii) above is 10 μsec, 15 μsec, 25 μsec, and 35 μsec. From this photograph, it can be seen that the size of the emulsion particles can be adjusted by changing the holding time of (ii) above. Furthermore, the volume of the connecting flow passage 170 (particularly each of the upstream connecting flow passage 170a, the connecting portion 170c, and the downstream connecting flow passage 170b) can be adjusted to adjust the pattern of the emulsion particles.

[0087] In the example described above, the first liquid flowing through the main flow path is prevented from entering the connecting flow path by supplying the second liquid from the liquid supply flow path to the connecting flow path. In the present technology, in order to prevent the first liquid flowing through the main flow path from entering the connecting flow path, a valve that prevents the first liquid from proceeding to the recovery flow path may be provided in the connecting flow path. Only when the recovery step is performed, the valve is opened to allow the first liquid to flow through the connecting flow path to the recovery flow path.

[0088] (2-4) Other processes

[0089] The microparticle recovery method of the present technology may further include other steps in addition to the flowing step, the determining step, and the recovery step described above. Examples of the other steps will be described below.

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

[0091] According to one preferred embodiment of the present technology, the microparticles are cells or cell clusters, and the first liquid is a culture solution for the microparticles. In this embodiment, an emulsion can be formed in which the culture solution is a dispersoid and the second liquid immiscible with the culture solution is a dispersion medium. One cell or cell cluster may be contained in each emulsion particle formed by the dispersoid. This allows one cell or cell cluster to be cultured in each emulsion particle. As described above, in this embodiment, the microparticle recovery method can further include a culture step of culturing the recovery target particles (i.e., cells or cell clusters) recovered in the recovery step in emulsion particles formed from the culture solution. The type of culture solution can be appropriately selected by a person skilled in the art depending on the cells to be cultured. This embodiment is suitable for cases where it is required to separately culture each cell or cell cluster in single cell analysis, for example.

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

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

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

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

[0096] According to one preferred embodiment of the present technology, after the recovery step, detection or analysis of a component contained in the particles to be recovered, or a reaction between the component contained in the particles to be recovered and another component may be performed. In order to carry out the detection or reaction, for example, emulsion particles formed according to the present technology may be merged with other emulsion particles, and after the merging, detection or analysis of a component contained in the target particles to be collected, or a reaction between the component and another component may be carried out within the merged emulsion particles. The merging enables, for example, Cellular Indexing of Transcriptomes and Epitopes by Sequencing (also called CITE-seq). For example, assume that the particles to be collected are cells bound to an antibody bound to an oligo barcode having a poly-A sequence. In the collection step, emulsion particles containing the cells are formed. Then, the emulsion particles are merged with emulsion particles containing beads or gels having a barcode sequence. The merging allows detection of cell surface proteins or intracellular mRNA of the cells. Alternatively, the emulsified cells or cell clusters (e.g., spheroids or organoids) may be reacted with a drug, allowing the response of the cells or cell clusters to the drug to be detected or analyzed. In addition, in the detection, a biomolecule may be detected. The detection of the biomolecule may be performed, for example, by a wash-free assay. In the wash-free assay, for example, the LOCI, FRET, BRET, or FlimPIA method may be used.

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

[0098] According to one preferred embodiment of the present technology, after the recovery step, the particles to be recovered may synthesize chemical substances. For example, the synthesis may be performed within the emulsion particles formed in the recovery step. For example, in vitro antibody production is possible by encapsulating a cell-free expression reagent within the emulsion particles. Examples of the cell-free expression reagent include linear DNA and E. coli S30 Extract system for linear DNA (Promega). Also, for example, protein synthesis may be performed within the emulsion particles using a cell-free protein synthesis system.

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

[0100] The microparticle sorting mechanism used in the microparticle sorting method of the present technology may be a structure or device having the above-mentioned flow path structure, for example, a chip having a microflow path, and in particular a microchip for sorting microparticles. In the present technology, "micro" means that at least a part of the flow path included in the microchip for sorting microparticles has a dimension on the order of μm, particularly a cross-sectional dimension on the order of μm. That is, in the present technology, "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 sorting section configured 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, the cross section of the main flow path 155 in the particle sorting section 157 is, for example, rectangular, and the width of the main flow path 155 can be, for example, 100 μm to 500 μm, particularly 100 μm to 300 μm, in the particle sorting section 157. The width of the branch flow path branching off 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 channel 170 is, for example, circular, and the diameter of the connection flow channel 170 at the connection portion between the connection flow channel 170 and the main flow channel 155 can be, for example, 10 μm to 60 μm, particularly 20 μm to 50 μm. These dimensions of the flow channel may be appropriately changed depending on the size of the microparticles, particularly the size of the particles to be collected.

[0101] The microchip 150 for sorting microparticles can be manufactured by a method known in the art. For example, the microchip 150 for sorting bioparticles can be manufactured by bonding two or more substrates on which a predetermined flow path is formed. The flow path may be formed, for example, 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 positions when bonding the substrates together, it is preferable that the flow path is formed only in one substrate.

[0102] Materials known in the art may be used to form the microchip 150 for sorting microparticles. Examples include, but are not limited to, polycarbonate, cycloolefin polymer, polypropylene, PDMS (polydimethylsiloxane), polymethylmethacrylate (PMMA), polyethylene, polystyrene, glass, and silicon. Polymer materials such as polycarbonate, cycloolefin polymer, and polypropylene are particularly preferred because they are easy to process and can be inexpensively manufactured using a molding device.

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

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

[0105] In the present technology, the microparticles may be particles having a size that allows them to flow through a flow path in the microparticle sorting mechanism (e.g., a microchip for sorting 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 clusters, microorganisms, and liposomes, as well as synthetic microparticles such as gel particles, beads, latex particles, polymer particles, and industrial particles. Biological microparticles (also called bioparticles) may include chromosomes, liposomes, mitochondria, organelles (cell organelles), and the like that constitute various cells. Cells may include animal cells (such as blood cells) and plant cells. Cells may be blood cells or tissue cells, in particular. The blood cells may be suspension cells, such as T cells and B cells. The tissue cells may be, for example, cultured adherent cells or adherent cells dissociated from tissues. Cell aggregates may include, for example, spheroids and organoids. Microorganisms may include bacteria such as E. coli, viruses such as tobacco mosaic virus, fungi such as yeast, and the like. 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. The synthetic microparticles may be, for example, microparticles made of organic or inorganic polymeric materials or metals. The organic polymeric materials may include polystyrene, styrene-divinylbenzene, and polymethylmethacrylate. The inorganic polymeric materials may include glass, silica, and magnetic materials. The 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 a combination of two or more selected from oligonucleotides, peptides, proteins, and enzymes are bound. The shape of the microparticles may be spherical or nearly spherical, or may be non-spherical. The size and mass of the microparticles may be appropriately selected by those skilled in the art depending on the size of the flow channel of the microchip. On the other hand, the size of the flow channel of the microchip may also be appropriately selected depending on the size and mass of the microparticles. In this technology, a chemical or biological label, such as a fluorescent dye or a fluorescent protein, may be attached to the microparticles as necessary. The label may make the detection of the microparticles easier. The label to be attached may be appropriately selected by those skilled in the art. A molecule (e.g., an antibody, an aptamer, DNA, or RNA) that specifically reacts with the microparticle may be bound to the label. According to one embodiment of the present technology, the microparticles may be biological particles, in particular cells.

[0106] In the present technology, the microparticles collected in the collection channel may be subjected to further microparticle sorting processing. The further microparticle sorting processing may be performed, for example, using the microparticle sorting microchip 150 described above, or may be performed using another microparticle sorting microchip. For example, in the further microparticle sorting process, 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 sorting process, in order to collect emulsion particles containing microparticles, the determination step may include a determination on microparticles, a determination on emulsion particles, or a determination on both microparticles and emulsion particles. In this way, in the further microparticle sorting process, it may be determined whether microparticles or emulsion particles are to be collected based on information obtained from the microparticles and / or emulsion particles. In addition, in the further microparticle sorting process, the emulsion containing the microparticle-containing emulsion particles may be subjected to the said sorting process.

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

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

[0109] In one embodiment of the present technology, the connection channel and the recovery channel do not have to be aligned in a straight line with the main channel. An example of a microchip for sorting microparticles in this embodiment will be described below with reference to FIG.

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

[0111] The microchip 350 for sorting microparticles includes, in addition to a main flow path 355, a recovery flow path 359 through which target particles are recovered, a connection flow path 370 that connects the main flow path 355 and the recovery flow path 359, and a liquid supply flow path 361 that is connected to the connection flow path 370 so as to be able to supply liquid.

[0112] The main channel 355 further includes a waste channel 358 through which microparticles that are not target particles for collection flow. In the microchip 150 for sorting microparticles described with reference to Fig. 1, two waste channels 158 branch off from the main channel 155, and the connection channel 170, the recovery channel 159, and the main channel 155 are aligned in a straight line. In contrast, the microchip 350 for sorting microparticles in Fig. 9 has a channel structure in which the main channel 355 and the waste channel 358 are aligned in a straight line, and the connection channel 370 and the recovery channel 359 branch off from the main channel 355. In this way, the microchip for sorting microparticles used in the microparticle recovery method of the present technology does not need to have the connection flow path and the recovery flow path aligned in a straight line with the main flow path. For example, the microchip may have a flow path structure in which the main flow path and the waste flow path are aligned in a straight line, and the connection flow path and the recovery flow path are branched off from the main flow path.

[0113] (3-2) Flow path structure having multiple recovery flow paths

[0114] In another embodiment of the present technology, the microchip for sorting microparticles may include one or more flow path structures including a main flow path through which microparticles flow, a recovery flow path through which recovery target particles 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 liquid. The microchip for sorting microparticles described above with reference to Fig. 1 has one such flow path structure. In contrast, as described below with reference to Figs. 10 and 11, the microchip for sorting microparticles used in the microparticle recovery method of the present technology may have two or more such flow path structures.

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

[0116] The microchip 450 for sorting microparticles includes, in addition to the main channel 455, a recovery channel 459-1 through which the particles to be collected are collected, a connection channel 470-1 that connects the main channel 455 and the recovery channel 459-1, and a liquid supply channel 461-1 that is connected to the connection channel 470-1 so as to be able to supply liquid. The microchip 450 for sorting microparticles further includes a recovery channel 459-2 through which the particles to be collected are collected, a connection channel 470-2 that connects the main channel 455 and the recovery channel 459-2, and a liquid supply channel 461-2 that is connected to the connection channel 470-2 so as to be able to supply liquid. That is, the microchip 450 for sorting microparticles has two of the above-mentioned channel structures. In the microchip 450 for sorting microparticles, the main channel 455 is shared by these two channel structures. The connection channel 470-1 is connected to the main channel 455 downstream of the connection channel 470-2.

[0117] By using the microchip 450 for sorting microparticles, for example, two emulsions containing different particles to be collected can be formed. In this case, in the determination step, it is determined whether the microparticle is, for example, one of two types of collection target particles A and B, or neither of the two types of collection target particles. The criteria for whether the microparticle belongs to collection target particle A or B may be appropriately selected by the user. In the recovery step, when the microparticle is a particle A to be recovered, the microparticle passes through the connection flow path 470-1 and is recovered into the recovery flow path 459-1. The microparticle A to be recovered is recovered in the second liquid in the recovery flow path 459-1 while being contained in the first liquid. In the recovery step, when the microparticles are recovery target particles B, the microparticles are recovered into recovery flow path 459-2 through connection flow path 470-2. The microparticles that are recovery target particles B are recovered in the second liquid in recovery flow path 459-2 while being contained in the first liquid. In the recovery step, if the microparticle is neither recovery target particle A nor recovery target particle B, the microparticle flows into the waste flow path 458. In this manner, an emulsion containing recovery target particles A and an emulsion containing recovery target particles B are formed.

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

[0119] The microchip 550 for sorting microparticles can also form two emulsions containing different particles to be collected. Even in this case, in the determination step, it is determined whether the microparticle is, for example, one of two types of collection target particles A and B, or neither of the two types of collection target particles. The criteria for whether the microparticle belongs to collection target particle A or B may be appropriately selected by the user. In the recovery step, when the microparticle is a particle A to be recovered, the microparticle passes through the connection flow path 570-1 and is recovered into the recovery flow path 559-1. The microparticle A to be recovered is recovered in the second liquid in the recovery flow path 559-1 while being contained in the first liquid. In the recovery step, when the microparticles are recovery target particles B, the microparticles are recovered through the connection flow path 570-2 to the recovery flow path 559-2. The microparticles that are recovery target particles B are recovered in the second liquid in the recovery flow path 559-2 while being contained in the first liquid. In the recovery step, if the microparticle is neither recovery target particle A nor recovery target particle B, the microparticle flows into the waste flow path 558. In this manner, an emulsion containing recovery target particles A and an emulsion containing recovery target particles B are formed.

[0120] 2. Second embodiment (microchip for sorting microparticles)

[0121] The present technology also provides a microchip for sorting microparticles, which has a flow path structure including a main flow path through which microparticles flow, a recovery flow path through which recovery target particles 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 liquid. The main flow path in the microchip has a determination area used to determine whether a microparticle flowing in a state contained in a first liquid is a particle to be collected, and a microparticle determined to be a particle to be collected is collected in a state contained in the first liquid into a second liquid in the recovery flow path that is immiscible with the first liquid. The microchip for sorting microparticles is the same as the microchip for sorting microparticles described in 1 above, and the description thereof also applies to this embodiment.

[0122] 3. Third embodiment (microparticle recovery device)

[0123] The present technology also provides a microparticle collection device including a microchip having a flow path structure including a main flow path through which microparticles flow, a collection flow path through which recovery 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, a first liquid supply unit supplying a first liquid containing the microparticles to the main flow path, a second liquid supply unit supplying a second liquid immiscible with the first liquid to the liquid supply flow path, and a determination unit determining whether the microparticles flowing in the main flow path are recovery target particles. The microparticle collection device is the same as the microparticle collection device described in 1 above, and the description thereof also applies to this embodiment.

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

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

[0126] 4. Fourth embodiment (method of producing emulsion)

[0127] The present technology also provides a method for producing an emulsion, including the flowing step, the determining step, and the collecting step described in "1. First embodiment (microparticle collecting method)" above. The producing method may include other steps described in "(2-4) Other steps" above. The explanation described in "1. First embodiment (microparticle collecting method)" above also applies to the emulsion producing method of the present technology.

[0128] The emulsion production method of the present technology can produce an emulsion containing a high content of emulsion particles containing one microparticle (particularly a particle to be collected). For example, the production method of the present technology can produce an emulsion in which the ratio of the number of emulsion particles containing one microparticle to the total number of emulsion particles is, for example, 70% or more, preferably 80% or more, more preferably 90% or more, and even more preferably 95% or more. In single cell analysis using an emulsion, it is important to increase the content ratio of emulsion containing one cell, and when the number of cells to be analyzed is small, it is particularly important to increase the content ratio. The production method of the present technology can produce an emulsion containing a high ratio of emulsion particles containing one microparticle, and is therefore 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 number ratio of emulsion particles containing one microparticle is 70% or more relative to 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. The emulsion of the present technology contains emulsion particles containing one microparticle at such a high ratio, so that it is suitable for, for example, single cell analysis. The emulsion particles containing one microparticle can be formed as described above in "1. First embodiment (microparticle recovery method)". The emulsion can be produced, for example, as described above in "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 dispersoid. The above description in "1. First embodiment (microparticle recovery method)" applies to these liquids. The above description in "1. First embodiment (microparticle recovery method)" also applies to the microparticles contained in the emulsion particles, and the microparticles may be, for example, cells, cell aggregates, or synthetic particles.

[0132] The present technology can also be configured as follows. [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. A method for recovering microparticles. [2] The method for recovering microparticles described in [1], wherein, by carrying out the method for recovering microparticles, an emulsion is formed in the recovery flow path, the emulsion having the second liquid as a dispersion medium and the first liquid as a dispersoid. [3] The method for recovering microparticles according to [1] or [2], in which an emulsion is formed by carrying out the method for recovering microparticles, and at least a portion of the droplets constituting the emulsion contain one of the particles to be recovered. [4] The method for recovering fine particles according to any one of [1] to [3], wherein the first liquid is hydrophilic and the second liquid is hydrophobic. [5] The method for recovering microparticles according to any one of [1] to [4], wherein the kinetic viscosity of the second liquid is 1 / 100 to 100 times the kinetic viscosity of the first liquid. [6] The microparticle recovery method described in any one of [1] to [5], wherein the flowing step, the determination step, and the recovery step are performed while the second liquid is 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 any one of [1] to [6]. [8] The method for recovering microparticles described in any one of [1] to [7], wherein the connecting flow path is provided with a valve that prevents the first liquid from proceeding to the recovery flow path. [9] The method for recovering fine particles according to any one of [1] to [8], 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 channel structure forms a laminar flow containing microparticles flowing in a line. The method for recovering microparticles according to any one of [1] to [9].

[11] The microparticle recovery method according to any one of [1] to

[10] , wherein in the determination step, light is irradiated onto a microparticle flowing in the main flow path, and it is determined whether the microparticle is a particle to be recovered based on the light generated by the irradiation.

[12] The method for recovering microparticles described in any one of [1] to

[11] , wherein in the recovery process, the target particles are recovered into the recovery flow path through the connecting flow path due to pressure fluctuations in the recovery flow path.

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

[12] , wherein the main flow path, the connection flow path, and the recovery flow path are arranged in a straight line.

[14] The method for recovering microparticles according to any one of [1] to

[13] , wherein the microparticles are cells or cell clusters, and the first liquid is a culture medium for the microparticles.

[15] The method for recovering microparticles according to any one of [1] to

[14] , wherein the microparticles are cells, cell clumps, 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 a further microparticle sorting process.

[17] The method for recovering microparticles according to any one of [1] to [6], wherein the microchip for sorting microparticles includes one or more of the flow channel 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 and is 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

[19] , wherein the microchip for sorting microparticles 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 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. [Explanation of symbols]

[0133] 100 Microparticle Collection Device 150 Microchip for Sorting Microparticles 155 Main channel 159 Recovery Channel 161 Liquid supply channel 170 Connection Channel

Claims

1. a flowing step of flowing a first liquid containing microparticles through a main flow path; 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 particles to be recovered into a recovery flow path; and In the recovery step, droplets containing a plurality of recovery target particles are recovered, the plurality of recovery target particles are recovered in the recovery flow path in a second liquid while being contained in the first liquid, the second liquid is immiscible with the first liquid; and In the recovery flow path, the second liquid becomes a dispersion medium and the first liquid becomes a dispersoid. A method for recovering microparticles.

2. 2. The method for recovering fine particles according to claim 1, wherein in the recovering step, the plurality of recovery target particles are recovered into the recovery flow path through a connection flow path that connects the main flow path and the recovery flow path.

3. 3. The method for recovering fine particles according to claim 2, wherein the second liquid is supplied into the connecting flow path from a liquid supply flow path that is connected to the connecting flow path so as to be able to supply liquid thereto.

4. The method for recovering microparticles according to claim 3 , wherein the supply of the second liquid from the liquid supply flow channel into the connecting flow channel forms a flow that flows from a connection position between the liquid supply flow channel and the connecting flow channel toward the main flow channel.

5. The method for recovering fine particles according to claim 1 , wherein a plurality of droplets are formed by carrying out the method for recovering fine particles, and at least a portion of the plurality of droplets includes the plurality of particles to be recovered.

6. The method for recovering fine particles according to claim 2 , 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.

7. The method for recovering fine particles according to claim 2 , wherein in the flowing step, the fine particles flow in a laminar flow through the main flow path toward the connecting flow path.

8. 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.

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

10. a flowing step of flowing a first liquid containing microparticles through a main flow path; 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 particles to be recovered into a recovery flow path; and In the recovery step, droplets containing a plurality of recovery target particles are recovered, the plurality of recovery target particles are recovered in the recovery flow path in a second liquid while being contained in the first liquid, the second liquid is immiscible with the first liquid; and In the recovery flow path, the second liquid becomes a dispersion medium and the first liquid becomes a dispersoid. Droplet production method.

11. a first liquid supply unit that supplies a first liquid containing microparticles to a main flow path in a microchip for sorting microparticles, the main flow path having a flow path structure including a main flow path through which microparticles flow and a recovery flow path through which recovery target particles among the microparticles are recovered; a determination unit that determines whether the microparticles flowing in the main flow path are particles to be collected; Equipped with Collecting droplets containing a plurality of collection target particles into the collection channel; the plurality of recovery target particles are recovered in the recovery flow path in a second liquid while being contained in the first liquid, the second liquid is immiscible with the first liquid; and In the recovery flow path, the second liquid becomes a dispersion medium and the first liquid becomes a dispersoid. Droplet collection device.

12. A microchip for sorting microparticles having a flow path structure including a main flow path through which microparticles flow and a recovery flow path through which target particles among the microparticles are recovered, The microchip is used to execute a microparticle recovery method, the method including: a flowing step of flowing a first liquid containing microparticles through the main flow path; a determining step of determining whether the microparticles flowing through the main flow path are particles to be recovered; and a recovery step of recovering the particles to be recovered into the recovery flow path, the recovery step recovering droplets containing a plurality of particles to be recovered, the plurality of particles to be recovered being contained in the first liquid, and being recovered in a second liquid in the recovery flow path, the second liquid being immiscible with the first liquid, and the second liquid being a dispersion medium and the first liquid being a dispersoid in the recovery flow path. The microchip.

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