Method for analyzing biological particles and reagent kit for analyzing biological particles

Abstract

The objective of the present invention is to provide a technique for analyzing biological particles in a state in which the influence of interactions between particles contained in a biological particle population is reflected. The present disclosure provides a biological particle analysis method including: a preparation step of preparing a biological particle population including biological particles to which a first capturing substance for secretory substance capture has been bound; a first capturing step of causing a secretory substance, generated by placing the biological particle population in a prescribed condition, and the first capturing substance to bind together; and a second capturing step of causing the secretory substance, bound to the first capturing substance, and a second capturing substance for secretory substance capture to bind together. Further, the present disclosure also provides a reagent kit for biological particle analysis, for use in the analysis method, and a biological particle analysis system employed to implement the analysis method.

Description

Technical Field

[0001] The present disclosure relates to a method for analyzing biological particles and a reagent kit for analyzing biological particles. More specifically, the present disclosure relates to a method for analyzing biological particles for performing single-cell analysis of each biological particle contained in a biological particle population, and a reagent kit for analyzing biological particles for use in the analysis method.

Background Art

[0002] In order to analyze the reactivity of cells, it has been proposed to measure secreted molecules. For example, Patent Document 1 below discloses a method for identifying a cell population containing effector cells having an extracellular effect. The document describes, as steps included in the method, a step of holding a cell population containing one or more effector cells in a microreactor containing a population of readout particles containing one or more readout particles, and a step of incubating the cell population and the one or more readout particles in the microreactor (Claim 1). The document describes that the extracellular effect is a direct or indirect effect of the readout particles outside the effector cells, and as a more specific example, the extracellular effect is the binding of a target biomolecule secreted by the effector cells to the readout particles, or a response such as apoptosis of the readout cells or accessory cells (paragraph 0183).

[0003] Also, Patent Document 2 below discloses a method for analyzing secreted proteins. The document describes that the method includes encapsulating cells in microdroplets containing predetermined components, retaining the molecules in the microdroplets by binding the molecules secreted from the cells to capture molecules, and detecting the secreted molecules (Claim 1).

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

[0005] This disclosure aims to provide a method for analyzing biological particles in a state contained within a population of biological particles, and in particular, a single-cell analysis method for cells in a state contained within a population of cells. [Means for solving the problem]

[0006] This disclosure includes a preparation step of preparing a bioparticle population containing bioparticles to which a first capture substance for capturing secreted substances is bound, A first capture step involves binding the secreted substance generated by placing the group of biological particles under predetermined conditions with the first capture substance, A second capture step involves combining the secreted substance bound to the first capture substance with a second capture substance for capturing the secreted substance, This invention provides a method for analyzing biological particles, including those mentioned above. The first capture step includes a processing step that places the group of biological particles under predetermined conditions, The processing step may be carried out while the collective state of the biological particle group is maintained. The first capture step and the second capture step may be performed while the state in which the first capture substance is bound to the biological particle is maintained. The biological particles included in the group of biological particles prepared in the aforementioned preparation step may have particle identifiers attached to them for identifying the biological particles. The second captured substance may have a captured substance identifier attached to it for the purpose of identifying the second captured substance. The first capture substance may include a secretion substance binding site and a biological particle binding site. The secretion-binding portion may be configured to bind one or more secretion-binding substances. The biological particle binding portion may include an antigen-binding substance that binds to an antigen on the surface of the biological particle, or a molecular-binding substance that binds to a molecule that forms a surface film on the biological particle. The secretion substance binding portion may be bound to the biological particle binding portion via a cross-linking portion. The first capture substance may include antibodies that bind to the surface of two or more cells of the same or different species. A biological particle analysis method according to this disclosure may further include, after the second capture step, an isolation step of isolating the biological particles contained in the biological particle population into single particles. A biological particle analysis method according to this disclosure may further include a destruction step of destroying the biological particles after the isolation step. The aforementioned destruction process may be carried out in an environment in which the components contained in one biological particle do not mix with the components contained in other biological particles. A biological particle analysis method according to this disclosure may further include an analysis step of performing an analysis on each biological particle after the destruction step.

[0007] Furthermore, this disclosure is, A first secretory scavenging substance comprising a first biological particle binding portion configured to bind to biological particles and a first secretory substance binding portion configured to bind to secretory substances produced by placing a group of biological particles containing the biological particles under predetermined conditions; and A substance for capturing a second secretion, comprising a second secretion binding portion configured to bind to the aforementioned secretion and a capture substance identifier for identifying the second capture substance; We also offer reagent kits for biological particle analysis, including [specific component / method]. The first secretion-capturing substance may further include a crosslinking portion that crosslinks the biological particle binding portion and the secretion-binding portion. The first biological particle binding portion may include an antigen-binding substance that binds to an antigen on the surface of the biological particle, or a molecular-binding substance that binds to a molecule that forms a surface film of the biological particle. The antigen-binding substance may include a substance selected from the group consisting of antibodies, antibody fragments, aptamers, and molecularly imprinted polymers. The molecularly binding substance may contain an oleyl group or a cholesteryl group. The reagent kit may further include a substrate having a surface on which a particle-capturing substance is immobilized, the substance comprising a second biological particle binding portion configured to bind to biological particles and a particle identifier for identifying biological particles. [Brief explanation of the drawing]

[0008] [Figure 1A] This is an example of a flowchart for the biological particle analysis method described herein. [Figure 1B] This is an example of a manufacturing process flow chart. [Figure 2A] This is a schematic diagram illustrating the manufacturing process. [Figure 2B] This is a schematic diagram illustrating the first capture process. [Figure 2C] This is a schematic diagram illustrating the second capture process. [Figure 2D] This is a schematic diagram illustrating the isolation, destruction, and analysis processes. [Figure 3A] This is a schematic diagram illustrating particle trapping material. [Figure 3B] This figure shows examples of molecularly bonded substances. [Figure 4] This is a schematic diagram illustrating the first captured substance. [Figure 5] This is a schematic diagram illustrating a biological particle formed by the binding of the primary capture substance and the particle capture substance. [Figure 6] This is a schematic diagram illustrating the second trapping substance. [Figure 7A] This figure shows an example of a microchip used to form emulsion particles. [Figure 7B] This is a schematic diagram illustrating how biological particles are sequestered within emulsion particles. [Figure 8] This is a schematic enlarged view of the particle sorting section. [Figure 9] This is a schematic enlarged view of the particle sorting section. [Figure 10] This is an example of a flowchart for a method of forming an emulsion. [Figure 11A]This is a schematic enlarged view of the connecting channel section. [Figure 11B] This is a schematic enlarged view of the connecting channel section. [Figure 12A] This is a schematic enlarged view of the connecting channel section. [Figure 12B] This is a schematic enlarged view of the connecting channel section. [Figure 13] This is a schematic diagram illustrating the state in which a container is connected to a microchip. [Figure 14] This is a schematic diagram of another example of a microchip. [Figure 15] Schematic diagram of an example of a well used to perform a particle isolation process. [Figure 16] This is a schematic diagram illustrating how droplets containing biological particles are generated by a nozzle on a microfluidic chip. [Figure 17] This is a schematic diagram illustrating an example of a state in which the first capture substance is bound to biological particles. [Figure 18] This is a schematic diagram illustrating an example of a state in which the first capture substance is bound to biological particles. [Figure 19] This is a schematic diagram illustrating an example of a state in which the first capture substance is bound to biological particles. [Figure 20] This is a schematic diagram showing a state in which two cells are trapped by one primary capture substance. [Figure 21] This is a schematic diagram showing an example of a first capture substance containing antibodies that bind to two or more biological particles. [Figure 22] This is a schematic diagram illustrating an example of crosslinking between two or more biological particles. [Figure 23] This is a schematic diagram illustrating an example of crosslinking between two or more biological particles. [Figure 24] This is a schematic diagram illustrating how surface molecular bonding materials are bound to biological particles. [Figure 25] This is a schematic diagram illustrating a surface molecular bonding material to which an identification substance is bonded. [Modes for carrying out the invention]

[0009] The following describes preferred forms for implementing this disclosure. The embodiments described below are representative of the disclosure, and the scope of the disclosure is not limited to these embodiments. The description of this disclosure will proceed in the following order. 1. First Embodiment (Method for Analyzing Biological Particles) (1) Explanation of the task (2) Description of the first embodiment (3) Example of the first embodiment (3-1) Preparation process (3-1-1) Surface preparation process (3-1-2) Surface capture process (3-1-3) Capture substance binding step (Modification 1: Embodiment containing multiple secretion-binding substances in the secretion-binding portion) (Modification 2: Embodiment in which the biological particle binding site is a multispecific antibody) (Modification 3: First capture substance containing antibodies that bind to two or more biological particles) (Modification 4: Crosslinking of 2 or more biological particles) (3-1-4) Cleavage process (3-1-4-1) Detection process (3-1-4-2) Linker rupture process (3-2) First capture step (3-3) Second capture step (Variation 5: Use of materials that bond to the surface molecules of biological particles) (3-4) Isolation process (3-4-1) Discrimination process (3-4-2) Particle isolation process (3-4-2-1) In the case of space within emulsion particles (3-4-2-2) In the case of space within a well (3-5) Destruction process (3-6) Analysis process 2. Second Embodiment (Reagent Kit for Biological Particle Analysis) 3. Third Embodiment (Bioparticle Analysis System)

[0010] 1. First Embodiment (Method for Analyzing Biological Particles)

[0011] (1) Explanation of the task

[0012] As described above, several methods have been proposed for analyzing cellular reactivity by measuring secreted molecules. However, these methods do not take into account the effects of intercellular interactions in cell populations containing multiple cell types (e.g., immune cell populations).

[0013] Furthermore, gene expression and secreted molecular weight can sometimes have a low correlation. Therefore, measuring only intracellular molecules may be insufficient for cell analysis. For a more detailed analysis of cells, it is desirable to measure intracellular and extracellular secreted molecules simultaneously, and even more so to measure cell surface molecules simultaneously in addition to these.

[0014] It is difficult to simultaneously identify the cell types of cells in a cell population, such as an immune cell population, analyze the intracellular molecules contained within those cells, and analyze the extracellular molecules (especially secreted molecules) related to those cells.

[0015] To identify and / or analyze these molecules, fluorescent dyes can be used as labels. However, due to the overlap of fluorescence spectra, the number of molecules that can be identified using fluorescent dyes is at most a few dozen. While cell types can be identified by flow cytometry, it is difficult to obtain other information (e.g., information about intracellular and / or extracellular secretory molecules) using fluorescent dyes alone.

[0016] Furthermore, beads configured to capture extracellular molecules secreted from cells are sometimes used to analyze these molecules. In this case, it is advisable to isolate the cells and the beads in a microspace before capturing the molecules. However, when multiple types of cells are present in the sample, it is difficult to identify the cell that secreted the molecule and to identify the molecule secreted from a particular cell.

[0017] Based on the above, the primary purpose of this disclosure is to provide a method for analyzing biological particles in a state contained within a group of biological particles. Furthermore, this disclosure also aims to provide a method for analyzing one or more substances (particularly secreted substances) present outside the biological particle and / or one or more substances present inside the biological particle. Such analysis may be performed, for example, on each individual biological particle contained within the group of biological particles.

[0018] (2) Description of the first embodiment

[0019] A method according to this disclosure includes a preparation step of preparing a bioparticle population including bioparticles to which a first capture substance for capturing secreted substances is bound; a first capture step of binding secreted substances, which are generated by placing the bioparticle population under predetermined conditions, to the first capture substance; and a second capture step of binding secreted substances bound to the first capture substance to a second capture substance for capturing secreted substances. This makes it possible to capture secreted substances generated when the bioparticle population is placed under predetermined conditions by the first and second capture substances, and to form a state in which these three substances (the secreted substances, the first capture substance, and the second capture substance) are bound to the bioparticles. This makes it possible to analyze the bioparticles in a state that reflects the interparticle interactions in the bioparticle population. That is, in a method according to this disclosure, the first capture step and the second capture step may be performed while the state in which the first capture substance is bound to the bioparticles is maintained.

[0020] This disclosure is suitable for analyzing cells in diverse cell populations, such as immune cell populations. For example, this disclosure can provide information about cells (cell type or state, e.g., degree of differentiation) and extracellular molecules (particularly secreted substances) that reflect the effects of intercellular interactions in a cell population. In addition to this information, this disclosure can also provide information about intracellular molecules. For example, this disclosure can directly or indirectly observe which cells in a cell population with a certain cellular composition are reacting by analyzing secreted substances (e.g., identifying the type or measuring the amount of secreted substances). This makes it possible to clarify the function of diverse cell populations.

[0021] In a method according to this disclosure, secreted substances may be captured by a first capture substance bound to the surface of a biological particle. Furthermore, in a method according to this disclosure, the biological particles do not need to be isolated in order to trigger the reaction that produces secreted substances, and the reaction may be carried out in an environment in which multiple types of biological particles are present. Furthermore, secreted substances captured on the surface of biological particles are reacted with a second capture substance (for example, a secreted substance-binding antibody to which a capture substance identifier such as an oligo barcode is bound). The second capture substance can be analyzed or measured using, for example, a particle identifier (including oligo barcodes) bound to the surface of the biological particles. Therefore, by pre-associating the secreted substance with the second capture substance, the secreted substance can also be analyzed or measured. In addition, in addition to the analysis or measurement of the secreted substance, analysis of surface antigens of the biological particles and / or analysis of gene expression within the biological particles can be performed simultaneously. Moreover, by placing a group of biological particles under conditions that promote the secretion of secreted substances, it is possible to confirm which biological particle the secreted substance originated from using, for example, a particle identifier bound to the surface of the biological particles.

[0022] In a preferred embodiment, the first capture step includes a processing step that places the bioparticle population under predetermined conditions, and the processing step is carried out while maintaining the collective state of the bioparticle population. This allows the reaction in which secreted substances are produced to be carried out while maintaining intercellular interactions in the cell population. After the reaction, it becomes possible to analyze cell type, cell state, intracellular gene expression, and extracellular secreted molecules with single-cell resolution.

[0023] A method according to this disclosure may further include, after the second capture step, an isolation step of isolating the biological particles contained in the group of biological particles into single particles. A method according to this disclosure may further include, after the isolation step, a destruction step of destroying the biological particles. The destruction step may be performed while the isolated state is maintained. That is, the destruction step may be performed in an environment in which the components contained in one biological particle do not mix with the components contained in other biological particles. A method according to this disclosure may further include, after the destruction step, an analysis step of performing an analysis on each biological particle. These processes enable the analysis of the reactivity of cells in a cell population containing multiple cell types at single-cell resolution. This analysis allows for the elucidation of the functionality of each cell in a given cell population. Furthermore, this analysis enables the identification of cells or cell populations optimal for treatment using in vitro assays. Therefore, this disclosure contributes to improving the response rate of cell populations (e.g., cell therapies) used to treat diseases such as cancer.

[0024] (3) Example of the first embodiment

[0025] The biological particle analysis method described herein will be explained below with reference to Figure 1A. Figure 1A is an example of a flowchart of the said biological particle analysis method.

[0026] The biological particle analysis method of this disclosure includes, for example, a preparation step S101, a first capture step S102, a second capture step S103, an isolation step S104, a disruption step S105, and an analysis step S106, as shown in Figure 1A. Each step will be described below.

[0027] (3-1) Preparation process

[0028] In preparation step S101, a group of biological particles containing biological particles to which a first capture substance for capturing secreted substances is bound is prepared. The group of biological particles may be, for example, a group of cells. The group of cells may be, for example, a group of immune cells or a group of blood cells.

[0029] The preparation process includes the manufacturing process for the bioparticle population. An example of this manufacturing process will be explained with reference to Figures 1B and 2A. Figure 1B is an example of a flow chart of the manufacturing process. Figure 2A is a schematic diagram illustrating the manufacturing process.

[0030] As shown in Figure 1B, the manufacturing process may include a surface preparation step S111, a surface capture step S112, a captured material bonding step S113, and a cracking step S114. These steps will be described below.

[0031] (3-1-1) Surface preparation process

[0032] In the surface preparation step S111, a surface on which particle-trapping material is fixed is prepared. For example, as shown in Figure 2Aa, multiple particle-trapping materials 120 are fixed to the surface 110 of the substrate 100.

[0033] The particle trapping material 120 is fixed to the surface 110 via a linker 126 which is included as part of the material.

[0034] In addition to the linker 126, the particle capture material 120 further includes a particle capture unit 121, a material recovery unit 122 (e.g., poly-T), a UMI (Unique Molecular Identifier) ​​unit 123, a particle identifier 124 (e.g., a cell barcode), and a recovered material amplification unit 125 (e.g., a nucleic acid amplification primer and / or a nucleic acid transcription promoter), as shown in Figure 3A. These will be described below.

[0035] The particle capture unit 121 is configured to capture biological particles, and in particular to capture cells. The particle capture unit 121 may be a biological particle-binding substance. This biological particle-binding substance may be an antigen-binding substance that binds to an antigen on the surface of the biological particle P, or a molecular-binding substance that binds to a molecule that forms the surface film of the biological particle P. The antigen-binding substance may include substances selected from the group including antibodies, antibody fragments, aptamers, and molecularly imprinted polymers. The antibody or antibody fragment may be an antibody or antibody fragment that binds to components (particularly surface antigens) present on the surface of biological particles such as cells. The aptamer may be a nucleic acid aptamer or a peptide aptamer. The aptamer and the molecularly imprinted polymer may also bind to components (particularly surface antigens) present on the surface of biological particles such as cells. The molecular-binding substance is, for example, a compound containing an oleyl group or a cholesteryl group. These groups can nonspecifically bind to molecules that form the surface membrane of biological particles P (e.g., cells). The oleyl group and cholesteryl group can bind to biological particles formed from lipid bilayers, such as cells. An example of a compound containing an oleyl group is oleylamine, shown on the left in Figure 3B. An example of a compound containing a cholesteryl group is Cholesterol-TEG (15 atom triethylene glycol spacer), shown on the right in Figure 3B. The upper right of Figure 3B shows Cholesterol-TEG bound to the 5' end of an oligonucleotide. The lower right of Figure 3B shows Cholesterol-TEG bound to the 3' end of an oligonucleotide. By modifying an oligonucleotide with a molecular-binding substance containing an oleyl group or a cholesteryl group, the oligonucleotide can capture biological particles.

[0036] The substance recovery unit 122 is configured to capture molecules contained in biological particles and / or the combined substance of the first captured substance, secreted substance and second captured substance formed in the second capture step (3-3) described below. The material recovery unit 122 may contain, for example, nucleic acids or proteins. The nucleic acid may be configured to comprehensively capture the conjugate and mRNA contained in biological particles (particularly cells), and may be, for example, a poly-T sequence. The poly-T sequence can bind to the poly-A sequence contained in the second capture substance that constitutes the conjugate. Furthermore, the poly-T sequence can bind to the poly-A sequence contained in mRNA within the biological particle. Alternatively, the nucleic acid may have a sequence complementary to the target sequence contained in the conjugate or to the target sequence of the nucleic acid in the biological particle. By having such a complementary sequence, the nucleic acid can bind to these target sequences. If the substance recovery unit is a protein, the protein may be, for example, an antibody. The substance recovery unit may also be an aptamer or a molecular imprinted polymer. The substance recovery unit 122 may include two or more components for capturing molecules contained in the conjugate or biological particles. The substance recovery unit 122 may include both proteins and nucleic acids, for example, both antibodies and polyT sequences. This allows for the simultaneous detection of both proteins and mRNA.

[0037] The UMI (Unique Molecular Identifier) ​​portion 123 may contain nucleic acids, particularly DNA or RNA, and more particularly DNA. The UMI portion 123 may have a sequence of, for example, 5 to 30 bases, particularly 6 to 20 bases, and more particularly 7 to 15 bases. The UMI portion 123 may be configured to have different sequences among the particle-trapping materials fixed to the surface 110. For example, if the UMI portion has a 10-base nucleic acid sequence, the number of UMI sequence types is 4 to the power of 10, or more than 1 million. The UMI section 123 can be used to quantify molecules contained in biological particles. For example, if the molecule to be quantified is mRNA, the UMI sequence may be added to the cDNA obtained by reverse transcribing the target mRNA in the analysis step described later. Numerous cDNAs obtained by amplifying cDNA reverse-transcribed from one mRNA molecule will have the same UMI sequence, but numerous cDNAs obtained by amplifying cDNA transcribed from other mRNA molecules having the same sequence as the mRNA will have different UMI sequences. Therefore, the copy number of mRNA can be determined by counting the number of different types of UMI sequences that have the same cDNA sequence. For this reason, the analysis step described later may include, for example, determining the copy number of mRNA, or it may include counting the number of different types of UMI sequences that have the same cDNA sequence.

[0038] For example, the UMI portion 123 may be configured to have different sequences among multiple particle-capturing materials containing the same particle identifier, fixed to a single region R (e.g., a spot or bead described later) as shown in Figure 2A a and b. That is, multiple target-capturing molecules fixed to the region R (e.g., a spot or bead described later) may have the same particle identifier, but may have different UMI portions (in particular, UMI portions having different base sequences).

[0039] The particle identifier 124 is used to identify or specify a biological particle to which the particle identifier is bound (more specifically, to which a particle-capturing material containing the particle identifier is bound). The particle identifier 124 includes, for example, a nucleic acid having a barcode sequence. The nucleic acid may be DNA or RNA in particular, and more particularly DNA. The barcode sequence may be used, for example, to identify a captured biological particle (particularly a cell), and may be used as an identifier to distinguish a biological particle isolated in one microspace from a biological particle isolated in another microspace. The barcode sequence may also be used as an identifier to distinguish a particle-capturing material containing one barcode sequence from a particle-capturing material containing another barcode sequence. The barcode sequence may be associated with a biological particle to which the particle-capturing material containing the barcode sequence is bound. The barcode sequence may also be associated with information about the position on the surface 110 to which the particle-capturing material containing the barcode sequence is fixed. Furthermore, the barcode sequence may be associated with a microspace to which a biological particle to which a particle-capturing material containing the barcode sequence is bound is isolated, and may also be associated with information regarding the location of the microspace. The position information mentioned above is, for example, information regarding XY coordinates, but is not limited to this. An ID number may be assigned to the barcode sequence associated with the position information. This ID number may be used in processes after the cracking process. This ID number may correspond one-to-one with the barcode sequence and may be used as data corresponding to the barcode sequence in processes after the cracking process. Thus, the biological particles included in the group of biological particles prepared in preparation step S101 may have particle identifiers attached to them for identifying the biological particles.

[0040] Multiple particle-capturing materials fixed within a certain region of the surface 110 may have the same particle identifier (particularly the same barcode sequence). This associates the region with the particle identifier. By setting the size of the region to be smaller than the size of a biological particle, the particle-capturing material containing the particle identifier can be associated with the location where a single biological particle exists. For example, as shown in Figures 2A a and b, the region R on which multiple particle-capturing materials 120 containing the same particle identifier are fixed may be smaller than the size of a biological particle P. Thus, the surface 110 used in the biological particle analysis method of this disclosure may have multiple regions on which multiple particle-capturing materials having the same particle identifier are fixed. The particle identifier may be different for each region. The size of each region (for example, the maximum dimension of the region, such as the diameter, major axis, or length of the major side) is preferably smaller than the size of the biological particle, for example, 50 μm or less, preferably 10 μm or less, and more preferably 5 μm or less. These multiple regions may be spaced apart, for example, so that a biological particle captured by a particle-capturing material fixed in one region is not captured by a particle-capturing material fixed in another region. This spacing may be, for example, greater than the size of the biological particle, and preferably greater than the size of the biological particle. The number of such multiple regions is preferably greater than the number of biological particles applied to the surface 110 in the capture step. This suppresses the capture of two or more biological particles in a single region.

[0041] In one embodiment of the present disclosure, a particle-capturing substance containing a known particle identifier (particularly a barcode sequence with a known sequence) can be fixed to a predetermined region. For example, the surface 110 has a plurality of regions, and a plurality of particle-capturing substances fixed to each of these regions may contain the same particle identifier. The plurality of regions may be set to be smaller than the size of the biological particles to be captured. With the surface 110 configured in this way, each of the plurality of regions can be associated with a particle identifier contained in a plurality of target-capturing molecules fixed to each region. In this specification, a region in which a particle-capturing material containing the same particle identifier is fixed is also referred to as a spot. That is, the size of the spot may be, for example, 50 μm or less, preferably 10 μm or less, and more preferably 5 μm or less. The surface 110 configured as described above allows for the association of a particle identifier contained in a particle-capturing substance with the location where that particle-capturing substance exists, once the particle-capturing substance is immobilized on the surface 110. For this immobilization, for example, biotin is bound to the linker 1 of the particle-capturing substance and streptavidin is bound to the surface 101 on which the particle-capturing substance is immobilized. The particle-capturing substance is then immobilized on the surface 110 by the binding of the biotin and streptavidin.

[0042] In other embodiments of this disclosure, particle-capturing material containing particle identifiers may be randomly arranged on the surface 110. In this case, after a particle-capturing material containing a particle identifier is fixed to the surface 110, the particle identifier contained in the fixed particle-capturing material is identified (particularly by reading the barcode sequence), thereby associating a particle identifier contained in a particular particle-capturing material with the location where that particle-capturing material exists. This reading can be performed by methods such as Sequencing By Synthesis, Sequencing by Ligation, or Sequencing by Hybridization. Furthermore, it is not necessary to associate a particle identifier contained in a particle-capturing material with the location where that particle-capturing material exists. In this case, for example, in the isolation step described later, the biological particle and the particle-capturing material are isolated in a microspace, thereby establishing a one-to-one correspondence between the biological particle and the particle-capturing material (particularly the barcode sequence contained in the particle-capturing material). In this embodiment, for example, beads (e.g., gel beads) to which multiple particle-capturing materials containing the same particle identifier are bound may be used. These beads (e.g., gel beads) can be fixed to a surface 110, for example. The size of the beads (e.g., gel beads) may be, for example, 50 μm or less, preferably 10 μm or less, and more preferably 5 μm or less. To bind the particle-capturing material to the beads (e.g., gel beads), a combination of biotin and streptavidin may be used, for example. For example, biotin is bound to the linker 126 of the particle-capturing material and streptavidin is bound to the beads, and the particle-capturing material is immobilized on the beads by the binding of the biotin and streptavidin.

[0043] The surface 110 may have a plurality of recesses. One spot or one bead may be placed in each of the plurality of recesses as in the embodiment. The plurality of recesses make it easier to place the spot or bead on the surface 110. The size of the recesses is preferably such that one bead can fit inside. The shape of the recesses may be, but is not limited to, circular, elliptical, hexagonal, or square.

[0044] Furthermore, the surface state of the portion of the surface 110 on which the spots or beads are placed may differ from that of other surface portions. For example, the portion of the surface on which the spots or beads are placed may be hydrophilic, while the other surface portions may be hydrophobic, or the other surface portions may be hydrophobic and have protrusions. Examples of methods for imparting hydrophilicity to the surface include reactive ion etching in the presence of oxygen and irradiation with deep ultraviolet light in the presence of ozone. In these methods, a mask with a perforated portion for imparting hydrophilicity may be used. Another example of a method for imparting hydrophobicity to the surface is spray-on-silicone, such as Techspray 2101-12S. In the case of imparting hydrophobicity, a mask with a perforated portion for imparting hydrophobicity may also be used.

[0045] For example, the particle-capturing material can be synthesized on a substrate using techniques such as DNA microarray fabrication. Alternatively, the particle-capturing material can be synthesized at a specific location using techniques such as DMDs (Digital Micromirror Devices), liquid crystal shutters, or spatial light phase modulators used in photolithography. The method for this synthesis is described, for example, in Basic Concepts of Microarrays and Potential Applications in Clinical Microbiology, CLINICAL MICROBIOLOGY REVIEWS, Oct. 2009, pp. 611-633. When the particle-capturing material is synthesized on a substrate using this method, information about the location where the particle-capturing material is synthesized is obtained, and the particle identifier is associated with the location information. In this case, an ID number may be assigned to each particle identifier.

[0046] In one embodiment of this disclosure, any of the particle-capturing materials fixed to the surface may contain a common oligo sequence. By using a nucleic acid having a sequence complementary to the oligo sequence and being fluorescently labeled, the position where the particle-capturing material is fixed (particularly the position of the spot or the bead) can be confirmed, and can be confirmed particularly in dark-field observation. Furthermore, if there are no recesses or protrusions on the surface as described above, it may be difficult to determine the position where the particle-capturing material is fixed. In this case, the fluorescent labeling makes it easier to determine the position where the particle-capturing material is fixed.

[0047] The recovered substance amplification unit 125 may include, for example, a nucleic acid having a primer sequence used for nucleic acid amplification and / or a promoter sequence used for nucleic acid transcription in the analytical step described later. The nucleic acid may be DNA or RNA, and is particularly DNA. The recovered substance amplification unit 125 may have both a primer sequence and a promoter sequence. The primer sequence may be, for example, a PCR handle. The promoter sequence may be, for example, a T7 promoter sequence. In this specification, the recovered substance amplification unit 125 is also called the first recovered substance amplification unit to distinguish it from the second recovered substance amplification unit 172 described later.

[0048] The linker 126 may be a linker that can be cleaved by stimulation, for example, a linker that can be cleaved by photostimulation or chemical stimulation. Photostimulation is particularly suitable for selectively stimulating a specific location in the cleavage process described later.

[0049] Linker 126 may contain, for example, one selected from an arylcarbonylmethyl group, a nitroaryl group, a coumarin-4-ylmethyl group, an arylmethyl group, a metal-containing group, and other groups, as a linker that can be cleaved by photostimulation. These groups may include, for example, those described in Photoremovable Protecting Groups in Chemistry and Biology: Reaction Mechanisms and Efficacy, Chem. Rev. 2013, 113, 119-191. For example, the arylcarbonylmethyl group may be a phenacyl group, an o-alkylphenacyl group, or a p-hydroxyphenacyl group. The nitroaryl group may be, for example, an o-nitrobenzyl group, an o-nitro-2-phenethyloxycarbonyl group, or an o-nitroanilide. The arylmethyl group may have a hydroxyl group introduced into it, or it may not have a hydroxyl group introduced.

[0050] If linker 126 is a linker that can be cleaved by light stimulation, it may preferably be cleaved by light with a wavelength of 360 nm or longer. The linker is preferably 0.5 μJ / μm 2 The linker may be cleaved at the following energies: (Light-sheet fluorescence microscopy for quantitative biology, Nat Methods. 2015 Jan;12(1):23-6. doi: 10.1038 / nmeth.3219.). By employing a linker cleaved at the above wavelengths of light or energies, it is possible to reduce cellular damage (especially DNA or RNA cleavage) that may occur when light stimulation is applied.

[0051] Particularly preferably, the linker may be a linker that is cleaved by light in the short wavelength region, specifically light in the wavelength region of 360 nm to 410 nm, or a linker that is cleaved by light in the near-infrared or infrared region, specifically light in the wavelength region of 800 nm or more. If the linker is a linker that is efficiently cut by light in the visible light region, handling of the analytical surface may become difficult. For this reason, it is preferable that the linker is a linker that is cleaved by light in the short wavelength region or light in the near-infrared or infrared region.

[0052] Linker 126 may contain, for example, a disulfide bond or a restriction endonuclease recognition sequence as a linker that can be cleaved by chemical stimulation. Reducing agents such as tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT), or 2-mercaptoethanol are used to cleave the disulfide bond. For example, when using TCEP, the reaction is carried out at 50 mM for approximately 15 minutes. For dissociation of restriction endonuclease recognition sequences, an appropriate restriction enzyme (http: / / catalog.takara-bio.co.jp / product / basic_info.php?unitid=U100003632) is used depending on the sequence. One unit of restriction enzyme activity is, in principle, the amount of enzyme that completely degrades 1 μg of λDNA per hour at 37°C in 50 μl of each enzyme reaction solution, and the amount of enzyme is adjusted according to the amount of restriction endonuclease recognition sequence.

[0053] To improve efficiency in the cracking process described later, the particle trapping material 120 may contain multiple crackable linkers. Preferably, these multiple linkers may be connected in series. For example, if the cracking probability of one linker is 0.8, by connecting three such linkers in series, the cracking probability becomes 0.992 (=1-0.2). 3 ) will improve to that.

[0054] (3-1-2) Surface capture process

[0055] In the surface capture step S112, biological particles P are captured by the particle capture material 120, for example, as shown in Figure 2Ab. In particular, the biological particles are captured by the particle capture section 121 of the particle capture material 120. In the surface capture step S112, the biological particles and the particle capture section 121 can be bound in a specific or nonspecific manner. For example, if the biological particle is a cell, the cell can be captured by the particle-capturing substance 120 by binding of the cell's surface antigen to an antibody, aptamer, or molecularly imprinted polymer contained in the particle-capturing unit 121. The antibody, aptamer, and molecularly imprinted polymer may be specific to the surface antigen or nonspecific. In this case, the cell may also be captured by the particle-capturing substance 120 by binding of the cell's lipid bilayer to an oleyl group or cholesteryl group contained in the particle-capturing unit 121.

[0056] The surface capture step S112 may include an application step of applying biological particles to the surface 110. The application method may be, for example, by bringing a sample containing a collection of biological particles (e.g., a liquid containing biological particles) into contact with the surface 110. For example, the sample containing a collection of biological particles may be dropped onto the surface 110.

[0057] In the surface capture step S112, preferably, multiple particle-capturing materials bound to a single biological particle may have the same particle identifier. This allows one particle identifier (particularly a barcode sequence) to be associated with one biological particle. Preferably, the UMI portions contained in the multiple particle-capturing materials may have different base sequences. This allows, for example, the determination of the copy number of mRNA.

[0058] The surface capture step S112 may include an incubation step for binding the biological particles with the particle-capturing material. Incubation conditions such as incubation time and temperature may be determined according to the type of biological particles and particle-capturing material used.

[0059] After performing the surface capture step S112, a removal step may be performed to remove unwanted substances, such as biological particles that did not bind to the particle capture material 120. The removal step may include, for example, washing the surface 110 with a liquid such as a buffer.

[0060] (3-1-3) Capture substance binding step

[0061] As shown in Figure 2Ab, each biological particle is bound to a first capture substance 130 for capturing secreted substances. The number of types of first capture substances 130 bound to a single biological particle may be one or more. Similarly, the number of first capture substances 130 bound to a single biological particle may be one or more, but preferably multiple.

[0062] The first capture substance 130 will be described with reference to Figure 4. This figure is a schematic diagram illustrating an example of the structure of the first capture substance 130. As shown in the figure, the first capture substance 130 includes a secretion substance binding portion 131 and a biological particle binding portion 133. The first capture substance 130 further includes a cross-linking portion 132. The secretion substance binding portion 131 is bound to the biological particle binding portion 133 via the cross-linking portion 132.

[0063] The secretion-binding portion 131 may be configured to bind one or more secretion-binding portions. The secretion-binding portion 131 may be appropriately designed or manufactured by a person skilled in the art depending on the secretion-binding portion intended to bind. For example, the secretion-binding portion 131 may be a substance selected from the group including, for example, antibodies, antibody fragments, aptamers, and molecularly imprinted polymers, and is particularly an antibody or antibody fragment. In Figure 4, an antibody is shown as the secretion-binding portion 131. The binding ability of the secretion-binding portion 131 may be specific or nonspecific, and is particularly specific. The number of types of secretion-binding portions 131 bound to a single biological particle P may be one or more.

[0064] The secretory substance to which the secretory substance binding portion 131 binds is a secretory substance produced by placing a group of biological particles containing biological particles P under predetermined conditions. The secretory substance may be a substance secreted from biological particles P, a substance secreted from other biological particles included in the group of biological particles, or a secretory substance derived from the environment constituting the predetermined conditions. The environment constituting the predetermined conditions may be appropriately selected by the user performing the biological particle analysis method of this disclosure, and may be an environment containing a material whose reactivity of the group of biological particles is analyzed. The environment may be, for example, an environment in which the group of biological particles is incubated, such as a culture medium or buffer environment. The material whose reactivity of the group of biological particles is analyzed may be selected according to the reactivity to be analyzed, and may be a biomaterial or a non-biological material. The biomaterial may be, for example, diseased tissue, diseased cells, microorganisms (bacteria, fungi, or viruses), or heterologous tissue. The non-biological material may be, for example, a drug or a toxic substance. The diseased tissue may be, for example, tumor tissue, and in particular, cancerous tissue or sarcoma tissue. The diseased cells may be, for example, tumor cells, and in particular may be cancer cells, sarcoma cells, or malignant lymphoma cells. For example, when analyzing the reactivity of a population of biological particles to diseased tissue or diseased cells, the environment constituting the predetermined conditions may be a liquid (particularly a culture medium or buffer) containing diseased tissue or diseased cells.

[0065] The crosslinking portion 132 is a substance that crosslinks the secretion substance binding portion 131 and the biological particle binding portion 133. Alternatively, the biological particle binding portion 133 may be directly bound to the secretion substance binding portion 131; in this case, the first capture substance 130 does not need to include a crosslinking portion.

[0066] The crosslinked portion 132 may be, for example, a compound described in International Publication No. 2017 / 177065, or a stereoisomer, salt, or tautomer thereof. Such a compound is described below. The bridge section 132 is The following structure (I): [ka] a compound having the same, or a stereoisomer, salt or tautomer thereof. R in structure (I) 2 and R 3 either one of which may be bound to the secretion substance binding site 131 and the other to the biological particle binding site 133. In structure (I): M is, in each occurrence, independently, a moiety containing two or more carbon-carbon double bonds and at least one degree of conjugation; L 1 is, in each occurrence, independently, either i) an optional alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, heteroalkynylene or heteroatom linker; or ii) a linker containing a functional group that can be formed by the reaction of two complementary reactive groups; L 2 and L 3 are, in each occurrence, independently, an optional alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, heteroalkynylene or heteroatom linker; L 4 is, in each occurrence, independently, a heteroalkylene, heteroalkenylene or heteroalkynylene linker having a length greater than 3 atoms, where the heteroatoms in the heteroalkylene, heteroalkenylene and heteroalkynylene linkers are selected from O, N and S; R 1 is, in each occurrence, independently, H, alkyl or alkoxy; R 2 and R 3 are each independently H, OH, SH, alkyl, alkoxy, alkyl ether, heteroalkyl, -OP(=R a )(R b )R c , Q, or a protected form thereof, or L'; R 4 is, in each occurrence, independently, OH, SH, O - S - OR d, SR d , or Q; R 5 In each existence, independently, it is either oxo, thioxo, or non-existent; R a is either O or S; R b OH, SH, O - S - , OR d or SR d and; R c OH, SH, O - S - , OR d ,OL',SR d , alkyl, alkoxy, heteroalkyl, heteroalkoxy, alkyl ether, alkoxyalkyl ether, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkyl ether or thiophosphoalkyl ether; R d is the counterion; Q is a moiety that, in each presence, independently contains a reactive group or a protected form thereof capable of forming a covalent bond with the analyte molecule, the targeting moiety, the solid support, or the complementary reactive group Q′; L' is, in each presence, independently a linker containing a covalent bond to Q, a linker containing a covalent bond to the targeting moiety, a linker containing a covalent bond to the analyte molecule, a linker containing a covalent bond to a solid support, a linker containing a covalent bond to a solid support residue, a linker containing a covalent bond to a nucleoside, or a linker containing a covalent bond to a further compound of structure (I); m is independently 0 or a greater integer in each existence, except that at least one existence of m is 1 or a greater integer; and n is an integer greater than or equal to 1. Also, R in structure (I) 4 The secretion substance binding portion 131 may be bound to it. For example, R 2 and R 3 The bioparticle binding portion 133 is bound to one of the two of them, and the other of these is R4 One or more of the selected secretion substance binding sites 131 may be bound to each of them. An example of a structure in which multiple secretion substance binding sites 131 are bound is also explained in Modification Example 1 below, so please refer to that as well. R 4 To attach multiple identical or different secretion-binding sites to structure (I), R 4 A part to which a secretion substance binding site has been attached (hereinafter referred to as R 4-1 Prepare (which we call), and similarly R 4-2 , R 4-3 , ...and R 4-i (where i can be any integer from 2 to 500, more particularly from 2 to 300, more particularly from 2 to 100, 2 to 50, 2 to 20, or 2 to 10, and more particularly from 2 to 4) and then, for example, as in DNA synthesis, R 4-1 ~R 4-i These may be incorporated sequentially into structure (I). R 4-1 ~R 4-i Between them, the R is not bound to the secretory binding site. 4 (R 4-0 (Also known as) it may be introduced as a spacer. For example, R having a secretion substance binding site. 4 R has one P atom bonded to it and a secretory substance binding site. 4 R that does not have a secretion substance binding site between it and other P atoms to which it is bonded. 4-0 There may be one or more P atoms bonded to it. Also, R 4 This may include spacer molecules such as PEG, meaning that the atom P and the secretion-binding site may be bonded via these spacer molecules.

[0067] Regarding compounds having structure (I), L 4 Each entity may independently be an alkylene oxide linker.

[0068] Regarding compounds having structure (I), L 4 It is polyethylene oxide, and the aforementioned chemical The object has the following structure (IA): [ka] The expression has such that z is an integer between 2 and 100, and may be an integer between 3 and 6, for example.

[0069] Regarding compounds having structure (I), L 1 The structure is as follows: [ka] It may have one of the following.

[0070] The compound described above has the following structure (IB): [ka] It may have, here: x 1 , x 2 , x 3 and x 4 In each existence, independently, is an integer from 0 to 6, and z can be an integer between 2 and 100. x 1 and x 3 In each existence, x is 0, and 2 and x 4 In each existence, each can be 1. x 1 , x 2 , x 3 and x 4 In each existence, each can be 1.

[0071] Regarding compounds having structure (I), R 4 In each existence, independently, OH, O - OR d And R 5 In each existence, it may be an oxo.

[0072] Regarding compounds having structure (I), R 1 In each existence, may be H.

[0073] Regarding compounds having structure (I), R 2 and R 3 Each of these is independent of -OP(=R a )(R b )R c That's fine. R c It's fine for it to be an office lady. L' may be a heteroalkylene linker to Q, the targeting moiety, the analyte molecule, the solid support, the solid support residue, the nucleoside, or a further compound of structure (I). L' may include an alkylene oxide or phosphodiester moiety, or a combination thereof. L' has the following structure: [ka] It has, and here: m" and n" are independently integers between 1 and 10; R e is H, an electron pair, or a counterion; L is R e Alternatively, it may be a direct bond, or a linkage to Q, the targeting moiety, the analyte molecule, the solid support, the solid support residue, the nucleoside, or a further compound of structure (I). The targeting portion may be an antibody or a cell surface receptor antagonist.

[0074] Regarding compounds having structure (I), R 2 or R 3 The structure is as follows: [ka] [ka] It may have one of the following.

[0075] With respect to compounds having structure (I), Q may include sulfhydryl, disulfide, activated ester, isothiocyanate, azide, alkyne, alkene, diene, dienophil, acid halide, sulfonyl halide, phosphine, α-haloamide, biotin, amino or maleimide functional groups. Q may contain a maleimide functional group.

[0076] With respect to a compound having structure (I), Q may include a portion selected from Table 1 (Tables 1-1 to 1-3) below. [Table 1-1] [Table 1-2] [Table 1-3]

[0077] With respect to a compound having structure (I), m may independently be an integer between 1 and 10 in each instance, and in particular may be an integer between 1 and 5.

[0078] For compounds having structure (I), n may be an integer from 1 to 10.

[0079] With respect to the compound having structure (I), M can independently be pyrene, perylene, perylene monoimide, or 6-FAM or a derivative thereof in each of these entities.

[0080] With respect to a compound having structure (I), M independently has the following structure in each instance: [ka] It is possible to possess one of them.

[0081] The compound having structure (I) may be any compound selected from, for example, the compounds listed in Table 2 of International Publication No. 2017 / 177065.

[0082] The biological particle binding portion 133 may be an antigen-binding substance that binds to an antigen on the surface of the biological particle P, or a molecular-binding substance that binds to a molecule forming the surface film of the biological particle P. The configuration of the biological particle binding portion 133 may be appropriately selected or designed by those skilled in the art depending on the type of biological particle P. The antigen-binding substance may include substances selected from the group including antibodies, antibody fragments, aptamers, and molecularly imprinted polymers. The antibody or antibody fragment may be an antibody or antibody fragment that binds to components (particularly surface antigens) present on the surface of biological particles such as cells. The aptamer may be a nucleic acid aptamer or a peptide aptamer. The aptamer and the molecularly imprinted polymer may also bind to components (particularly surface antigens) present on the surface of biological particles such as cells. The molecular-binding substance is, for example, a compound containing an oleyl group or a cholesteryl group. These groups can nonspecifically bind to molecules that form the surface film of biological particles P (e.g., cells). Oleyl and cholesteryl groups can bind to biological particles formed from lipid bilayers, such as cells. Examples of these compounds are described above in "(3-1-1) Surface Preparation Step" with reference to Figure 3B.

[0083] The capture substance binding step S113 may include an incubation step for binding the biological particles with the first capture substance. Incubation conditions such as incubation time and temperature may be determined according to the type of biological particles and first capture substance used.

[0084] After performing the capture substance binding step S113, a removal step may be performed to remove unwanted substances, such as first capture substances that did not bind to the particle capture substance 120. The removal step may include, for example, washing the surface 110 with a liquid such as a buffer.

[0085] In the flow chart of Figure 1B, the captured substance binding step S113 is shown to occur after the surface capture step S112 and before the cracking step S114, but the timing of the captured substance binding step S113 is not limited to this. The captured substance bonding step S114 may be performed before the surface capture step S112, or it may be performed while the surface capture step S112 is being performed. For example, the biological particle-containing sample and the first capture substance are mixed to bind the first capture substance to the biological particles contained in the sample. Then, the biological particle-containing sample on which the first capture substance has been bound is used in the surface capture step S112, and the biological particles to which the first capture substance has been bound are captured on the surface 110. Alternatively, the biological particle-containing sample and the first capture substance may be applied to the surface 110, and the first capture substance may bind to each biological particle while the biological particles contained in the sample are captured on the surface 110. Alternatively, the bioparticle-containing sample is applied to the surface 110, and the bioparticles contained in the sample are captured by the surface 110. After the capture is complete, the first capturing substance is applied to the surface 110, and the first substance can be bound to each bioparticle.

[0086] (Modification 1: Embodiment containing multiple secretion-binding substances in the secretion-binding portion) The secretory binding portion may contain one secretory binding substance, as described with reference to Figure 4, or it may contain multiple identical or different secretory binding substances. The first capture substance when the secretory binding portion contains multiple identical or different secretory binding substances will be described with reference to Figure 17.

[0087] Figure 17 is a schematic diagram showing an example of a state in which the first capture substance is bound to a biological particle (cell) P. The first capture substance 330 shown in Figure 17 includes a secretion substance binding portion 331, a crosslinking portion 332, and a biological particle binding portion 333.

[0088] The secretion-binding region 331 contains four secretion-binding substances (antibodies) 331-1, 331-2, 331-3, and 331-4. These four antibodies may be the same or they may be different from each other. For example, the four antibodies may be configured to capture the same secretion (such as cytokines) or they may be configured to capture different secretions. Thus, the multiple secretion-binding substances contained in the secretion-binding region may be the same or they may be different from each other. For example, the multiple secretion-binding substances may be antibodies that bind to different antigens. Furthermore, the multiple secretion-binding substances do not have to be antibodies; they may be, for example, antibody fragments, aptamers, and molecularly imprinted polymers. Also, the multiple secretion-binding substances contained in the secretion-binding region may have the same secretion-binding properties or they may have different secretion-binding properties.

[0089] The crosslinked portion 332 is bound to multiple secretion-binding substances 333-1 to 333-4 and is also bound to the bioparticle-binding portion 333. The crosslinked portion 332 having such multiple binding sites may be, but is not limited to, a compound having structure (I) as described above as an example of the crosslinked portion 132. The crosslinked portion 132 may be selected from known compounds in the art that have multiple binding sites, such as the compound having structure (I).

[0090] Although the biological particle binding portion 333 is shown as an antibody in Figure 17, as described above with respect to 133, it may be an antigen-binding substance or molecular-binding substance other than an antibody.

[0091] Furthermore, the multiple secretion-binding substances do not necessarily have to be bound to a single linear compound as shown in Figure 17, but may be bound to a substance bound to the cross-linking portion 332. An example of this is shown in Figure 18. In the first capture substance 335 shown in Figure 18, a granular substance 336 is bound to one end of the cross-linking portion 332, and multiple secretion-binding substances 331-1 to 331-4 are bound to the granular substance 336.

[0092] (Modification 2: Embodiment in which the biological particle binding site is a multispecific antibody) The biological particle binding portion may be an antigen-binding substance that binds to an antigen on the surface of the biological particle P. Furthermore, the antigen-binding substance may be a multispecific antibody, particularly a bispecific or trispecific antibody. This modification will be explained with reference to Figure 19.

[0093] Figure 19 is a schematic diagram showing an example of a state in which the first capture substance is bound to a biological particle (cell) P. The first capture substance 430 shown in Figure 19 includes a secretion substance binding portion 431, a crosslinking portion 432, and a biological particle binding portion 433.

[0094] The secretion-binding portion 431 contains one secretion-binding substance (antibody). Note that this secretion-binding substance does not have to be an antibody; for example, it may be an antibody fragment, an aptamer, or a molecular imprinted polymer.

[0095] The crosslinked portion 432 may be, but is not limited to, a compound having structure (I) as described above as an example of the crosslinked portion 132.

[0096] The biological particle binding site 433 may be a bispecific antibody, as shown in Figure 19. This bispecific antibody may be, for example, an antibody that (specifically) binds to the surface antigen of cell P and (specifically) binds to cells other than cell P.

[0097] Figure 20 shows a state in which two cells, P1 and P2, are captured by a single primary capture substance 430. The bioparticle binding site 433 of the primary capture substance 430 is a bispecific antibody, bound to the surface antigen of cell P1 (black circle) and the surface antigen of cell P2 (black square). Unlike the surface antigen of cell P2, the surface antigen of cell P1 is different from that of cell P2, and these two distinct antigens are captured by a single bioparticle binding site (antibody) 433.

[0098] (Modification 3: First capture substance containing antibodies that bind to two or more biological particles) In the modified versions of this disclosure, the first capture substance may include antibodies that bind to the surfaces of two or more identical or heterogeneous biological particles (particularly cells), and more particularly, antibodies that bind to the surfaces of two or more heterogeneous biological particles. The antibodies may be antibodies that bind to two or more different antigens. The antibodies may be, for example, so-called multispecific antibodies, and more specifically, bi-specific antibodies or tri-specific antibodies. In the modified versions, the first capture substance may include the antibodies separately from the secretion-binding site and the biological particle-binding site.

[0099] In the modified example described above, the antibody captures, for example, two or more cells, particularly two or more cells that are different from each other. That is, in the first capture step S102, the antibody contained in the first capture substance captures two or more cells, and these cells are held in very close proximity. Therefore, intercellular interactions between the two or more cells can be intentionally induced. For example, such intercellular interactions may be interactions between one immune cell and one tumor cell, interactions between one immune cell and another immune cell, or interactions between one immune cell, another immune cell and one tumor cell. That is, the antibody may be an antibody that captures two or more identical or different immune cells, or an antibody that captures one or more immune cells and one or more tumor cells. By including the antibody in the first capture substance, the intercellular interactions can be analyzed more efficiently, which is extremely useful in the research and development of antibody drugs or cell therapies.

[0100] This modified example will be explained with reference to Figure 21. As shown in Figure 21, the first capture substance 530 includes a secretion-binding portion 531, a cross-linking portion 532, and a bioparticle-binding portion 533 that binds to cell P1. The first capture substance 530 further includes antibodies 535-1 and 535-2 that bind to the surface of the cell. Antibody 535-1 binds to the surface antigen of cell P2 (black star). Antibody 535-2 binds to the surface antigen of cell P3 (black circle). By binding antibodies 535-1 and 535-2 to cells P2 and P3, respectively, cells P2 and P3 are kept in close proximity to each other. As cells P2 and P3 are in close proximity to each other, interactions occur between these cells. These interactions cause, for example, the release of secretions (black squares) from these cells. These secretions are captured by the secretion-binding portion 531. In this way, intercellular interactions can be analyzed.

[0101] In this modified example, the secretory substance binding portion of the first capture substance may be configured to bind to the secretory substance produced by the intercellular interaction. Also in this modified example, the second secretory substance binding portion of the second capture substance, described later, may be configured to bind to the secretory substance at a site different from the site to which the secretory substance binding portion binds.

[0102] (Modification 4: Crosslinking of 2 or more biological particles) In yet another modification of the present disclosure, two or more biological particles may be crosslinked in the first capture step. Such crosslinking maintains, for example, a state in which two or more cells are in close proximity, thereby enabling intercellular interactions.

[0103] In this modified example, a crosslinking material similar to that of the first capture material may be used to perform the crosslinking. This crosslinking material will be described with reference to Figure 22. The crosslinking material 670 shown in Figure 22 includes two bioparticle binding sites 672 and 673 and a crosslinking site 671. The bioparticle binding sites 672 and 673 may be the same as the other bioparticle binding sites described above. The crosslinking site 671 may be the same as the crosslinking site described above. The bioparticle binding sites 672 and 673 bind to the surface antigens of cells P2 and P3, respectively. Therefore, the crosslinking material 670 maintains a state in which cells P1 and P2 are in close proximity. This results in an interaction between cells P1 and P2. Secretions produced by this interaction are captured, for example, by the first capture materials 630-1 and 630-2 according to this disclosure. Because the bioparticle binding sites 672 and 673 are substances that bind to bioparticles in a specific mode, such as antibodies, it is possible to crosslink specific groups of bioparticles (cells). This makes it possible to analyze interactions between specific cells.

[0104] The crosslinking material may bind in a non-specific manner. For example, the crosslinking material 770 shown in Figure 23 includes two bioparticle binding sites 772 and 773 and a crosslinking site 771. The bioparticle binding site 772 is a substance that binds to various cells in a non-specific manner, such as a compound containing an oleyl group or cholesteryl group as described above. The bioparticle binding site 773 is an antibody that binds to cells in a specific manner. The crosslinking site 771 may be the same as the crosslinking site described above. The crosslinking material 770 allows specific cells to be crosslinked with various other cells. This makes it possible to analyze the interactions between specific cells and various other cells.

[0105] (3-1-4) Cleavage process

[0106] In the cleavage step S114, the linker 126 is cleaved, and the biological particles captured in the surface capture step S112 are released from the surface 110. Preferably, in the cleavage step S114, the state in which the biological particles P are captured by the particle capture unit 121 is maintained. This captured state may be maintained until the environmental transfer of the biological particles in the environmental transfer step S104 described later is completed, or for example, until the destruction of the biological particles in the destruction step S105 described later is completed.

[0107] The cracking may be performed over the entire surface 110, or on only a portion of the surface 110. In the latter case, the portion may be selected, for example, based on the detection results of the detection process described below.

[0108] Furthermore, the cleavage may be performed in such a way that all of the biological particles trapped on the surface 110 are released from the surface 110, or in such a way that only some of the biological particles trapped on the surface 110 are released from the surface 110. In the latter case, the portion of biological particles may be selected, for example, based on the detection results of the detection process described below.

[0109] For example, the biological particles released from the surface 110 may be selected based on the label of the biological particle P, the label of the particle capturing substance 120, or the label of the first capturing substance 130. The label on the biological particle P may be, for example, a fluorescent dye that constitutes a fluorescently labeled antibody, or a label (particularly a fluorescent dye) present inside the biological particle. The label on the particle-capturing substance 120 is, for example, a fluorescent dye. A portion of the nucleic acid contained in the particle-capturing substance 120 may be nucleic acid labeled with a fluorescent dye. Alternatively, the antibody contained in the particle-capturing substance 120 may be labeled with a fluorescent dye. The label on the first capture substance 130 is, for example, a fluorescent dye. A portion of the nucleic acid contained in the first capture substance 130 may be nucleic acid labeled with a fluorescent dye. Alternatively, the antibody contained in the first capture substance 130 may be labeled with a fluorescent dye.

[0110] The biological particles included in the group of biological particles obtained in the cracking process S114 will be explained with reference to Figure 5. Figure 5 is a schematic diagram of the biological particles.

[0111] As shown on the left of Figure 5, the biological particle P has multiple primary capture substances 130, 130-2, and 130-3 for capturing secreted substances, and multiple particle capture substances 120 bound to it. Each biological particle in the biological particle population may have a different particle identifier bound to it. The biological particle shown on the left of Figure 5 has particle identifier 124 bound to it, while the biological particle shown on the right of Figure 5 has particle identifier 124-2 bound to it, which is different from particle identifier 124. The difference in these particle identifiers may be, for example, a difference in the base sequence that constitutes the particle identifier. In this way, the biological particles in the biological particle population obtained in the preparation step may have different particle identifiers from each other. Also, multiple particle identifiers bound to a single biological particle may be the same. Such a biological particle population is suitable for performing single-cell analysis in the analysis step described later.

[0112] In one embodiment of this disclosure, the cleavage step S114 may include a detection step of detecting light generated from biological particles or light from a substance bound to biological particles, and a linker cleavage step of cleaving the linker and releasing biological particles from the surface 111 based on the detection result in the detection step. This makes it possible to select biological particles to be released from the surface 110, for example, according to the detection result. This makes it possible to exclude unintended biological particles from the analysis steps described later, thereby improving the efficiency of the analysis.

[0113] In other embodiments of this disclosure, the linker cracking step S114 may be performed without performing the detection step. By omitting the detection step, the number of steps in the analytical method of this disclosure can be reduced.

[0114] The detection process and the linker cracking process will be explained below.

[0115] (3-1-4-1) Detection process

[0116] The cleavage step S114 may include a detection step that detects one or more of the following: light originating from biological particles (e.g., scattered light and / or autofluorescence), light originating from target capture molecules (e.g., fluorescence), light originating from antibodies bound to biological particles (e.g., fluorescence), morphology of biological particles (e.g., morphology (morphology characterized by images acquired in bright-field, phase-contrast, or dark-field imaging and image processing, particularly morphology acquired by morphology processing) or the state in which two or more biological particles (such as cells) are bound together), and characteristics of biological particles predicted from the morphological information of biological particles (e.g., cell type or cell state (living cells or dead cells)). These lights, morphologies, and characteristics can be detected, for example, by an observation device including an objective lens, particularly by a microscope. These lights, morphologies, and characteristics may be detected, for example, by an image sensor or by a photodetector. Based on the detection results in the detection step, such as light, morphology, and characteristics, a target capture molecule to be cleaved in the linker cleavage step described later may be selected, or biological particles to be released from the surface 110 in the cleavage step S114 may be selected. For example, the image sensor may acquire an image of the surface 110 or an image of biological particles captured on the surface 110, and based on the acquired image, the biological particles to be released may be selected.

[0117] (3-1-4-2) Linker rupture process

[0118] The cleavage step S114 includes a linker cleavage step that cleaves the linker 126. The cleavage of the linker 126 releases the biological particles to which the first capture material and the particle capture material are bound from the surface 110. The cleavage of the linker 1 of the particle capture material 120 releases the particle capture material 120 from the surface 110, as shown in, for example, Figure 2Ac, and consequently the biological particles are also released from the surface 110.

[0119] In the cleavage step S114, the linker may be cleaved by a stimulus such as chemical stimulation or optical stimulation. Optical stimulation is particularly suitable for selectively stimulating a specific narrow area.

[0120] The stimulation in the cracking process S114 may be performed by a stimulation device. The drive of the stimulation device may be controlled by an information processing device, such as a general-purpose computer. For example, the information processing device may drive the stimulation device to selectively apply stimulation to the location of the biological particles to be released. Examples of stimulation devices that may be used are described below.

[0121] To selectively apply light stimulation to the location of cells, a light irradiation device may be used as a stimulation device. The light irradiation device may be, for example, a DMD (Digital Micromirror Device) or a liquid crystal display device. By using micromirrors that make up the DMD, light can be irradiated to a selected location on the surface 110. The liquid crystal display device may be, for example, a reflective liquid crystal display, and a specific example is SXRD (Sony Corporation). By controlling the liquid crystal of the liquid crystal display device, light can be irradiated to a selected location on the surface 110. Furthermore, a liquid crystal shutter or a spatial light modulator may be used to selectively apply light stimulation to the location of the cells. These methods also allow light stimulation to be applied to the selected location. The wavelength of the irradiated light may be appropriately selected by those skilled in the art depending on the type of linker contained in the particle trapping material.

[0122] Chemical stimulation may be applied by bringing a reagent that cleaves the linker 126 into contact with the surface 110. The reagent may be determined according to the type of linker 126, as described above. For example, if linker 126 contains a disulfide bond, the reagent may be a reducing agent capable of cleaving the bond, such as Tris(2-carboxyethyl)phosphine (TCEP), Dithiothreitol (DTT), or 2-Mercaptoethanol. For example, if TCEP is used, the reaction is carried out for about 15 minutes at, for example, 50 mM. For example, if linker 126 is a nucleic acid containing restriction enzyme recognition sequences, the reagent may be a restriction enzyme corresponding to each restriction enzyme recognition sequence. One unit of restriction enzyme activity is, in principle, the amount of enzyme that completely degrades 1 μg of λDNA per hour at 37°C in 50 μl of each enzyme reaction solution, and the amount of enzyme can be adjusted according to the amount of restriction enzyme recognition sequences.

[0123] At least one biological particle released by the cleavage in the cleavage step S114 may be recovered in a liquid such as a buffer or culture medium. The liquid may be, for example, a hydrophilic liquid. The biological particle-containing liquid obtained by this recovery may be used in the environment transfer step S104 described later. To recover the released biological particles, fluid force may be used by flowing a liquid such as a buffer, or the biological particles may be suspended in the liquid by vibration, or the biological particles may be suspended in the liquid by gravity, etc. The vibration may be, for example, vibration of the substrate 100, or vibration of the liquid containing the biological particles. In addition, the substrate 110 may be moved so that the surface 110 faces the direction of gravity in order to suspend the biological particles in the liquid by gravity.

[0124] (3-2) First capture step

[0125] The first capture step S102 includes a processing step that places the group of biological particles prepared in the preparation step S101 under predetermined conditions. The processing step may be performed while the group state of the group of biological particles is maintained. The secreted substance produced by placing the group of biological particles under the predetermined conditions is bound to the first capture substance bound to each biological particle contained in the group of biological particles. The first capture step S102 may be performed while the state in which the first capture substance is bound to the biological particles is maintained.

[0126] The predetermined conditions may be conditions under which the reactivity of a group of biological particles is analyzed, and may be appropriately selected by the user performing the biological particle analysis method of this disclosure. The predetermined conditions may be, for example, conditions under which secreted substances are produced, or conditions under which it is analyzed whether or not secreted substances are produced. The produced secreted substances are captured by the first capture substance.

[0127] The predetermined conditions are, more specifically, the incubation environment for the biological particle population (particularly the cell population), for example, an environment in a culture medium or buffer. In the first capture step S102, secreted substances generated when the biological particle population is placed in this environment are captured by the first capture substance.

[0128] The incubation environment may contain, for example, biomaterials or non-biomaterials. In this disclosure, the reactivity of a population of bioparticles in the environment in which such materials are present may be analyzed. The materials may be, for example, diseased tissue, diseased cells, microorganisms (bacteria, fungi, or viruses), substances that cause disease or increase the risk of disease (e.g., carcinogens, amyloid-beta, prions, etc.), drugs, toxic substances, or heterogeneous tissues. The non-biomaterials may be, for example, drugs or toxic substances. The diseased tissue may be, for example, tumor tissue, and in particular cancerous tissue or sarcoma tissue. The diseased cells may be, for example, tumor cells, and in particular cancer cells, sarcoma cells, or malignant lymphoma cells.

[0129] The secreted substance may be a secreted substance secreted from biological particles included in the group of biological particles, or a secreted substance secreted from materials used to constitute the predetermined conditions. For example, the secreted substance may be a secreted substance secreted from diseased tissue, diseased cells, microorganisms, or other tissues.

[0130] The biological particles may be cells as described above, and the secreted substances secreted from the biological particles may be secreted substances from cells. For example, the secreted substances may be substances secreted by immune cells, and may be one or more selected from cytokines, hormones, antibodies, and exosomes, but are not limited to these. The secreted substances may also be substances secreted by nerve cells, muscle cells, skin cells, or glandular cells. The secreted substances may also be exosomes.

[0131] From a material standpoint, the secreted substance may be, for example, a protein, peptide, exosome, or other biomolecule. From the standpoint of cell type, the secreted substance may be, for example, an exosome, cytokine, hormone, or neurotransmitter.

[0132] Furthermore, the secreted substances produced when the bioparticle population is placed under the predetermined conditions are not limited to substances secreted from cells contained in the bioparticle population. For example, they may be secreted from materials constituting the predetermined conditions. These materials may be materials contained in the incubation environment described above, and may be biological tissues, cells (particularly diseased cells), microorganisms, or heterologous tissues, particularly diseased tissues, and more particularly tumor tissues or neurodegenerative tissues. The cells are, for example, diseased cells, and particularly tumor cells.

[0133] A specific example of the first capture step S102 will be explained with reference to Figure 2B.

[0134] To perform the first capture step S102, a predetermined condition is prepared, for example, an incubation environment. This incubation environment may be the environment inside a container 140, as shown in Figure 2Bd. The container 140 is, for example, a petri dish, a well plate, or a tube, but is not limited to these. The container 140 contains an incubation medium, such as culture medium or buffer solution. As a material constituting the incubation environment, the container 140 further contains a group of diseased cells (tumor cells) 145. The group of diseased cells 145 may consist of one or more types of cells. In Figure 2Bd, the group of diseased cells 145 includes two types of cells (cells 145a and 145b).

[0135] As shown in Figure 2B, d, the bioparticle population prepared in the preparation step is added to container 140. The bioparticle population is then incubated in the container. The incubation time and / or temperature may be appropriately selected by those skilled in the art to produce secreted substances.

[0136] The incubation process generates secreted substances within container 140. These secreted substances may be substances derived from biological particles contained in the biological particle population, substances derived from materials constituting the incubation environment (the diseased cell population in Figure 2B), or both of these substances, as described above.

[0137] In Figure 2Be, it is shown that secretions 160, 161, and 162 have been produced. These secretions are captured by the first capture substance 130, as shown in the same figure. In Figure 2Be, multiple different types of secretions are produced, but only one type of secretion may be produced.

[0138] (3-3) Second capture step

[0139] In the second capture step S103, the secreted substance bound to the first capture substance is bound to a second capture substance for capturing the secreted substance. This forms a compound of the first capture substance, the secreted substance, and the second capture substance. Preferably, the second capture substance is configured to bind to a site different from the site to which the first capture substance is bound. The second capture step S103 may be performed while the state in which the first capture substance is bound to the biological particle is maintained. In a preferred embodiment of this disclosure, both the first capture step S102 and the second capture step S103 are performed while the state in which the first capture material is bound to the biological particles is maintained. This forms a sandwich structure, as described below, on the biological particles. Forming this structure is useful, for example, for analyzing the interactions between biological particles contained in a group of biological particles.

[0140] The second capture step S103 may be performed in the incubation environment in which the first capture step S102 was performed, or in an environment separate from the incubation environment. Preferably, from the viewpoint of efficiency of conjugate formation, the second capture step S103 is performed in the latter separate environment. For example, after the completion of the first capture step S102, a group of biological particles containing biological particles having the first capture substance to which the secreted substance has been bound is recovered from the incubation environment and transferred to an incubation environment in which the second capture step S103 is performed (hereinafter also referred to as the "second incubation environment"). The second incubation environment may be an environment that allows the binding of the second secreted substance binding portion, described later, to the secreted substance, and may be an environment inside a container. The container may be, for example, a petri dish, a well plate, or a tube, but is not limited to these. The container may contain an incubation medium such as a culture medium or a buffer.

[0141] An example of the composition of the second capture substance will be described with reference to Figure 6. As shown in Figure 6, the second capture substance 170 includes a second secretion binding portion 171, a second recovered substance amplification portion 172, a capture substance identifier 173, and a poly(A) sequence 174. The second capture substance is, for example, a complex of nucleic acid and protein, as will be described later, and can be appropriately manufactured by those skilled in the art.

[0142] The second secretion-binding portion 171 may be appropriately designed or manufactured by those skilled in the art depending on the secretion-binding substance to which it is intended. For example, the second secretion-binding portion 171 may be a substance selected from the group including, for example, antibodies, antibody fragments, aptamers, and molecularly imprinted polymers, and is particularly an antibody or antibody fragment. In Figure 6, an antibody is shown as the second secretion-binding portion 171. The binding ability of the second secretion-binding portion 171 may be specific or nonspecific, and is particularly specific.

[0143] The second secretory binding portion 171 is configured to bind to the secretory to which the first capture substance 130 binds, and in particular, is configured to bind to a portion of the secretory to which the first capture substance 130 binds that is different from the portion to which the first capture substance 130 binds.

[0144] In the second capture step S103, a state is formed in which the second capture substance 170 is bound to the secreted substance to which the first capture substance 130 is bound. A state in which two different antibodies are bound to one substance is also called a sandwich structure. Such a sandwich structure may be formed in the second capture step S103. More specifically, a structure may be formed in which a secreted substance is bound to a secreted substance binding site 131 (e.g., an antibody) contained in the first capture substance 130 and a second secreted substance binding site 171 (e.g., an antibody) contained in the second capture substance 170. Note that one type of second secreted substance binding site may be bound to one secreted substance, or two or more types of second secreted substances may be bound.

[0145] The second recovered material amplification unit 172 includes, for example, a nucleic acid amplification primer and / or a nucleic acid transcription promoter, and in particular may include a nucleic acid having a primer sequence used for nucleic acid amplification or a promoter sequence used for nucleic acid transcription in the analytical step described later. The nucleic acid may be DNA or RNA, and is particularly DNA. The second recovered material amplification unit 172 may have both a primer sequence and a promoter sequence. The primer sequence may be, for example, a PCR handle. The promoter sequence may be, for example, a T7 promoter sequence.

[0146] The capture substance identifier 173 is used to identify or specify a second capture substance or second secretion binding site containing the capture substance identifier. The capture substance identifier 173 includes, for example, a nucleic acid having a barcode sequence. The nucleic acid may be particularly DNA or RNA, and more particularly DNA. The barcode sequence may be used to specify, for example, a second capture substance or second secretion binding site bound to a secretion. For this specification, the barcode sequence may be associated with a second capture substance or second secretion binding site containing the barcode sequence. Therefore, the barcode sequence may be associated with a second capture substance or second secretion binding site. For example, the sequence information of the barcode sequence may be associated with the type of second capture substance or second secretion binding site. The barcode sequence may be associated with a second capture substance or second secretion binding site in a one-to-one relationship, for example. Thus, the second captured substance 173 may have a captured substance identifier attached to it for identification. This makes it possible to identify the captured substance that was bound to the biological particle in the analysis step described later.

[0147] The poly(A) sequence 174 stabilizes the amplified product of the barcode sequence when reading the barcode sequence in the analysis process described later.

[0148] A specific example of the second capture step S103 will be explained with reference to Figure 2C.

[0149] In order to perform the second capture step S103, an incubation environment is prepared in which the secreted substance captured by the first capture substance 130 in the first capture step S102 is bound to the second capture substance 170. This incubation environment may be the environment inside a container 150, as shown in Figure 2Cf. The container 150 is, for example, a petri dish, a well plate, or a tube, but is not limited to these. The container 150 contains an incubation medium, such as a culture medium or a buffer.

[0150] As shown in Figure 2Cf, a group of biological particles containing the biological particles P after the capture treatment of secreted substances in the first capture step S102, and the second capture substance 170 are added to the container 150. The group of biological particles is then incubated in the container. The incubation time and / or temperature may be appropriately selected by those skilled in the art to generate secreted substances. Through incubation, the secreted substances 160 are captured by the second capture substance 170. This creates a state in which the secreted substances 160 are captured by both the first capture substance 130 and the second capture substance 170.

[0151] (Variation 5: Use of materials that bond to the surface molecules of biological particles) In modifications of this disclosure, in the second capture step, in addition to capturing the secreted substance by the second capture substance, a binding step may be performed to bind a surface molecule-binding substance to the surface molecules of the biological particles. The surface molecule-binding substance may be, for example, an antibody, an antibody fragment, an aptamer, or a molecularly imprinted polymer. The surface molecule-binding substance may be bound to, for example, a fluorescent label or an identification substance. The incubation is performed with the surface molecule-binding substance added to the incubation medium. As a result, in addition to the second capture substance binding to the secreted substance, the surface molecule-binding substance binds to the surface molecules (particularly surface antigens) of the biological particles.

[0152] The fluorescent label can be used, for example, in the isolation step described later to determine whether to isolate biological particles in a microspace. The identification substance is released from the surface of the biological particles when the biological particles are destroyed in the destruction step described later, and then binds to a material recovery part, such as a poly-T array, to form a conjugate. This conjugate is used in the analysis step described later to identify the surface molecule-binding substance that was bound to the surface of the biological particles.

[0153] This modified example will be explained with reference to Figure 24. In addition to the first capture substance 130, the secreted substance 160, and the second capture substance 170, the biological particle P shown in Figure 24 has a binding substance 190 bound to it, which is a binding substance 180 labeled with a fluorescent label 181 and an identification substance 191. When the surface molecule binding substance is used, the state shown in this figure is formed in the second capture step.

[0154] As the binding substance (e.g., antibody) 180 labeled with the fluorescent label 181, any substance known in the art may be used. The binding substance (e.g., antibody) 190 to which the identification substance 191 is bound will be described below with reference to Figure 25.

[0155] As shown in Figure 25, the identification substance 191 bound to the binding substance 190 includes a third recovered substance amplification unit 192, a binding substance identifier 193, and a poly(A) sequence 194.

[0156] The same explanation as for the second recovered material amplification unit 172 described above applies to the third recovered material amplification unit 192.

[0157] The binding substance identifier 193 is used to identify or specify the binding substance 190. The binding substance identifier 193 includes, for example, a nucleic acid having a barcode sequence. The nucleic acid may be particularly DNA or RNA, and more particularly DNA. The barcode sequence may be used, for example, to specify the binding substance 190. For such specification, the barcode sequence may be associated with the binding substance 190. For example, the sequence information of the barcode sequence may be associated with the type of binding substance 190. The barcode sequence may be associated with the binding substance 190 in a one-to-one relationship, for example.

[0158] The poly(A) sequence 194 stabilizes the amplified product of the barcode sequence when reading the barcode sequence in the analysis process described later.

[0159] (3-4) Isolation process

[0160] In isolation step S104, the biological particles included in the group of biological particles are isolated into single particles. In this specification, the term "isolate" may mean that, when the destruction step described later is performed, the components contained in one biological particle and the substances bound to that one biological particle (e.g., the first capture substance, the second capture substance, and the particle identifier) ​​are placed in a state where they do not mix with the components contained in other biological particles and the substances bound to those other biological particles. For example, the term "isolate" may mean being confine to a microspace as described later.

[0161] In one embodiment of the present disclosure, in isolation step S104, each of the biological particles included in the group of biological particles is isolated in a single microspace. This microspace may be a space within an emulsion particle or a space within a well. By performing the destruction step described later within this microspace, as described above, the components contained in one biological particle, as well as the first capture substance, the second capture substance, and the particle identifier bound to that biological particle, do not mix with the components contained in other biological particles and the substances bound to those other particles.

[0162] Furthermore, by performing the isolation step S104, a one-to-one correspondence can be established between a single biological particle and a substance bound to that biological particle (for example, the first capture substance, the second capture substance, and the particle identifier).

[0163] In one embodiment of this disclosure, the isolation step S104 may include a discrimination step of determining whether to isolate biological particles in a microspace, and a particle isolation step of isolating the biological particles determined to be isolated in the discrimination step in a microspace. This makes it possible to isolate only the target biological particles in a microspace. Therefore, for example, unintended biological particles can be excluded from the analysis steps described later, thereby improving the efficiency of the analysis.

[0164] The discrimination may be performed, for example, based on light emitted from the biological particle (e.g., scattered light and / or autofluorescence), light emitted from a substance bound to the biological particle, or a morphological image. The substance bound to the biological particle may be, for example, a target capture molecule, or an antibody (particularly a fluorescently labeled antibody) bound to the biological particle. The scattered light emitted from the biological particle may be, for example, forward scattered light and / or side scattered light. Doublet detection can be performed from the signal height and / or area value obtained by scattered light detection. Single cell determination is also possible using morphological image information. Whether a biological particle is a dead cell can be determined from the scattered light and / or morphological image, or from the fluorescence after staining with a dead cell staining reagent, thereby allowing the removal of dead cells. In this disclosure, the discrimination step may be performed immediately before the isolation step, thereby ensuring the isolation of only single cells with barcodes.

[0165] In other embodiments of the present disclosure, the particle isolation step may be performed without performing the discrimination step. By omitting the discrimination step, the number of steps in the analytical method of the present disclosure can be reduced.

[0166] The discrimination process and the particle separation process will be described below.

[0167] (3-4-1) Discrimination process

[0168] In the aforementioned discrimination step, a determination is made as to whether to isolate each biological particle contained in the group of biological particles into a microspace. This determination may be made based on light emitted from the biological particles or light emitted from a substance bound to the biological particles, as described above.

[0169] The discrimination step may include, for example, an irradiation step of irradiating biological particles with light, and a detection step of detecting the light generated by the irradiation.

[0170] The irradiation step may be performed, for example, by a light irradiation unit that irradiates the biological particles with light. The light irradiation unit may include, for example, a light source that emits light. The light irradiation unit may also include an objective lens that focuses light onto the biological particles. 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, SHG laser, solid-state laser, gas laser, high-brightness LED, or halogen lamp, or a combination of two or more of these. In addition to the light source and objective lens, the light irradiation unit may include other optical elements as needed.

[0171] The detection step may be performed by a detection unit that detects light generated from, for example, biological particles or substances bound to biological particles. The detection unit detects, for example, the light generated from biological particles or substances bound to biological particles by light irradiation by the light irradiation unit, which may be, for example, scattered light and / or fluorescence. The detection unit may include, for example, a focusing lens for focusing light generated from biological particles and a detector. The detector may be, but is not limited to, PMTs, photodiodes, CCDs, and CMOSs. In addition to the focusing lens and detector, the detection unit may include other optical elements as needed. The detection unit may further include, for example, a spectrometer. Examples of optical components constituting the spectrometer include gratings, prisms, and optical filters. The spectrometer can, for example, separate and detect light of a wavelength to be detected from light of other wavelengths. The detection unit may convert the detected light into an analog electrical signal by photoelectric conversion. The detection unit may further convert the analog electrical signal into a digital electrical signal by AD conversion.

[0172] In the discrimination step, a determination unit may perform a determination process to determine whether or not to distinguish biological particles based on the light detected in the detection step. The processing by the determination unit can be implemented by an information processing device such as a general-purpose computer, and in particular by a processing unit included in said information processing device.

[0173] (3-4-2) Particle isolation process

[0174] The isolation step includes a particle isolation step in which biological particles are isolated in a microspace. In this disclosure, a microspace may mean a space having dimensions that can accommodate one biological particle to be analyzed. Such dimensions may be appropriately determined depending on factors such as the size of the biological particle. The microspace may have dimensions that can accommodate two or more biological particles to be analyzed, in which case, in addition to cases where one biological particle is contained in a single microspace, there may also be cases where two or more biological particles are contained. Biological particles in a microspace containing two or more biological particles may be excluded from destruction in the destruction step described later, or may be excluded from analysis in the analysis step described later.

[0175] Furthermore, in the disruption step described later, the conjugate formed in the second disruption step, consisting of the first disruption substance, the secreted substance, and the second disruption substance, is released from the biological particle. Also, in the disruption step described later, a complex of, for example, a substance within the biological particle and a particle identifier (particularly a complex formed by the binding of mRNA within the biological particle to the poly-T sequence of the particle identifier) ​​may be generated. In this disclosure, it is preferable that each of the microspaces is separated from one another so that the conjugate (and optionally the complex) generated in one microspace does not migrate to other microspaces. Examples of such separated microspaces include spaces within emulsion particles and spaces within wells. That is, in a preferred embodiment of this disclosure, the microspace may be a space within an emulsion particle or a space within a well. Below, examples of particle isolation steps when the microspace is one of these spaces will be described.

[0176] (3-4-2-1) In the case of space within emulsion particles

[0177] Emulsion particles can be generated, for example, using a microfluidic system. The apparatus includes, for example, a channel through which a first liquid flows that forms the dispersed phase of the emulsion, and a channel through which a second liquid flows that forms the dispersion medium. The first liquid may contain biological particles. The apparatus further includes a region where these two liquids come into contact to form an emulsion.

[0178] Hereinafter, an example of an apparatus for efficiently forming an emulsion containing emulsion particles each containing one biological particle will be described with reference to FIGS. 7A and 7B. With this emulsion forming apparatus, one biological particle can be isolated within one emulsion particle with a very high probability, and the number of empty emulsion particles can be reduced. Furthermore, the probability of isolating one biological particle and one barcode array within one emulsion particle is also increased by the emulsion forming apparatus.

[0179] FIG. 7A is an example of a microchip used for forming emulsion particles in the apparatus. The microchip 250 shown in FIG. 7A includes a main flow path 255 through which biological particles flow, and a recovery flow path 259 from which target particles to be recovered among the biological particles are recovered. The microchip 250 is provided with a particle sorting section 257. An enlarged view of the particle sorting section 257 is shown in FIG. 9. As shown in A of FIG. 8, the particle sorting section 257 includes a connection flow path 270 that connects the main flow path 255 and the recovery flow path 259. A liquid supply flow path 261 capable of supplying liquid to the connection flow path 270 is connected to the connection flow path 270. As described above, the microchip 250 has a flow path structure including the main flow path 255, the recovery flow path 259, the connection flow path 270, and the liquid supply flow path 261. FIG. 7B is a schematic diagram for explaining the formation of emulsion particles in the microchip 250 shown in FIG. 7A and the isolation of biological particles within the formed emulsion particles.

[0180] Also, as shown in FIG. 7A, in addition to the microchip, the microchip 250 constitutes a part of a biological particle sorting apparatus 200 including a light irradiation section 291, a detection section 292, and a control section 293. As shown in FIG. 9, the control section 293 may include a signal processing section 294, a determination section 295, and a sorting control section 296. The biological particle sorting apparatus 200 is used as the emulsion forming apparatus described above.

[0181] As shown in FIG. 10, in order to form an emulsion containing emulsion particles including one target biological particle (biological particle P to which the first capture substance, the secretion substance, and the second capture substance are bound), for example, in the microchip 250, a flowing process S201 of flowing a first liquid containing a population of biological particles including the target biological particle through the main flow path 255, a discrimination process S202 of determining whether the biological particles flowing through the main flow path 255 are particles to be collected, and a collection process S203 of collecting the particles to be collected into the collection flow path 259 can be executed. The discrimination process S202 corresponds to the discrimination process described in the above (3-4-1). The collection process S203 corresponds to the particle separation process described in the above (3-4-2). Each process will be described below.

[0182] (Flowing Process)

[0183] In the flowing process S201, the first liquid containing the population of biological particles is flowed through the main flow path 255. The first liquid flows in the main flow path 255 from the confluence part 262 toward the particle separation part 257. The first liquid may be a laminar flow formed from a sample liquid containing biological particles and a sheath liquid, and in particular, may be a laminar flow in which the periphery of the sample liquid is surrounded by the sheath liquid. The flow path structure for forming the laminar flow will be described below. The sheath solution may contain, for example, a component for destroying biological particles, such as a cell-lysing component. This allows the component to be incorporated into the emulsion particles, enabling the destruction of biological particles within the emulsion particles during the destruction step described later. The cell-lysing component may be a cell-lysing enzyme, such as proteinase K. For example, after capturing cells in emulsion particles containing proteinase K, the cells are lysed by placing the emulsion particles at a predetermined temperature (e.g., 37°C to 56°C) for, for example, less than 1 hour, particularly less than 1 hour. Although proteinase K is active even below 37°C, if such a lower temperature is used, the cell-lysing properties of proteinase K will decrease, and the mixture may be incubated overnight, for example. The sheath solution may also contain a surfactant (e.g., SDS, Sarkosyl, Tween 20, or Triton X-100). This surfactant can enhance the activity of proteinase K. Furthermore, the sheath fluid does not need to contain biological particle-destroying components. In this case, the biological particles may be physically destroyed. As physical destruction methods, for example, optical treatment (e.g., optical cell lysis) or thermal treatment (e.g., thermal cell lysis) may be employed. Optical treatment can be performed, for example, by irradiating emulsion particles with laser light to form plasma or cavitation bubbles within the particles. Thermal particle destruction can be performed by heating the emulsion particles.

[0184] The microchip 250 is provided with a sample liquid inlet 251 and a sheath liquid inlet 253. The sample liquid containing the bioparticle population and the sheath liquid not containing bioparticles are introduced from these inlets into the sample liquid channel 252 and the sheath liquid channel 254, respectively.

[0185] The microchip 250 has a flow channel structure in which the sample channel 252 through which the sample liquid flows and the sheath liquid channel 254 through which the sheath liquid flows merge at a confluence 262 to form a main channel 255. The sample liquid and the sheath liquid merge at the confluence 262 to form a laminar flow in which, for example, the sample liquid is surrounded by the sheath liquid. A schematic diagram of the formation of this laminar flow is shown in Figure 7B. As shown in Figure 7B, the laminar flow is formed such that the sheath liquid introduced from the sheath liquid channel 254 surrounds the sample liquid introduced from the sample channel 252. Preferably, the biological particles are arranged in a substantially straight line in the laminar flow. For example, as shown in Figure 7B, the biological particles P may be arranged in a substantially straight line in the sample liquid. Thus, in this disclosure, the flow channel structure forms a laminar flow containing biological particles flowing in a substantially straight line.

[0186] The laminar flow flows through the main channel 255 toward the particle sorting section 257. Preferably, the biological particles flow in a single line within the main channel 255. This makes it easier to distinguish between the light generated by the irradiation of one microparticle and the light generated by the irradiation of other microparticles during the light irradiation in the detection region 256 described below.

[0187] (Discrimination process)

[0188] In the discrimination step S202, it is determined whether the biological particles flowing through the main channel 255 are particles to be recovered. This discrimination can be performed by the determination unit 295. The determination unit 295 may perform this discrimination based on the light generated by the light irradiation unit 291 on the biological particles. An example of the discrimination step S202 will be described in more detail below.

[0189] In the discrimination step S202, the light irradiation unit 291 irradiates the biological particles flowing in the main channel 255 (particularly the detection region 256) in the microchip 250 with light (e.g., excitation light), and the detection unit 292 detects the light generated by the light irradiation. Based on the characteristics of the light detected by the detection unit 292, the determination unit 295 included in the control unit 293 determines whether the biological particles are to be recovered. For example, the discrimination unit 295 can perform determination based on scattered light, determination based on fluorescence, or determination based on an image (e.g., one or more of dark-field images, bright-field images, and phase-contrast images). In the recovery step S203 described later, the control unit 293 controls the flow in the microchip 250 so that the particles to be recovered are recovered into the recovery channel 259.

[0190] The light irradiation unit 291 irradiates biological particles flowing in the channels within the microchip 250 with light (e.g., excitation light). The light irradiation unit 291 may include a light source that emits light and an objective lens that focuses the excitation light onto minute particles flowing in the detection area. The light source may be appropriately selected by those skilled in the art depending on the purpose of the analysis, and may be, for example, a laser diode, SHG laser, solid-state laser, gas laser, high-brightness LED, or halogen lamp, or a combination of two or more of these. In addition to the light source and objective lens, the light irradiation unit may include other optical elements as needed.

[0191] (Discrimination of objects to be sorted based on fluorescence signals and / or scattered light signals)

[0192] In one embodiment of this disclosure, the detection unit 292 detects scattered light and / or fluorescence generated from the minute particles by light irradiation by the light irradiation unit 291. The detection unit 292 may include a focusing lens and a detector for focusing the fluorescence and / or scattered light generated from the biological particles. The detector may be, but is not limited to, a PMT, a photodiode, a CCD, and a CMOS. In addition to the focusing lens and the detector, the detection unit 292 may include other optical elements as needed. The detection unit 292 may further include, for example, a spectroscopic unit. Examples of optical components constituting the spectroscopic unit include a grating, a prism, and an optical filter. The spectroscopic unit can, for example, separate and detect light of a wavelength to be detected from light of other wavelengths. The detection unit 292 may convert the detected light into an analog electrical signal by photoelectric conversion. The detection unit 292 may further convert the analog electrical signal into a digital electrical signal by AD conversion.

[0193] The signal processing unit 294 included in the control unit 293 can process the waveform of the digital electrical signal obtained by the detection unit 292 to generate information (data) regarding the characteristics of the light used for determination by the determination unit 295. As information regarding the characteristics of the light, the signal processing unit 294 may obtain one, two, or three of the following from the waveform of the digital electrical signal: the width of the waveform, the height of the waveform, and the area of ​​the waveform. The information regarding the characteristics of the light may also include, for example, the time when the light was detected. The processing by the signal processing unit 294 described above can be performed in an embodiment in which scattered light and / or fluorescence is detected.

[0194] The determination unit 295 included in the control unit 293 determines whether a biological particle is a particle to be recovered based on the light generated by the irradiation of the biological particle flowing in the channel. In the embodiment in which scattered light and / or fluorescence are detected, the waveform of the digital electrical signal obtained by the detection unit 292 is processed by the control unit 293, and the determination unit 295 determines whether the biological particle is a particle to be recovered based on the information regarding the characteristics of the light generated by the processing. For example, in the determination based on scattered light, the external shape and / or internal structure characteristics of the biological particle may be identified, and it may be determined whether the biological particle is a particle to be recovered based on these characteristics. Furthermore, by pre-treating the biological particle, such as a cell, it is also possible to determine whether the biological particle is a particle to be recovered based on characteristics similar to those used in flow cytometry. In addition, by labeling the biological particle, such as a cell, with an antibody or dye (particularly a fluorescent dye), it is also possible to determine whether the biological particle is a particle to be recovered based on the characteristics of the surface antigen of the biological particle.

[0195] (Discrimination of objects to be sorted based on brightfield images and / or phase-contrast images)

[0196] In other embodiments of this disclosure, the detection unit 292 may acquire a bright-field image and / or a phase-contrast image generated by light irradiation by the light irradiation unit 291. In this embodiment, the light irradiation unit 291 may include, for example, a halogen lamp, and the detection unit 292 may include a CCD or CMOS. For example, light is irradiated onto biological particles by a halogen lamp, and the CCD or CMOS may acquire a bright-field image and / or a phase-contrast image of the irradiated biological particles.

[0197] In the embodiment in which the bright-field image and / or phase-contrast image are acquired, the determination unit 295 included in the control unit 293 determines whether the biological particles are to be recovered based on the acquired bright-field image and / or phase-contrast image. For example, whether the biological particles are to be recovered can be determined based on one or more combinations of the morphology, size, and color of the biological particles (especially cells).

[0198] (Target sorting based on dark-field imaging)

[0199] In yet another embodiment of this disclosure, the detection unit 292 may acquire a dark-field image generated by light irradiation by the light irradiation unit 291. In this embodiment, the light irradiation unit 291 may include, for example, a laser light source, and the detection unit 292 may include a CCD or CMOS. For example, light is irradiated onto biological particles by a laser, and a dark-field image (e.g., a fluorescence image) of the irradiated minute particles may be acquired by the CCD or CMOS.

[0200] In the embodiment in which the dark-field image is acquired, the determination unit 295 included in the control unit 293 determines whether the biological particle is a particle to be recovered based on the acquired dark-field image. For example, whether the biological particle is a particle to be recovered can be determined based on one or more combinations of the morphology, size, and color of the biological particle (especially cells).

[0201] In any of the above-mentioned methods of "discrimination of target to be collected based on fluorescence signals and / or scattered light signals," "discrimination of target to be collected based on bright-field images," and "discrimination of target to be collected based on dark-field images," the detection unit 292 may be an image sensor in which a substrate incorporating a CMOS sensor and a substrate incorporating a DSP (Digital Signal Processor) are stacked. By operating the DSP of the image sensor as a machine learning unit, the image sensor can operate as a so-called AI sensor. The detection unit 292 including the image sensor can determine, for example, whether a biological particle is a target particle to be collected based on a learning model. Furthermore, the learning model may be updated in real time while the method according to this disclosure is being performed. For example, the DSP can perform machine learning processing during the reset of the pixel array in the CMOS sensor, during the exposure of the pixel array, or during the reading of pixel signals from each unit pixel of the pixel array. An example of an image sensor operating as an AI sensor is the imaging device described in International Publication No. 2018 / 051809. When using an AI sensor as an image sensor, the raw data acquired from the image array is used directly for learning, resulting in faster sorting and discrimination processing.

[0202] The determination can be made, for example, based on whether information regarding the characteristics of the light satisfies a preset criterion. The criterion can be a criterion indicating that the biological particle is a particle to be collected. The criterion may be appropriately set by those skilled in the art and can be a criterion regarding the characteristics of light, such as a criterion used in technical fields such as flow cytometry.

[0203] One beam of light may be shone at one location within the detection area 256, or light may be shone at each of multiple locations within the detection area 256. For example, the microchip 250 may be configured such that light is shone at each of two different locations within the detection area 256 (i.e., there are two locations within the detection area 256 where light is shone). In this case, for example, it may be determined whether a biological particle is a particle to be recovered based on the light (e.g., fluorescence and / or scattered light) produced by the light shone on the biological particle at one location. Furthermore, the velocity of the biological particle in the flow path can be calculated based on the difference between the detection time of the light produced by the light shone at the one location and the detection time of the light produced by the light shone at the other location. For this calculation, the distance between the two shone locations may be determined in advance, and the velocity of the biological particle can be determined based on the difference between the two detection times and the distance. Furthermore, based on this velocity, the arrival time at the particle sorting unit 257 described below can be accurately predicted. By accurately predicting the arrival time, the timing of the flow formation entering the recovery channel 259 can be optimized. Furthermore, if the difference between the arrival time of a certain biological particle at the particle sorting unit 257 and the arrival time of a biological particle preceding or succeeding that biological particle is below a predetermined threshold, it can be determined not to recover that biological particle. When the distance between a certain biological particle and the biological particles preceding or succeeding it is small, the likelihood of the preceding or succeeding microparticles being recovered together with the biological particle increases during the aspiration of the biological particle. By determining not to recover the biological particle when there is a high probability of them being recovered together, it is possible to prevent the preceding or succeeding biological particles from being recovered. This makes it possible to increase the purity of the target biological particle among the recovered biological particles. Specific examples of a microchip in which light is irradiated at two different positions in the detection region 256 and an apparatus including the microchip are described, for example, in Japanese Patent Application Publication No. 2014-202573.

[0204] The control unit 293 may also control light irradiation by the light irradiation unit 291 and / or light detection by the detection unit 292. Furthermore, the control unit 293 may control the drive of a pump for supplying fluid into the microchip 250. The control unit 293 may consist of, for example, a hard disk containing a program and an OS for causing the device to perform the isolation process, a CPU, and memory. For example, the functions of the control unit 293 can be realized in a general-purpose computer. The program may be recorded on a recording medium such as a microSD memory card, an SD memory card, or flash memory. A drive (not shown) provided in the biological particle sorting device 200 may read the program recorded on the recording medium, and the control unit 293 may cause the biological particle sorting device 200 to perform the isolation process according to the read program.

[0205] (Recovery process)

[0206] In the recovery step S203, the biological particles identified as target particles in the discrimination step S202 are recovered into the recovery channel 259. In the recovery step S203, the target particles are recovered into a second liquid in the recovery channel, which is immiscible with the first liquid, while still contained in the first liquid. This allows for the formation of an emulsion in the recovery channel 259, with the second liquid as the dispersion medium and the first liquid as the dispersed phase, and each emulsion particle in the emulsion contains one target particle. This isolates the target biological particles within the space of the emulsion particles. For example, as shown in Figure 7B, the recovered particles P are recovered into the second liquid (shown in gray) while still contained in the first liquid (shown in white). This forms emulsion particles 290, and one recovered particle P is isolated within the space of one emulsion particle 290. The recovery process will be explained in more detail below.

[0207] The recovery process S203 is performed in the particle sorting section 257 of the microchip 250. In the particle sorting section 257, the laminar flow that has flowed through the main channel 255 splits and flows into two waste channels 258. The particle sorting section 257 shown in Figure 7A has two waste channels 258, but the number of branch channels is not limited to two. The particle sorting section 257 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 a single plane, as shown in Figure 7A, or they may be configured to branch in three dimensions.

[0208] In the particle sorting section 257, a flow is formed from the main channel 255 through the connecting channel 270 to the recovery channel 259 only when particles to be recovered are flowing in, and the particles to be recovered are recovered into the recovery channel 159. An enlarged view of the particle sorting section 257 is shown in Figure 8. As shown in Figure 8A, the main channel 255 and the recovery channel 259 are connected via the connecting channel 270, which is coaxial with the main channel 255. As shown in Figure 8B, the particles to be recovered flow through the connecting channel 270 to the recovery channel 259. Fine particles that are not to be recovered flow to the waste channel 258, as shown in Figure 8C.

[0209] Enlarged views of the vicinity of the connecting channel 270 are shown in Figures 11A and 11B. Figure 11A is a schematic perspective view of the vicinity of the connecting channel 270. Figure 11B is a schematic cross-sectional view in a plane passing through the center line of the liquid supply channel 261 and the center line of the connecting channel 270. The connecting channel 270 includes a channel 270a on the detection area 256 side (hereinafter also referred to as the upstream connecting channel 270a), a channel 270b on the recovery channel 159 side (hereinafter also referred to as the downstream connecting channel 270b), and a connection portion 270c between the connecting channel 270 and the liquid supply channel 261. The liquid supply channel 261 is provided so as to be substantially perpendicular to the axis of the flow path of the connecting channel 270. In Figures 11A and 11B, two liquid supply channels 261 are provided so as to face each other at approximately the center position of the connecting channel 270, but only one liquid supply channel may be provided.

[0210] The shape and dimensions of the cross-section of the upstream connecting channel 270a may be the same as those of the downstream connecting channel 270b. For example, as shown in Figures 11A and 11B, both the cross-section of the upstream connecting channel 220a and the cross-section of the downstream connecting channel 220b may be approximately circular with the same dimensions. Alternatively, both of these cross-sections may be rectangular (e.g., square or rectangle) with the same dimensions.

[0211] From the two liquid supply channels 261, a second liquid is supplied to the connecting channel 270 as shown by the arrows in Figure 11B. This second liquid flows from the connection part 270c to both the upstream connecting channel 270a and the downstream connecting channel 270b.

[0212] If the recovery process is not performed, the second liquid will flow as follows. The second liquid that flows into the upstream connecting channel 270a exits from the connection surface between the connecting channel 270 and the main channel 255, and then splits and flows into the two waste channels 258. By having the second liquid exit from this connection surface, it is possible to prevent the first liquid and fine particles, which do not need to be recovered into the recovery channel 259, from entering the recovery channel 259 through the connecting channel 270. The second liquid that flows into the downstream connecting channel 270b flows into the recovery channel 259. As a result, the recovery channel 259 is filled with the second liquid, which then acts as a dispersion medium for emulsion formation, for example.

[0213] Even when the recovery process is performed, the second liquid can be supplied from the two liquid supply channels 261 to the connecting channel 270. However, pressure fluctuations within the recovery channel 259, particularly by generating negative pressure within the recovery channel 259, create a flow from the main channel 255 through the connecting channel 270 to the recovery channel 259. That is, a flow is formed from the main channel 255, passing through the upstream connecting channel 270a, the connection section 270c, and the downstream connecting channel 270b in that order to the recovery channel 259. As a result, the particles to be recovered are recovered into the second liquid in the recovery channel 259 while encased in the first liquid. By performing this recovery process, an emulsion, for example, may be formed within the recovery channel 259 or in a container connected to the end of the recovery channel 263, for example, via a channel.

[0214] The cross-sectional shape and / or dimensions of the upstream connecting channel 220a may differ from those of the downstream connecting channel 220b. Examples of these two channels with different dimensions are shown in Figures 12A and 12B. As shown in Figures 12A and 12B, the connecting channel 280 includes a channel 280a on the detection area 256 side (hereinafter also referred to as the upstream connecting channel 280a), a channel 280b on the recovery channel 259 side (hereinafter also referred to as the downstream connecting channel 280b), and a connection portion 280c between the connecting channel 280 and the liquid supply channel 261. Both the cross-sections of the upstream connecting channel 280a and the downstream connecting channel 280b have a substantially circular shape, but the diameter of the latter cross-section is larger than the diameter of the former cross-section. By making the diameter of the latter cross-section larger than that of the former, it is possible to more effectively prevent the recovered particles already separated into the recovery channel 259 from being released into the main channel 255 through the connecting channel 280 immediately after the negative pressure-induced particle separation operation described above, compared to the case where the diameters of both are the same. For example, if both the cross-section of the upstream connecting channel 280a and the cross-section of the downstream connecting channel 280b are rectangular, by making the area of ​​the latter cross-section larger than the area of ​​the former cross-section, it is possible to more effectively prevent already collected fine particles from being released into the main channel 255 through the connecting channel 280, as described above.

[0215] In the recovery process S203, the particles to be recovered are recovered into the recovery channel through the connecting channel due to pressure fluctuations within the recovery channel 259. This recovery may be performed, for example, by generating negative pressure within the recovery channel 259, as described above. This negative pressure can be generated by, for example, an actuator 297 (particularly a piezo actuator) attached to the outside of the microchip 250, which deforms the wall defining the recovery channel 259. This negative pressure can form the flow entering the recovery channel 259. To generate this negative pressure, the actuator 297 can be attached to the outside of the microchip 250 so as to be able to deform the wall of the recovery channel 259. This deformation of the wall changes the internal environment of the recovery channel 259, thereby generating negative pressure. The actuator 297 may be, for example, a piezo actuator. When the particles to be recovered are drawn into the recovery channel 259, 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 259. In this way, the particles to be recovered are separated in the particle separation unit 257 and recovered into the recovery channel 259.

[0216] The particles to be recovered are encapsulated in the first liquid and then recovered into a second liquid, which is immiscible with the first liquid, within the recovery channel 259. As a result, as described above, an emulsion is formed within the recovery channel 259, with the second liquid as the dispersion medium and the first liquid as the dispersed phase.

[0217] To prevent biological particles that are not to be recovered from entering the recovery channel 259 through the connecting channel 270, the connecting channel 270 is equipped with a liquid supply channel 261. A second liquid, which is immiscible with the liquid (sample liquid and sheath liquid) flowing through the main channel 255, is introduced into the connecting channel 270 from the liquid supply channel 261. A portion of the second liquid introduced into the connecting channel 270 forms a flow from the connecting channel 270 toward the main channel 255, preventing biological particles other than those to be recovered from entering the recovery channel 259. The second liquid formed by the flow from the connecting channel 270 toward the main channel 255 flows through the waste channel 258, just like the first liquid, without flowing through the main channel 255, due to the flow of the first liquid flowing through the main channel 255 toward the waste channel 258. Furthermore, any remaining second liquid introduced into the connecting channel 270 flows into the recovery channel 259. As a result, the recovery channel 259 can be filled with the second liquid.

[0218] The recovery channel 259 may be filled with a second liquid that is immiscible with the first liquid. In order to fill the recovery channel 259 with the second liquid, the second liquid may be supplied from the liquid supply channel 261 to the connecting channel 270. Upon this supply, the second liquid flows from the connecting channel 270 to the recovery channel 259, thereby filling the recovery channel 259 with the second liquid.

[0219] The laminar flow that flows into the waste channel 258 can be discharged to the outside of the microchip at the end of the waste channel 260. Furthermore, the recovered particles collected into the recovery channel 259 can be discharged to the outside of the microchip at the end of the recovery channel 261.

[0220] A container 271 may be connected to the end 263 of the recovery channel via a channel such as a tube 272, as shown in Figure 13. As shown in the same figure, an emulsion is recovered into the container 271, in which the first liquid containing the particles to be recovered is used as the dispersion phase and the second liquid as the dispersion medium. In this way, an emulsion is obtained that contains emulsion particles to which the first capture substance, the secreted substance, and the biological particles P to which the second capture substance are bound are isolated. Figure 2D g shows the state in which the biological particles P are isolated within the emulsion particles E. The obtained emulsion may then be subjected to the destruction and analysis steps described later.

[0221] As described above, according to one embodiment of the present disclosure, the biological particle sorting device 200 may be equipped with a channel for collecting the emulsion containing the particles to be recovered into a container. Furthermore, by closing the end 263 of the recovery channel and performing the recovery operation, multiple emulsion particles can be retained within the recovery channel 259. After the completion of the recovery operation, assays such as single-cell analysis can be performed continuously within the recovery channel 259. For example, the disruption step described later may be performed within the recovery channel 259. In conjunction with the disruption step, binding of the target capture molecule to the target substance may occur.

[0222] As described above, in the microchip used in this disclosure, the main channel may branch into the connecting channel and the at least one waste channel. The at least one waste channel is a channel through which biological particles other than the particles to be recovered flow.

[0223] Furthermore, as shown in Figures 7A and 7B and Figure 8, in the microchip used in this disclosure, the main channel, the connecting channel, and the recovery channel may be arranged in a straight line. When these three channels are arranged in a straight line (particularly coaxially), the recovery process can be performed more efficiently compared to, for example, when the connecting channel and the recovery channel are arranged at an angle to the main channel. For example, the amount of suction required to guide the particles to be recovered into the connecting channel can be reduced. Furthermore, as shown in Figures 7A and 7B, in the microchip used in this disclosure, the biological particles are arranged in a nearly single line within the main channel and flow toward the connecting channel. Therefore, the amount of material aspirated during the recovery process can be reduced. The flow path configuration of the microchip used in this disclosure is not limited to that shown in Figure 7A. For example, the microchip used in this disclosure may have, for example, two or more inlets and / or outlets, preferably all inlets and / or outlets, formed on a single surface. A microchip with inlets and outlets formed in this manner is shown in Figure 14. In the microchip 350 shown in Figure 14, both the recovery channel end 263 and the two branch channel ends 260 are formed on the same surface as the sample liquid inlet 251 and the sheath liquid inlet 253. Furthermore, the introduction channel inlet 264 for introducing liquid into the introduction channel 261 is also formed on the same surface. Thus, in the microchip 350 for biological particle sorting, all of the inlets for liquid introduction and outlets for liquid discharge are formed on a single surface. This facilitates the attachment of the chip to the biological particle sorting device 200. For example, compared to a case where inlets and / or outlets are formed on two or more surfaces, the connection between the flow channels provided in the biological particle sorting device 200 and the flow channels of the biological particle sorting microchip 350 becomes easier. In Figure 14, a portion of the sheath liquid channel 254 is shown with a dotted line. This portion is located at a lower position (shifted in the direction of the optical axis indicated by the arrow) than the sample liquid channel 252 shown with a solid line, and it is the point where the dotted and solid channels intersect; these channels are not connected. This explanation also applies to a portion of the recovery channel 259 shown with a dotted line and the branch channel 258 that intersects with this portion.

[0224] Furthermore, in this disclosure, the liquid supply channel supplies liquid (particularly a second liquid) to the connecting channel. This creates a flow within the connecting channel that flows from the connection point between the liquid supply channel and the connecting channel toward the main channel, preventing the liquid flowing in the main channel from entering the connecting channel and preventing fine particles other than the particles to be recovered from flowing through the connecting channel into the recovery channel. When performing the recovery process, as described above, for example, the negative pressure generated in the recovery channel causes the first liquid containing one particle to be recovered to pass through the connecting channel and be recovered into the second liquid in the recovery channel. As a result, emulsion particles containing one particle to be recovered are formed in the second liquid.

[0225] Furthermore, in this disclosure, when a biological particle determined to be a particle to be recovered in the determination step is driven, for example, by driving a piezo actuator at an appropriate timing (for example, when it reaches the particle sorting unit 257), the hydrophilic solution containing the particle to be recovered is recovered into the recovery channel 259 and emulsion particles are formed. In the determination step, for example, by using the peak signal and area signal to determine whether it is a particle to be recovered, it is also possible to determine whether it is a single microparticle (singlet), a doublet of two biological particles bound together, or a triplet of three biological particles bound together. This method avoids the formation of emulsion particles containing two or more biological particles. Therefore, emulsion particles containing one biological particle can be formed with high probability and efficiency. Furthermore, since it avoids the formation of emulsion particles containing two or more bound biological particles, the operation of removing bound biological particles before the emulsion formation operation, for example by using a cell sorter, can be omitted.

[0226] In this disclosure, emulsion particles may be formed in the isolation step as described above. The biological particle P to which the first capture substance, the secreted substance, and the second capture substance are bound is sequestered within the emulsion particles.

[0227] (3-4-2-2) In the case of space within a well

[0228] A schematic diagram of an example of a well used to perform a particle isolation process. 16 As shown in the figure. 16 As shown in Figure, for example, a plurality of wells 40 having dimensions capable of containing one biological particle may be formed on the surface of the substrate 41. By applying the liquid containing the group of biological particles that have undergone the second capture step described in (3-3) above to the surface of the substrate 41, for example, from any nozzle 42, 16 As shown, the biological particle 43 is isolated in the space within the well 40. In this way, one biological particle may be placed in the space within one well, and the biological particle may be isolated in a microspace.

[0229] figure 16 When a liquid containing multiple biological particles is applied to a substrate in which wells are formed, as in the example shown, the particle isolation step may be performed without carrying out the discrimination step described above (3-4-2-1).

[0230] Furthermore, when carrying out the discrimination process described above (3-4-2-1), a device that places one biological particle in each well may be used, such as a cell sorter or a single-cell dispenser. In this device as well, a substrate (such as a plate) with multiple wells formed thereon may be used to isolate the biological particles. A commercially available device may be used as this device. This device may, for example, have a light irradiation unit that irradiates the biological particles with light, a detection unit that detects the light from the biological particles, a discrimination unit that determines whether to place the biological particles in the wells based on the detected light, and a distribution unit that distributes the biological particles determined to be placed in the wells into the wells.

[0231] The light irradiation unit and the detection unit perform the detection step, and the discrimination unit performs the discrimination step. The distribution unit includes, for example, a microfluidic chip having a nozzle that forms droplets containing biological particles.

[0232] The apparatus manipulates the position of the microfluidic chip according to the determination result by the discrimination unit to place one biological particle-containing droplet into a predetermined well. Alternatively, the apparatus controls the direction of travel of the biological particle-containing droplet exiting the nozzle using the charge applied to the droplet, according to the determination result by the discrimination unit. This control places one biological particle-containing droplet into a predetermined well. In this way, one biological particle is distributed into each well.

[0233] For example, Figure 15 As shown, droplets containing biological particles are ejected from a nozzle 52 provided on the microfluidic chip of the apparatus. Light (e.g., laser light L) is irradiated onto the biological particles contained in the droplets by a light irradiation unit 54, and a detection process is performed by a detection unit 55, detecting light (fluorescence F). Then, a discrimination unit (not shown) performs a determination process based on the detected light. Then, according to the determination result, the distribution unit controls the direction of movement of the droplets by utilizing the charge attached to the droplets. Through this control, droplets containing the target biological particles are collected in predetermined wells. As a result, one biological particle is distributed to each well.

[0234] By performing the aforementioned discrimination step, it is possible to identify, for example, the cell population to which the biological particles belong, the biological particles to which a barcode has been assigned, or the droplets containing singlet biological particles, according to the detection signal. This allows only the droplets containing the target biological particles to be recovered. As a result, it becomes unnecessary to exclude data in the analysis step described later, improving analytical efficiency.

[0235] The number of wells provided on a single substrate (plate) may be, for example, 1 to 1000, more particularly 10 to 800, and more particularly 30 to 500, but the number of wells may be appropriately selected by those skilled in the art.

[0236] As described above, in this disclosure, the first capture substance, the secreted substance, and the biological particle P to which the second capture substance is bound may be sequestered within the well.

[0237] (3-5) Destruction process

[0238] In the destruction step S105, the biological particles are destroyed within a microspace. The destruction step may be carried out in an environment in which the components contained in one biological particle do not mix with the components contained in other biological particles. As a result of this destruction, the composite of the first captured substance, the secreted substance, and the second captured substance formed in the second capture step S103 is dissociated from the biological particles. In addition, as a result of this destruction, the particle capturing substance 120 is also dissociated from the biological particles. Here, the second capture substance in the conjugate contains a poly-A sequence, and the particle capture substance 120 contains a substance recovery unit 122 (e.g., poly-T). Therefore, the poly-A and the substance recovery unit 122 bind together. The second capture substance contains a capture substance identifier as described above, and the particle capture substance contains a particle identifier as described above. Therefore, the capture substance identifier and the particle identifier are bound together through the binding of the poly-A and the substance recovery unit. This allows for analysis, for example, in the analysis step described later, with the capture substance identifier and the particle identifier associated. More specifically, the capture substance identifier can identify the secreted substance captured by the second capture substance, and the particle identifier can identify the biological particle to which the particle capture substance containing the particle identifier was bound. Therefore, the secreted substance and the biological particle can be associated. As a result, information (information on type and / or amount) of the secreted substance captured by the biological particle can be associated with the biological particle, and analysis of the secreted substance becomes possible at the single-cell level.

[0239] Furthermore, in the destruction step S105, the substance recovery unit 122 contained in the particle capturing material 120 can capture the target substance constituting the biological particle or the target substance bound to the biological particle. As a result, a composite of the particle capturing material 120 and the target substance is formed, and in the analysis step described later, the target substance can be associated with the particle identifier 124 contained in the particle capturing material 120. The composite formed in this way is analyzed in the analysis step described later. Therefore, information about the target substance (information about its type and / or quantity) can be associated with the biological particle, and the analysis of the target substance becomes possible at the single-cell level.

[0240] The disruption step S105 is preferably performed while maintaining the isolation of the biological particles within the microspace. This allows for the efficient formation of the conjugate and / or the complex. Furthermore, it prevents the constituent molecules of the conjugate and / or the complex from binding with molecules outside the microspace. If the aforementioned microspace refers to a space within an emulsion particle, then maintaining the isolation state may mean maintaining the emulsion particle, and in particular, it may mean that the emulsion particle is not destroyed. If the aforementioned microspace refers to a space within a well, then maintaining the isolation state may mean that the components within the well (particularly the biological particles within the well, the aggregates, the complexes, and the constituent molecules of the aggregates and / or the complexes) remain within that well, and may further mean that components from other wells do not enter the well.

[0241] The destruction step S105 may be carried out by chemically or physically destroying the biological particles.

[0242] For the chemical destruction of biological particles, a biological particle-destroying substance and the biological particles may be brought into contact within a microspace. The biological particle-destroying substance may be appropriately selected by those skilled in the art depending on the type of biological particle. When the biological particle is a cell, for example, a lipid bilayer-destroying component may be used as the biological particle-destroying substance, specifically a surfactant, an alkaline component, or an enzyme. As a surfactant, anionic surfactants, nonionic surfactants, amphoteric surfactants, or cationic surfactants may be used. Examples of anionic surfactants include sodium dodecyl sulfate (SDS) and sodium lauroyl sarcosinate. Examples of nonionic surfactants include Triton X-100, Triton X-114, Tween 20, Tween 80, NP-40, Brij-35, Brij-58, octyl glucoside, octyl thioglucoside, and octylphenoxypolyethoxyethanol. Examples of amphoteric surfactants include CHAPS and CHAPSO. An example of the cationic surfactant is cetyltrimethylammonium bromide (CTAB). An example of the alkaline component is the OH- ion. Examples of the enzymes include Proteinase K, streptolin, lysozyme, lysostaffin, zymolase, cellulase, glycanase, and protease. The type of enzyme can be appropriately selected depending on the type of cell (e.g., animal cells, plant cells, bacteria, and yeast).

[0243] If the aforementioned microspace is a space within a well, the destruction process can be carried out, for example, by adding a bioparticle-destroying substance to each well. Since each well is isolated from the others, even if destruction occurs, the components within the well are maintained within that well.

[0244] If the aforementioned microspace is a space within an emulsion particle, for example, a bioparticle-destroying substance can be introduced into the emulsion particle simultaneously with the formation of the emulsion particle. Then, after the emulsion particle is formed, a process of destroying the bioparticle with the bioparticle-destroying substance can be carried out.

[0245] To physically destroy biological particles, a physical stimulus that destroys the biological particles can be applied to them. For example, optical treatment, thermal treatment, electrical treatment, acoustic treatment, freeze-thaw treatment, or mechanical treatment may be employed as the process for applying such physical stimulus to the biological particles. These treatments can destroy cells or exosomes. Examples of optical treatments include plasma formation or cavitation bubble formation by laser irradiation. Examples of thermal treatments include heat treatment. Examples of acoustic treatments include sonication using ultrasound. Examples of mechanical treatments include treatment using a homogenizer or bead mill. The physical destruction of biological particles by these treatments can be applied to both cases where the microspace is a space within a well and a space within an emulsion particle. When the microspace is a space within an emulsion particle, optical treatment, thermal treatment, electrical treatment, and freeze-thaw treatment are particularly suitable among these treatments. Furthermore, in order to destroy biological particles while preventing the destruction of emulsion particles by the acoustic treatment, a surfactant may be added to the emulsion particles, and the concentration of the surfactant may be adjusted.

[0246] In the destruction step S105, by using the substance recovery unit 122 contained in the particle capture material 120, it becomes possible to analyze secreted substances and intracellular target substances, and furthermore, the results of these analyses can be correlated with biological particles. Therefore, single-cell analysis of secreted substances and intracellular substances can be performed simultaneously.

[0247] The destruction step S105 includes a step of recovering the aggregate and / or the particle trapping material 120 (particularly the target material bound to the particle trapping material 120) using the material recovery unit 122. The material recovery unit 122 can recover the aggregate, and further recover the particle trapping material 120, particularly the target material bound to the particle trapping material 120.

[0248] For example, as shown in Figure 2D g, a biological particle P to which the first capture substance, the secreted substance, and the second capture substance are bound is sequestered within the emulsion particle E. By performing a destruction treatment on the biological particle P, the first capture substance, the secreted substance, and the second capture substance are released from the biological particle P.

[0249] Then, as shown in Figure 2D, for example, the second capture substance 170 binds to the particle capture substance 120. This binding may be based on the binding of the poly-A sequence 173 of the second capture substance 170 to the substance recovery portion 122 (in this case, the poly-T sequence) of the particle capture substance 120. Although the first capture substance and the secreted substance are not depicted in Figure 2D, after this binding, the secreted substance and the first capture substance may continue to be bound to the second capture substance 170, or they may not be bound to the second capture substance 170. For example, when a biological particle P is destroyed, the secreted substance may be released from the second capture substance 170 or may be destroyed. Consequently, the first capture substance may also be released from the second capture substance 170.

[0250] Furthermore, the destruction of biological particle P releases the mRNA inside biological particle P into the emulsion particle. This mRNA then binds to the material recovery portion (poly T sequence) 122 of the particle capture material 120.

[0251] As described above, during the disruption step, a conjugate of the particle-capturing substance 120 and the second-capturing substance 170 is formed within the emulsion particles. Furthermore, during the disruption step, a complex of the particle-capturing substance 120 and a substance contained in the biological particle (the target substance mentioned above, particularly mRNA) may also be formed within the emulsion particles. The conjugate and / or the complex conjugate are the targets of analysis in the analysis step described later.

[0252] (3-6) Analysis process

[0253] In analysis step S106, each biological particle is analyzed. This analysis may be performed, for example, on the conjugates and / or complexes released by the destruction of the biological particles in destruction step S105. For example, as shown in i in Figure 2D, the analysis may be performed on the conjugate of the particle capturing substance 120 and the second capturing substance 170. In addition, the analysis may be performed on the complex of the particle capturing substance 120 and the substance contained in the biological particle (the target substance mentioned above, particularly mRNA).

[0254] The conjugate and the complex each include the recovered substance amplification unit and the second recovered substance amplification unit. Therefore, the analysis in analysis step S106 may include a nucleic acid amplification step in which the nucleic acids contained in the conjugate and / or the complex are amplified using the recovered substance amplification unit and / or the second recovered substance amplification unit. In this nucleic acid amplification step, the capture substance identifier (particularly nucleic acid, more particularly mRNA) contained in the conjugate is amplified, and / or the target substance (particularly nucleic acid, more particularly mRNA) contained in the complex is amplified. Then, nucleic acid sequence information is obtained by performing a sequencing process on the amplified nucleic acid. Here, the capture substance identifier (particularly the sequence information contained in the identifier) ​​included in the conjugate is associated with the secreted substance. Therefore, the secreted substance can be identified from the nucleic acid sequence information. Furthermore, the target substance included in the complex is a substance contained in or bound to a biological particle, and this is amplified. Therefore, the target substance can be identified from the nucleic acid sequence information. Consequently, the secreted substance and the target substance can be identified by the sequencing process.

[0255] Furthermore, as described above, the captured substance identifier contained in the conjugate is bound to the particle identifier via the binding between PolyA and the substance recovery unit. The particle capturing substance contained in the complex is also bound to the particle identifier. Therefore, the amplified nucleic acid also includes the sequence of the particle identifier; that is, the nucleic acid sequence information obtained by the sequencing process also includes information about the particle identifier. Therefore, among multiple types of nucleic acid sequence information, nucleic acid sequence information containing the same particle identifier sequence can be identified as originating from a conjugate (secreted substance) that was bound to the same biological particle or a target substance contained in the same biological particle. Thus, in the analysis step S106, information regarding the identified secreted substance and / or target substance based on the sequence of particle identifiers may be associated with a single biological particle.

[0256] Since the aggregate and / or complex includes a particle identifier in the destruction step, even when the aggregate and / or complex originating from different biological particles present in multiple microspaces are analyzed together in the analysis step S106, the results of the analysis of the aggregate and / or complex can be associated with the biological particles from which the aggregate and / or complex originated, based on the particle identifier.

[0257] For example, if the microspace is a space within a well, each biological particle disruption product within the well may be analyzed separately, or the biological particle disruption products from multiple wells may be combined into a single sample and analyzed collectively. In the former case, it is easy to associate the biological particles with the analysis results. In the latter case as well, since the secreted substances or target substances contained in each biological particle disruption product exist as components of a conjugate or complex containing a particle identifier, the analysis results for the conjugate or complex can be associated with the biological particles from which they originated.

[0258] Furthermore, if the microspace is a space within an emulsion particle, multiple emulsion particles may be analyzed together, for example, the entire resulting emulsion may be analyzed together. Since the secreted substances or target substances contained in each biological particle destruction product exist as components of a conjugate or complex containing a particle identifier, the analysis results for the conjugate or complex can be correlated with the biological particles from which they originated. This improves the efficiency of the analysis.

[0259] The analysis step S106 may be performed using the analyzer 1000 as shown in i in Figure 2D. The analyzer 1000 may be, for example, an analyzer that performs sequencing on the conjugate and / or the complex. The sequencing process provides sequence information of nucleic acids, particularly DNA or RNA, and more particularly mRNA. The sequencing process may be performed by a sequencer, and may be performed by a next-generation sequencer or a Sanger sequencer. To perform comprehensive analysis of multiple biological particles (particularly cell populations) at a faster rate, the sequencing process may be performed by a next-generation sequencer.

[0260] In order to perform sequencing in analysis step S106, the analysis step may further include a preparation step for nucleic acids (e.g., cDNA) to be sequenced and a purification step for nucleic acids. These preparation and purification steps may prepare, for example, a library for next-generation sequencing.

[0261] The preparation step may include, for example, a cDNA synthesis step in which cDNA is synthesized from mRNA. The preparation step may also include an amplification step in which the synthesized cDNA is amplified. After the preparation step, a purification step may be performed to purify the nucleic acid obtained in the preparation step. The purification step may include a degradation treatment of components other than nucleic acid using an enzyme such as proteinase K. A nucleic acid recovery treatment may also be performed in the purification step. In the nucleic acid recovery treatment, commercially available nucleic acid purification reagents may be used, for example, magnetic beads such as AMPure XP. Although intracellular dsDNA may also be recovered in the purification step, it is possible to prevent dsDNA from being sequenced in the sequencing treatment. For example, by including an adapter sequence for sequencing treatment (particularly for next-generation sequencing treatment) in the sequence to be amplified (for example, in the second capture substance and particle capture substance), only nucleic acids containing the adapter sequence can be sequenced.

[0262] In analysis step S106, secreted substances and / or target substances can be analyzed for each biological particle based on the sequencing results. For example, in analysis step S106, the type of secondary captured substance (particularly the sequence of captured substance identifiers) and / or the number of secondary captured substances may be determined. This determination may be based on the sequence of captured substance identifiers in the sequence determined by the sequencing process. This determines the type and / or number of secreted substances captured by the secondary captured substances. Furthermore, in analysis step S106, the sequence of the target substance (such as mRNA contained in cells) and / or the copy number of each target substance may be determined. Analysis of secreted substances and / or target substances for each biological particle can be performed based on particle identifiers in sequences determined by sequencing. For example, from among a large number of base sequences determined by sequencing, base sequences containing the same particle identifier are selected. Base sequences containing the same particle identifier are based on a secondary capture substance that captures secreted substances bound to a single cell and / or a particle capture substance that binds to components contained in that cell. Therefore, by summarizing the analysis results of secreted substances and / or target substances for each particle identifier, these substances can be analyzed for each biological particle.

[0263] 2. Second Embodiment (Reagent Kit for Biological Particle Analysis)

[0264] This disclosure also provides a reagent kit for biological particle analysis, comprising: a first secretion-capturing substance comprising a first biological particle binding portion configured to bind to biological particles and a first secretion-binding portion configured to bind to secretions produced by placing a group of biological particles containing the biological particles under predetermined conditions; and a second secretion-capturing substance comprising a second secretion-binding portion configured to bind to the secretions and a capture-substance identifier for identifying the second capture-substance substance.

[0265] The first secretion-capturing substance is the first capture substance 130 described in 1. above, and the description of the first capture substance 130 also applies to the first secretion-capturing substance in this embodiment. The first biological particle binding portion and the first secretion-capturing substance are the biological particle binding portion 133 and the secretion-binding portion 131 described in 1. above. The description of the biological particle binding portion 133 and the secretion-binding portion 131 also applies to the first biological particle binding portion and the first secretion-capturing substance in this embodiment. The first secretion-capturing substance may further include a crosslinking portion that crosslinks the first biological particle binding portion and the first secretion-binding portion.

[0266] For example, the first biological particle binding portion may include an antigen-binding substance that binds to an antigen on the surface of the biological particle, or a molecular-binding substance that binds to a molecule forming the surface film of the biological particle. The antigen-binding substance may include a substance selected from the group including antibodies, antibody fragments, aptamers, and molecularly imprinted polymers. The molecular-binding substance may include an oleyl group or a cholesteryl group.

[0267] The second secretion-capturing substance is the second capture substance 170 described in 1. above, and the description of the second capture substance 170 also applies to the second secretion-capturing substance in this embodiment. The second secretion-binding portion and the capture substance identifier are the second secretion-binding portion 171 and the capture substance identifier 173 described in 1. above. The description of the second secretion-binding portion 171 and the capture substance identifier 173 also applies to the second secretion-binding portion and the capture substance identifier in this embodiment.

[0268] The reagent kit may further include a substrate having a surface on which a particle-capturing substance is immobilized, the substance comprising a second biological particle binding portion configured to bind to biological particles and a particle identifier for identifying biological particles. The surface and the substrate are the surface 110 and the substrate 100 described in 1. above, and the particle-capturing substance is the particle-capturing substance 120 described in 1. above. Therefore, the descriptions of the surface 110, the substrate 100, and the particle-capturing substance 120 also apply to the surface, substrate, and particle-capturing substance in this embodiment.

[0269] A reagent kit for biological particle analysis according to this disclosure may be used in a biological particle analysis method according to this disclosure. As described in 1. above, among the materials contained in the reagent kit, the combination of the first secretion-capturing substance and the second secretion-capturing substance is used to capture secretions. In particular, this combination is used to capture secretions while they are bound to biological particles.

[0270] The first secretion-capturing substance may further include a crosslinking portion that crosslinks the biological particle binding portion and the secretion-binding portion. The crosslinking portion is the crosslinking portion 132 described in 1. above. The description of the crosslinking portion 132 also applies to the crosslinking portion in this embodiment.

[0271] The reagent kit may further include a substrate having a surface on which a particle-capturing substance is immobilized, the substance comprising a second biological particle binding portion configured to bind to biological particles and a particle identifier for identifying biological particles. The surface and the substrate are the surface 110 and the substrate 100 described in 1. above, and the particle-capturing substance is the particle-capturing substance 120 described in 1. above. Therefore, the descriptions of the surface 110, the substrate 100, and the particle-capturing substance 120 also apply to the surface, substrate, and particle-capturing substance in this embodiment.

[0272] 3. Third Embodiment (Bioparticle Analysis System)

[0273] This disclosure also provides a biological particle analysis system. The system may include: a first container in which a group of biological particles containing biological particles bound to a first capture substance for capturing secretory substances is placed under predetermined conditions to induce the secretion of secretory substances; a second container in which the secretory substances bound to the first capture substance are bound to a second capture substance for capturing secretory substances; and a biological particle processing device for isolating the biological particles to which the first capture substance, the secretory substances, and the second capture substance are bound into single particles.

[0274] The first container corresponds to container 140 in which the first capture step S102 described in 1 above is performed. The second container corresponds to container 150 in which the second capture step S103 described in 1 above is performed.

[0275] The biological particle processing device may be configured to perform the isolation step S104 described in 1. above. The biological particle processing device may be, for example, the biological particle sorting device 200 described in 1. above.

[0276] The biological particle analysis system of this disclosure may further include an apparatus configured to perform the cleavage step S114 (particularly the detection step and / or linker cleavage step) described in 1. above. The apparatus may be the stimulation device described in 1. above.

[0277] Furthermore, according to one embodiment, the biological particle analysis system of the present disclosure may include a biological particle processing device for isolating the biological particles to which the first capture substance, the secreted substance, and the second capture substance are bound into single particles. In addition to the biological particle processing device, the biological particle analysis system may also include a biological particle analysis reagent kit according to the present disclosure (or one or more of the materials contained in the reagent kit).

[0278] The biological particle analysis system of this disclosure may include an analytical instrument that performs the analytical steps described in 1. above. Such analytical instrument may be, for example, a sequencer.

[0279] Furthermore, this disclosure may also take the following form. [1] Preparation steps include preparing a group of biological particles containing biological particles to which a first capture substance for capturing secreted substances is bound, A first capture step involves binding the secreted substance generated by placing the group of biological particles under predetermined conditions with the first capture substance, A second capture step involves combining the secreted substance bound to the first capture substance with a second capture substance for capturing the secreted substance, A method for analyzing biological particles, including the analysis of such particles. [2] The first capture step includes a processing step that places the group of biological particles under predetermined conditions, The processing step is carried out while the collective state of the biological particle collection is maintained. The method for analyzing biological particles described in [1]. [3] The method for analyzing biological particles according to [1] or [2], wherein the first capture step and the second capture step are performed while the state in which the first capture substance is bound to the biological particles is maintained. [4] A method for analyzing biological particles according to any one of [1] to [3], wherein a particle identifier for identifying the biological particles is attached to the biological particles included in the group of biological particles prepared in the preparation step. [5] A method for analyzing biological particles according to any one of [1] to [4], wherein a capture substance identifier for identifying the second capture substance is attached to the second capture substance. [6] The method for analyzing biological particles according to any one of [1] to [4], wherein the first capture substance includes a secretion substance binding site and a biological particle binding site. [7] The method for analyzing biological particles according to [6], wherein the secretion substance binding portion is configured to be able to bind one or more secretion substances. [8] The method for analyzing biological particles according to [6] or [7], wherein the biological particle binding portion includes an antigen-binding substance that binds to an antigen on the surface of the biological particle or a molecular-binding substance that binds to a molecule that forms a surface film of the biological particle. [9] The method for analyzing biological particles according to any one of [6] to [8], wherein the secretion substance binding portion is bound to the biological particle binding portion via a cross-linking portion.

[10] The method for analyzing biological particles according to any one of [1] to [9], wherein the first capture substance includes an antibody that binds to the surface of two or more cells of the same or different species.

[11] A method for analyzing biological particles according to any one of [1] to

[10] , further comprising an isolation step of isolating the biological particles contained in the group of biological particles into single particles after the second capture step.

[12] The method for analyzing biological particles according to

[11] , further comprising a destruction step of destroying the biological particles after the isolation step.

[13] The method for analyzing biological particles according to

[12] , wherein the destruction step is performed in an environment in which the components contained in one biological particle do not mix with the components contained in other biological particles.

[14] The method for analyzing biological particles according to

[13] , further comprising an analysis step of performing an analysis of each biological particle after the destruction step.

[15] A first secretory scavenging substance comprising a first biological particle binding portion configured to bind to biological particles and a first secretory substance binding portion configured to bind to secretory substances produced by placing a group of biological particles containing the biological particles under predetermined conditions; and A substance for capturing a second secretion, comprising a second secretion binding portion configured to bind to the aforementioned secretion and a capture substance identifier for identifying the second capture substance; A reagent kit for biological particle analysis, including [specific component].

[16] The reagent kit for biological particle analysis according to

[15] , wherein the first secretion substance capturing substance further comprises a crosslinking portion that crosslinks the biological particle binding portion and the secretion substance binding portion.

[17] The reagent kit for biological particle analysis according to

[15] or

[16] , wherein the first biological particle binding portion includes an antigen-binding substance that binds to an antigen on the surface of a biological particle or a molecular-binding substance that binds to a molecule that forms a surface film of the biological particle.

[18] The reagent kit for biological particle analysis according to

[17] , wherein the antigen-binding substance comprises a substance selected from the group including antibodies, antibody fragments, aptamers, and molecularly imprinted polymers.

[19] The reagent kit for biological particle analysis according to

[17] , wherein the molecular binding substance comprises an oleyl group or a cholesteryl group.

[20] A substrate having a surface on which a particle-capturing substance is immobilized, the substance comprising a second biological particle binding portion configured to bind to biological particles and a particle identifier for identifying biological particles; A reagent kit for biological particle analysis described in any one of

[15] to

[19] , further including the above. [Explanation of Symbols]

[0280] 100 substrates 110 surface 120 Particle Capture Material 130 First Capture Material 160 secretions 170 Second Capture Material

Claims

1. Preparation steps include preparing a group of biological particles containing biological particles to which a first capture substance for capturing secreted substances is bound, A first capture step involves binding the secreted substance generated by placing the bioparticle group under predetermined conditions with the first capture substance, A second capture step involves combining the secreted substance bound to the first capture substance with a second capture substance for capturing the secreted substance, Includes, The first capture substance has a secretion-binding portion designed according to the secretion-binding portion and a biological particle-binding portion, A method for analyzing biological particles, wherein the secreted substance binding portion specifically binds to the secreted substance.

2. The first capture step includes a processing step that places the group of biological particles under predetermined conditions, The processing step is carried out while the collective state of the biological particle collection is maintained. The method for analyzing biological particles according to claim 1.

3. The method for analyzing biological particles according to claim 1, wherein the first capture step and the second capture step are performed while the state in which the first capture substance is bound to the biological particles is maintained.

4. The method for analyzing biological particles according to claim 1, wherein a particle identifier for identifying biological particles is attached to the biological particles included in the group of biological particles prepared in the preparation step.

5. The method for analyzing biological particles according to claim 1, wherein a capture substance identifier for identifying the second capture substance is bound to the second capture substance.

6. The method for analyzing biological particles according to claim 1, wherein the secretion substance binding portion is configured to bind one or more secretion substances.

7. The method for analyzing biological particles according to claim 1, wherein the biological particle binding portion includes an antigen-binding substance that binds to an antigen on the surface of a biological particle or a molecular-binding substance that binds to a molecule that forms a surface film of the biological particle.

8. The method for analyzing biological particles according to claim 1, wherein the secreted substance binding portion is bound to the biological particle binding portion via a cross-linking portion.

9. The method for analyzing biological particles according to claim 1, wherein the first capture substance includes antibodies that bind to the surface of two or more cells of the same or different species.

10. The method for analyzing biological particles according to claim 1, further comprising an isolation step of isolating the biological particles contained in the group of biological particles into single particles after the second capture step.

11. The method for analyzing biological particles according to claim 10, further comprising a destruction step of destroying the biological particles after the isolation step.

12. The method for analyzing biological particles according to claim 11, wherein the destruction step is performed in an environment in which the components contained in one biological particle do not mix with the components contained in other biological particles.

13. The method for analyzing biological particles according to claim 12, further comprising an analysis step of performing an analysis on each biological particle after the destruction step.

14. A first secretory scavenging substance comprising a first biological particle binding portion configured to bind to biological particles and a first secretory substance binding portion configured to bind to secretory substances generated by placing a group of biological particles containing the biological particles under predetermined conditions; and A secondary secretion-capturing substance comprising a secondary secretion-binding portion configured to bind to the aforementioned secretion-containing substance and a capture-containing substance identifier for identifying the secondary capture-containing substance; Includes, The first secretion binding portion is designed according to the secretion, The first secretion-binding site specifically binds to the secretion-binding site, and the reagent kit is for the analysis of biological particles.

15. The reagent kit for biological particle analysis according to claim 14, wherein the first secretion substance capturing substance further comprises a crosslinking portion that crosslinks the first biological particle binding portion and the first secretion substance binding portion.

16. The reagent kit for biological particle analysis according to claim 14, wherein the first biological particle binding portion includes an antigen-binding substance that binds to an antigen on the surface of a biological particle or a molecular-binding substance that binds to a molecule that forms a surface film of the biological particle.

17. The reagent kit for biological particle analysis according to claim 16, wherein the antigen-binding substance comprises a substance selected from the group comprising antibodies, antibody fragments, aptamers, and molecularly imprinted polymers.

18. The reagent kit for biological particle analysis according to claim 16, wherein the molecular binding substance comprises an oleyl group or a cholesteryl group.

19. A substrate having a surface on which a particle-capturing material is immobilized, the material comprising a second biological particle binding portion configured to bind to biological particles and a particle identifier for identifying biological particles; The reagent kit for biological particle analysis according to claim 14, further comprising:

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