Single cell sample pretreatment device

Abstract

A single cell sample pretreatment device is disclosed. The single cell sample pretreatment device, according to one embodiment of the present disclosure, may comprise: a housing; a cartridge which is inserted into the housing and comprises a sample receiving unit comprising at least one injection container for receiving a sample therein, a microfluidic chip comprising a plurality of microwells, and a sample transport unit connected to the sample receiving unit and the microfluidic chip and transporting the sample inside the at least one injection container to an inlet of the microfluidic chip; a pressure control unit which is disposed inside the housing, is connected to the at least one injection container, and controls the pressure inside the at least one injection container; and a sample storage unit which is connected to an outlet of the microfluidic chip and stores the sample that is discharged from the outlet of the microfluidic chip. A cartridge for single cell sample pretreatment is disclosed.

Description

Single-cell sample pretreatment device

[0001] The present disclosure relates to a single-cell sample pretreatment device, and more specifically, to a single-cell sample pretreatment device that transfers a sample inside an injection vessel to a microfluidic chip by controlling the pressure inside the injection vessel, and enables pretreatment of the single-cell sample to be performed on the microfluidic chip.

[0002] The present disclosure relates to a cartridge for single-cell sample pretreatment, and more specifically, to a cartridge for single-cell sample pretreatment in which a sample inside an injection vessel is transferred to a microfluidic chip by controlling the pressure inside the injection vessel, and pretreatment of the single-cell sample is performed on the microfluidic chip.

[0003] Single-Cell Multi-Omics analysis technology is a technology that enables the analysis of various data generated from the genome, transcriptome, proteome, and epigenome within the same cell.

[0004] For multi-omics analysis of single cells, a single-cell sample pretreatment process including a process of separating the cell sample to be analyzed into single cells may be required.

[0005] According to one example of prior art regarding a single-cell sample pretreatment device, a technology has been presented in which a user manually injects a sample into a microfluidic chip using an injection tool such as a pipette, and the injected sample flows within the microfluidic chip by flowing due to gravity, thereby performing single-cell sample pretreatment.

[0006] However, according to these conventional technologies, the user must determine the sample injection speed and volume by manually adjusting hand gripping pressure, and there was a problem where the consistency and reproducibility of the analysis were reduced due to disturbances such as laboratory temperature.

[0007] Therefore, there is a need for single-cell sample pretreatment technology that can enhance user convenience through automated pretreatment and ensure high reproducibility.

[0008] [Prior Art Literature]

[0009] Published Patent Application No. 10-2020-0002705

[0010] The present disclosure aims to solve all the problems of the aforementioned prior art.

[0011] Additionally, the present disclosure has another objective of providing a single-cell sample pretreatment device comprising a housing, a sample receiving portion including at least one injection vessel for receiving a sample, a microfluidic chip including a plurality of microwells, and a sample transfer portion connected to the sample receiving portion and the microfluidic chip and transferring a sample inside at least one injection vessel to an inlet of the microfluidic chip, a cartridge inserted into the housing, a pressure control portion disposed inside the housing and connected to at least one injection vessel and controlling the pressure inside at least one injection vessel, and a sample storage portion connected to an outlet of the microfluidic chip and storing a sample discharged from the outlet of the microfluidic chip.

[0012] In addition, the present disclosure has another objective of enabling the automatic inflow and outflow of a sample within a microfluidic chip by controlling the pressure inside at least one injection vessel by a pressure control unit.

[0013] In addition, the present disclosure has another objective of increasing the pretreatment efficiency and performance of a single cell by maintaining the temperature of at least one injection vessel and / or microfluidic chip within a set range.

[0014] Additionally, the present disclosure has another objective of providing a cartridge comprising a sample receiving portion including at least one injection vessel for receiving a sample, a microfluidic chip including a plurality of microwells, and a sample transfer portion connected to the sample receiving portion and the microfluidic chip and transferring a sample inside at least one injection vessel to an inlet of the microfluidic chip.

[0015] In addition, another objective of the present disclosure is to enable the automatic inflow and outflow of a sample within a microfluidic chip by controlling the pressure inside at least one injection vessel.

[0016] In addition, another objective of the present disclosure is to maintain the temperature of at least one injection vessel and / or microfluidic chip within a set range so as to increase the pretreatment efficiency and performance of a single cell.

[0017] In addition, the present disclosure has another objective of providing a single-cell sample pretreatment method.

[0018] In addition, another objective of the present disclosure is to maintain the temperature of the microfluidic chip within a set range so that the pretreatment efficiency and performance of a single cell can be increased.

[0019] In addition, the present disclosure has another objective of providing an automated single-cell sample pretreatment method.

[0020] The purposes of the present disclosure are not limited to those mentioned above, and other purposes and advantages of the present disclosure not mentioned may be understood from the following description and will be more clearly understood from the embodiments of the present disclosure. Furthermore, it will be readily apparent that the purposes and advantages of the present disclosure can be realized by the means and combinations thereof set forth in the claims.

[0021] A single-cell sample pretreatment device according to one embodiment of the present disclosure may include a housing, a sample receiving unit comprising at least one injection vessel for receiving a sample, a microfluidic chip comprising a plurality of microwells, and a sample transfer unit connected to the sample receiving unit and the microfluidic chip and transferring a sample inside the at least one injection vessel to an inlet of the microfluidic chip, a cartridge inserted into the housing, a pressure control unit disposed inside the housing and connected to the at least one injection vessel and controlling the pressure inside the at least one injection vessel, and a sample storage unit connected to an outlet of the microfluidic chip and storing a sample discharged from the outlet of the microfluidic chip.

[0022] Additionally, the pressure control unit can control at least one injection vessel valve connected to the inlet of each of the at least one injection vessel. Here, when the at least one injection vessel valve is opened, the pressure inside the injection vessel connected to the opened injection vessel valve may increase.

[0023] Additionally, the sample transfer unit may include at least one sample injection channel connected to each of the at least one injection vessel and a sample transfer valve connected to the at least one sample injection channel and the inlet of the microfluidic chip. Here, the pressure control unit may control the injection vessel valve to increase the internal pressure of the selected injection vessel based on a control signal regarding the selection of an injection vessel containing a sample to be supplied to the microfluidic chip. And, the sample transfer valve may be controlled so that the sample moves along the sample injection channel connected to the selected injection vessel to the inlet of the microfluidic chip.

[0024] Additionally, the sample storage unit may include at least one storage container for storing a sample discharged from an outlet of the microfluidic chip, a rotating member to which the at least one storage container is detachably coupled, and a rotary driving module for driving the rotary member. Here, the rotary driving module may drive the rotary member so that the selected storage container is connected to the outlet based on a control signal regarding the selection of a storage container for storing the discharged sample.

[0025] Meanwhile, the single-cell sample pretreatment device may include an optical unit for photographing a plurality of microwells of the cartridge. Here, the optical unit may include a light source positioned lower than the cartridge and irradiating light in a direction toward the cartridge, an objective lens through which light emitted from the light source and transmitted through the cartridge is transmitted, and an image sensor that senses the light transmitted through the objective lens.

[0026] Meanwhile, the single-cell sample pretreatment device may include a cooling unit for cooling at least one cooling injection vessel among the at least one injection vessel that requires cooling. Here, the cooling unit may include a first temperature sensor for sensing the temperature of the at least one cooling injection vessel, a Peltier module for cooling the at least one cooling injection vessel, and a cooling fan.

[0027] In addition, the Peltier module can be in contact with the at least one cooling injection vessel on two sides.

[0028] Meanwhile, the single-cell sample pretreatment device may include a heating unit for heating the microfluidic chip. The heating unit may include a second temperature sensor for sensing the temperature of the microfluidic chip, an air heating module for heating air, and a blower module for discharging the heated air toward the microfluidic chip.

[0029] In addition, the heating unit can heat the microfluid chip for a preset reaction time such that the temperature of the microfluid chip becomes 37 to 42 degrees Celsius after the dissolved buffer is transferred from the injection vessel containing the dissolved buffer to the microfluid chip and a preset dissolution time has elapsed.

[0030] Meanwhile, the single-cell sample pretreatment device described above may include magnetic force control for controlling the position of a magnet. Here, the magnetic force control unit may control the magnet to be positioned lower than the microfluidic chip based on a control signal regarding the fixation of magnetic beads, and control the magnet to be positioned higher than the microfluidic chip based on a control signal regarding the retrieval of magnetic beads.

[0031] A cartridge for single-cell sample pretreatment according to one embodiment of the present disclosure may include a sample receiving portion comprising at least one injection vessel for receiving a sample, a microfluidic chip comprising a plurality of microwells, and a sample transfer portion connected to the sample receiving portion and the microfluidic chip, and transferring a sample inside the at least one injection vessel to an inlet of the microfluidic chip.

[0032] Additionally, the sample receiving portion may be provided in a predetermined area of ​​the upper surface of the sample transfer portion. Here, the at least one injection container may include at least one cooling injection container that contacts the cooling portion of the single-cell sample pretreatment device when the cartridge is inserted into the single-cell sample pretreatment device.

[0033] Additionally, the sample transfer unit may include at least one sample injection channel connected to each of the at least one injection vessel and a sample transfer valve connected to the at least one sample injection channel and the inlet of the microfluidic chip. Here, whether or not to transfer a sample in the at least one sample injection channel may be determined in correspondence with the operating state of the sample transfer valve.

[0034] In addition, the microfluidic chip may include a fluid channel formed inside the microfluidic chip to allow a sample to flow.

[0035] Additionally, the fluid channel may include a main fluid channel formed at the center of the upper surface of the fluid channel and auxiliary fluid channels formed on both sides of the main fluid channel.

[0036] Additionally, the fluid channel may include a plurality of microwells formed on the lower surface of the fluid channel. Here, the plurality of microwells may be formed to capture a single magnetic bead.

[0037] In addition, the microfluidic chip may be configured to come into contact with the heating portion of the single-cell sample pretreatment device when the cartridge is inserted into the single-cell sample pretreatment device.

[0038] In addition, the outlet of the microfluidic chip may be connected to the sample storage section of the single-cell sample pretreatment device and a tube when the cartridge is inserted into the single-cell sample pretreatment device.

[0039] Meanwhile, the cartridge may include a microfluidic chip cover formed integrally with the microfluidic chip and covering the microfluidic chip. Here, the sample transfer unit and the microfluidic chip can be connected as the sample transfer unit and the microfluidic chip cover are detachably coupled.

[0040] Meanwhile, the cross-sectional area of ​​the fluid channel at each of the inlet and outlet of the microfluidic chip may be smaller than the average value of the total cross-sectional area of ​​the fluid channel.

[0041] A single-cell sample pretreatment method according to one embodiment of the present disclosure comprises: (1) a priming step of treating the surface of a fluid channel; (2) a cell loading step of positioning cells inside a microwell; (3) a bead loading step of positioning magnetic beads inside a microwell; (4) a bead washing step of removing magnetic beads not positioned inside a microwell; (5) a lysis step of lysing cells using a lysis buffer; (6) a cDNA synthesis step of heating the microwell to proceed with cDNA synthesis; (7) a molecule removal step of removing residual molecules by treating with an exonuclease; and (8) a bead recovery step.

[0042] The above single-cell sample pretreatment method may be performed by a single-cell sample pretreatment device comprising: a housing; a sample receiving portion including at least one injection vessel for receiving a sample; a microfluidic chip including a plurality of microwells; a cartridge inserted into the housing, which includes a sample transfer portion connected to the sample receiving portion and the microfluidic chip and transferring a sample inside the at least one injection vessel to an inlet of the microfluidic chip; a heating portion for heating the microfluidic chip; and a magnetic force control portion for controlling the position of a magnet.

[0043] The heating unit may heat the microfluidic chip for a preset reaction time such that the temperature of the microfluidic chip becomes 37 to 42 degrees Celsius after the dissolved buffer is transferred from the injection vessel containing the dissolved buffer to the microfluidic chip and a preset dissolution time has elapsed.

[0044] The magnetic force control unit may control the magnet to be positioned lower than the microfluidic chip based on a control signal regarding the fixation of the magnetic bead, and control the magnet to be positioned higher than the microfluidic chip based on a control signal regarding the retrieval of the magnetic bead.

[0045] The heating unit may include a second temperature sensor for sensing the temperature of the microfluidic chip; an air heating module for heating air; and a blower module for discharging the heated air toward the microfluidic chip.

[0046] The above (1) priming step may include a step of treating the fluid channel surface using ethanol; and then a step of washing the fluid channel using a washing buffer.

[0047] The above (2) cell loading step may be a step in which a fluid containing cells is injected so that the cells are positioned inside the microwell by gravity.

[0048] The above (5) lysis step may be a step of injecting a lysis buffer and reacting for 1 to 5 minutes to lyse the cells.

[0049] The above (6) cDNA synthesis step may be performed at a temperature of 37 to 42 degrees Celsius for 10 to 60 minutes.

[0050] The above (7) molecular removal step may be performed at a temperature of 37 to 42 degrees Celsius for 10 to 60 minutes.

[0051] According to the present disclosure, a single-cell sample pretreatment device may be provided, comprising: a housing; a sample receiving portion including at least one injection vessel for receiving a sample; a microfluidic chip including a plurality of microwells; a sample transfer portion connected to the sample receiving portion and the microfluidic chip and transferring a sample inside at least one injection vessel to an inlet of the microfluidic chip; a cartridge inserted into the housing; a pressure control portion disposed inside the housing and connected to at least one injection vessel and controlling the pressure inside at least one injection vessel; and a sample storage portion connected to an outlet of the microfluidic chip and storing a sample discharged from the outlet of the microfluidic chip.

[0052] In addition, according to the present disclosure, the pressure control unit controls the pressure inside at least one injection vessel, thereby enabling the automatic inflow and outflow of a sample inside a microfluidic chip.

[0053] In addition, according to the present disclosure, the pretreatment efficiency and performance of a single cell can be increased by maintaining the temperature of at least one injection vessel and / or microfluidic chip within a set range.

[0054] According to the present disclosure, a cartridge may be provided comprising a sample receiving portion including at least one injection vessel for receiving a sample, a microfluidic chip including a plurality of microwells, and a sample transfer portion connected to the sample receiving portion and the microfluidic chip and transferring a sample inside at least one injection vessel to an inlet of the microfluidic chip.

[0055] According to the present disclosure, a single-cell sample pretreatment method can be provided.

[0056] In addition, according to the present disclosure, a single-cell sample pretreatment method can be provided in which the temperature of the microfluidic chip is maintained within a set range, thereby increasing the pretreatment efficiency and performance of the single cell.

[0057] In addition, according to the present disclosure, an automated single-cell sample pretreatment method can be provided.

[0058] FIG. 1 is a diagram exemplarily illustrating a flowchart of a single-cell sample pretreatment method according to one embodiment of the present disclosure.

[0059] FIGS. 2a, 2b, 2c, 2d, 2e, 2f, 2g and 2h are drawings illustrating, exemplarily, each step of a single-cell sample pretreatment method according to one embodiment of the present disclosure.

[0060] FIG. 3 is a drawing illustrating an exemplary single-cell sample pretreatment apparatus according to one embodiment of the present disclosure.

[0061] FIG. 4 is a drawing illustrating the internal configuration of a single-cell sample pretreatment device according to one embodiment of the present disclosure.

[0062] FIGS. 5A and 5B are drawings illustrating an exemplary pressure control unit according to one embodiment of the present disclosure.

[0063] FIGS. 6a and FIGS. 6b are drawings illustrating an exemplary cartridge according to one embodiment of the present disclosure.

[0064] FIGS. 7a and 7b are drawings illustrating an exemplary microfluidic chip according to one embodiment of the present disclosure.

[0065] FIG. 8a is a drawing exemplarily illustrating a cross-sectional view of a microfluidic chip according to one embodiment of the present disclosure, and FIG. 8b is a drawing exemplarily illustrating a cross-sectional view of the upper surface of a microfluidic chip according to one embodiment of the present disclosure in an inverted manner.

[0066] FIGS. 9a and 9b are drawings illustrating the fluid transport direction in a fluid channel of a microfluidic chip according to one embodiment of the present disclosure.

[0067] FIG. 10 is a drawing that exemplarily illustrates a cross-sectional view of the lower surface of a microfluidic chip according to one embodiment of the present disclosure.

[0068] FIGS. 11a and FIGS. 11b are drawings illustrating a microwell according to one embodiment of the present disclosure in an exemplary manner.

[0069] FIGS. 12a and FIGS. 12b are drawings exemplarily illustrating the flow of a sample in a fluid channel according to one embodiment of the present disclosure.

[0070] FIGS. 13a and FIGS. 13b are drawings illustrating an exemplary sample storage unit according to one embodiment of the present disclosure.

[0071] FIGS. 14a, FIGS. 14b and FIGS. 14c are drawings illustrating an optical part according to one embodiment of the present disclosure.

[0072] FIGS. 15a, FIGS. 15b, and FIGS. 15c are drawings illustrating an exemplary cooling unit according to one embodiment of the present disclosure.

[0073] FIGS. 16a, FIGS. 16b, FIGS. 16c and FIGS. 16d are drawings exemplarily illustrating a heating unit according to one embodiment of the present disclosure.

[0074] FIGS. 17a and FIGS. 17b are drawings illustrating an exemplary magnetic control unit according to one embodiment of the present disclosure.

[0075] The embodiments described herein are subject to various modifications and may have various forms; specific embodiments are illustrated in the drawings and described in detail in the detailed description. However, this is not intended to limit the scope of specific embodiments and should be understood to include various modifications, equivalents, and / or alternatives of the embodiments of the present disclosure. In relation to the description of the drawings, similar reference numerals may be used for similar components.

[0076] In describing the present disclosure, if it is determined that a detailed description of related known functions or configurations could unnecessarily obscure the essence of the present disclosure, such detailed description is omitted.

[0077] Additionally, the following embodiments may be modified in various other forms, and the scope of the technical concept of the present disclosure is not limited to the following embodiments. Rather, these embodiments are provided to make the present disclosure more faithful and complete and to fully convey the technical concept of the present disclosure to those skilled in the art.

[0078] The terms used in this disclosure are used merely to describe specific embodiments and are not intended to limit the scope of the rights. The singular expression includes the plural expression unless the context clearly indicates otherwise.

[0079] In the present disclosure, expressions such as “have,” “may have,” “include,” or “may include” indicate the presence of such features (e.g., numerical values, functions, actions, or components such as parts) and do not exclude the presence of additional features.

[0080] In the present disclosure, expressions such as “A or B,” “at least one of A or / and B,” or “one or more of A or / and B” may include all possible combinations of items listed together. For example, “A or B,” “at least one of A and B,” or “at least one of A or B” may refer to cases including (1) at least one A, (2) at least one B, or (3) both at least one A and at least one B.

[0081] Expressions such as "first," "second," "first," or "second" used in this disclosure may modify various components regardless of order and / or importance, and are used only to distinguish one component from another and do not limit said components.

[0082] Where it is stated that a component (e.g., Component 1) is "(operatively or communicatively) coupled with / to" or "connected to" another component (e.g., Component 2), it should be understood that the component may be directly connected to the other component or connected through the other component (e.g., Component 3).

[0083] On the other hand, when it is stated that a certain component (e.g., a first component) is "directly connected" or "directly coupled" to another component (e.g., a second component), it may be understood that no other component (e.g., a third component) exists between the certain component and the other component.

[0084] The expression “configured to” as used in this disclosure may be replaced, depending on the context, with, for example, “suitable for,” “having the capacity to,” “designed to,” “adapted to,” “made to,” or “capable of.” The term “configured to” may not necessarily mean only “specifically designed to” in hardware.

[0085] In the embodiment, the 'module' or 'part' performs at least one function or operation and may be implemented in hardware or software, or a combination of hardware and software.

[0086] Meanwhile, the various elements and areas in the drawings are depicted schematically. Accordingly, the technical concept of the present invention is not limited by the relative sizes or spacing depicted in the attached drawings.

[0087] Hereinafter, embodiments according to the present disclosure are described in detail with reference to the attached drawings so that those skilled in the art can easily implement them.

[0088] FIG. 1 shows a flowchart of a single-cell sample pretreatment method according to one embodiment of the present disclosure.

[0089] As shown in FIG. 1, a single-cell sample pretreatment method according to one embodiment of the present disclosure comprises: (1) a priming step of treating the surface of a fluid channel; (2) a cell loading step of positioning cells inside a microwell; (3) a bead loading step of positioning magnetic beads inside a microwell; (4) a bead washing step of removing magnetic beads not positioned inside a microwell; (5) a lysis step of lysing cells using a lysis buffer; (6) a cDNA synthesis step of heating the microwell to proceed with cDNA synthesis; (7) a molecule removal step of removing residual molecules by treating with an exonuclease; and (8) a bead recovery step.

[0090] (1) Priming step

[0091] The priming step is a step of treating the fluid channel and / or microwell. It may include a step of treating the surface of the fluid channel using ethanol; and then a step of washing the fluid channel using a washing buffer.

[0092] More specifically, as shown in FIG. 2a, 100% ethanol is injected, followed by injecting air to remove the ethanol from the fluid channel, and a first wash buffer is injected and maintained for 30 seconds to 3 minutes, preferably 1 minute, followed by injecting air to remove the first wash buffer from the fluid channel, and then injecting the first wash buffer again and maintaining for 3 to 15 minutes, preferably 10 minutes, followed by injecting air to remove the first wash buffer from the fluid channel, and then injecting a second wash buffer. Here, the reason for injecting 100% ethanol is to ensure that the wash buffer is sufficiently injected between the wells. Here, the reason for injecting the wash buffer is to prevent unnecessary binding and adsorption of substances and to remove residual ethanol. The reason for injecting the first wash buffer again is to perform surface pretreatment. For the second wash buffer, a buffer that is not toxic to cells may be used.

[0093] (2) Cell loading step

[0094] The cell loading step may be a step in which a fluid containing cells is injected so that the cells are positioned inside the microwell by gravity.

[0095] More specifically, as shown in FIG. 2b, air is injected to remove the second wash buffer, a fluid containing cells is injected, and the mixture is left for 5 to 20 minutes, preferably 15 minutes, so that the cells are positioned inside the microwell by gravity. The fluid containing cells may be a liquid or cell suspension in which the cells are suspended.

[0096] (3) Bead loading step

[0097] The bead loading step may be a bead loading step in which magnetic beads are positioned inside the microwell.

[0098] More specifically, as shown in FIG. 2c, air is injected to remove the fluid containing the cells, and a fluid containing magnetic beads is injected and left to stand for 1 to 5 minutes, preferably 3 minutes, to allow the magnetic beads to settle. If necessary, the magnetic beads can be positioned evenly inside the microwell by shaking. The fluid containing the magnetic beads may be a magnetic bead suspension.

[0099] (4) Bead washing step

[0100] The bead washing step is a step to remove magnetic beads that are not located inside the microwells.

[0101] More specifically, as shown in FIG. 2d, air is injected, CS buffer is injected, and air is injected to remove the fluid containing magnetic beads not contained in the microwell. The air injection and buffer injection can be repeated several times.

[0102] (5) Dissolution step

[0103] The lysis step is the step of lysing cells using a lysis buffer.

[0104] More specifically, as shown in FIG. 2e, a lysis buffer is injected and left for 1 to 5 minutes, preferably 2 minutes, to lyse the cells. At this time, a magnetic bead can be fixed using a magnet located at the bottom of the microwell. The magnet can prevent the magnetic bead from detaching from the microwell during the cell lysis process. After lysis, a wash buffer is injected for 1 to 5 minutes, preferably 1 minute, to remove the lysis buffer, and then air is injected to remove the wash buffer.

[0105] (6) cDNA synthesis step

[0106] The cDNA synthesis step is the step of proceeding with cDNA synthesis by heating the microwell.

[0107] More specifically, as shown in FIG. 2f, cDNA synthesis proceeds while the mRNA of the lysed cells is captured by magnetic particles. At this time, the process can be carried out at a temperature of 37 degrees Celsius or higher. For example, it is carried out for 10 to 60 minutes at a temperature of 37 to 80 degrees Celsius, preferably 37 to 55 degrees Celsius, and more preferably 37 to 42 degrees Celsius. Heating is performed by a heating unit included in a single-cell sample pretreatment device.

[0108] (7) Molecular removal step

[0109] The molecule removal step is a step of removing residual molecules by treating with an exonuclease.

[0110] More specifically, as shown in FIG. 2g, the step involves removing small-sized nucleic acids remaining after cDNA synthesis using an exonuclease. At this time, the process is performed at a temperature of 37 to 42 degrees Celsius for 10 to 60 minutes, preferably at 37 degrees Celsius for 30 minutes. Heating is performed by a heating unit included in a single-cell sample pretreatment device. After molecular removal, the exonuclease can be removed by washing.

[0111] (8) Bead recovery step

[0112] The bead recovery step is a step of recovering magnetic beads in which mRNA and cDNA have been captured.

[0113] More specifically, as shown in FIG. 2h, magnetic beads are floated by a magnet located at the top of the microwell, and bead wash buffer is injected to recover the magnetic beads. The mRNA and cDNA obtained from the recovered beads can be used for single-cell multi-omics analysis.

[0114] Below, a single-cell sample pretreatment device used in the single-cell sample pretreatment method is described.

[0115] FIG. 3 is a drawing exemplarily illustrating a single-cell sample pretreatment device (10) according to one embodiment of the present disclosure. FIG. 4 is a drawing exemplarily illustrating the internal configuration of a single-cell sample pretreatment device (10) according to one embodiment of the present disclosure.

[0116] Referring to FIGS. 3 and 4, a single-cell sample pretreatment device (10) according to one embodiment of the present disclosure may include a housing (100), a cartridge (300), a pressure control unit (200), and a sample storage unit (400). Additionally, in some cases, the single-cell sample pretreatment device (10) may further include at least one of an input interface and a communication interface.

[0117] The housing (100) forms the outer shape of the single-cell sample pretreatment device (10). Here, an opening (101) may be formed in the housing (100).

[0118] For example, a cartridge (300) can be inserted into the opening (101).

[0119] For example, a microfluidic chip (320) and / or a microfluidic chip cover (340) included in a cartridge (300) may be inserted into the opening (101).

[0120] However, the opening (101) in the housing (100) does not necessarily have to be formed, and it is also possible to insert the cartridge (300) into the single-cell sample pretreatment device (10) by disassembling the housing (100).

[0121] Additionally, a manual injection channel may be separately formed in the housing (100) for a user to manually inject a sample. Here, the manual injection channel may be connected to a cartridge (300) along a tube. Meanwhile, the manual injection channel may be connected to an injection port (312) of the cartridge (300).

[0122] The cartridge (300) is configured to react the sample in the fluid channel (350) of the microfluidic chip (320). The cartridge (300) may be provided so as to be detachably attached to the single-cell sample pretreatment device (10), but may also be formed fixedly.

[0123] The cartridge (300) may include a sample receiving section (310), a microfluidic chip (320), and a sample transfer section (330). Here, the sample receiving section (310) is configured to include at least one injection container (311) for receiving a sample. The microfluidic chip (320) is configured to include a plurality of microwells (353). Additionally, the sample transfer section (330) is configured to transfer a sample inside at least one injection container (311) to an inlet (321) of the microfluidic chip (320).

[0124] The cartridge (300) will be described in detail below in FIG. 6.

[0125] The pressure control unit (200) is configured to control the pressure inside at least one injection container (311). To this end, the pressure control unit (200) may be connected to at least one injection container (311) and may be placed inside the housing (100).

[0126] The pressure control unit (200) will be described in detail later in Fig. 5.

[0127] The sample storage unit (400) is configured to store a sample discharged from the outlet (322) of the microfluidic chip (320). To this end, the sample storage unit (400) can be connected to the outlet (322) of the microfluidic chip (320).

[0128] The sample storage unit (400) will be described in detail later in FIG. 13a and FIG. 13b.

[0129] The input interface may include a button, a lever, a switch, a touch interface, etc., and the touch interface may be implemented in a way that receives input by touching the user on a display screen.

[0130] The communication interface may include a wireless communication interface, a wired communication interface, or an input interface. The wireless communication interface may perform communication with various external devices using wireless communication technology or mobile communication technology. Such wireless communication technologies may include, for example, Bluetooth, Bluetooth Low Energy, CAN communication, Wi-Fi, Wi-Fi Direct, ultrawide band (UWB), Zigbee, infrared data association (IrDA), or near field communication (NFC), and mobile communication technologies may include 3GPP, Wi-Max, LTE (Long Term Evolution), 5G, etc. The wireless communication interface may be implemented using an antenna, a communication chip, a substrate, etc., capable of transmitting electromagnetic waves to the outside or receiving electromagnetic waves transmitted from the outside.

[0131] A wired communication interface can communicate with various external devices based on a wired communication network. Here, the wired communication network can be implemented using physical cables, such as, for example, pair cables, coaxial cables, fiber optic cables, or Ethernet cables.

[0132] Depending on the embodiment, either the wireless communication interface or the wired communication interface may be omitted. Accordingly, the single-cell sample pretreatment device (10) may include only a wireless communication interface or only a wired communication interface. In addition, the single-cell sample pretreatment device (10) may be equipped with an integrated communication interface that supports both wireless connection via the wireless communication interface and wired connection via the wired communication interface.

[0133] The single-cell sample pretreatment device (10) is not limited to having one communication interface that performs a communication connection in one manner, but may include multiple communication interfaces that perform communication connections in multiple manners.

[0134] The lower components of the single-cell sample pretreatment device (10) according to various embodiments of the present disclosure (e.g., pressure control unit (200), cartridge (300), sample storage unit (400), optical unit (500), cooling unit (600), heating unit (700) and magnetic control unit (800), etc.) can communicate with an external electronic device or server through a communication interface.

[0135] FIGS. 5A and FIGS. 5B are drawings illustrating an exemplary pressure control unit (200) according to one embodiment of the present disclosure.

[0136] Referring to FIG. 5a and FIG. 5b, a pressure control unit (200) according to one embodiment of the present disclosure can control the pressure inside at least one injection container (311).

[0137] Specifically, at least one injection container (311) may be at least one container for receiving a sample. Here, the at least one injection container (311) may be implemented as a vial, pipette, burette, flask, syringe, etc., and is not limited to the examples described above. Also, the material of the at least one injection container (311) may be acrylic, plastic, glass, etc., but is not limited to the examples described above, and various materials may be used.

[0138] A sample may be contained in at least one injection container (311). This sample may be injected into at least one injection container (311) in advance prior to the operation of the single-cell sample pretreatment device (10). However, the sample does not necessarily have to be injected into at least one injection container (311) in advance prior to the operation of the single-cell sample pretreatment device (10), and may be supplied from an external system (e.g., a fluid supply device) through at least one pipe connected to the inlet of each of the at least one injection container (311).

[0139] The sample that can be contained in at least one injection container (311) may include a fluid sample such as a liquid, a gas, or a mixture of a liquid and a fine powder (e.g., magnetic bead powder).

[0140] For example, the sample may include a washing solution, cell buffer, cell lysate, magnetic bead (M) buffer, cell capture magnetic bead (M), nucleic acid capture magnetic bead, ethanol, air, etc. However, the sample according to the present disclosure is not limited to the examples described above, and various samples for achieving the purpose of the present disclosure may be included herein.

[0141] A pressure control unit (200) according to one embodiment of the present disclosure can control at least one injection container valve connected to the inlet of each of at least one injection container (311). Here, the injection container valve can be connected to the inlet of at least one injection container (311).

[0142] For example, the injection container valve can be connected to the inlet of at least one injection container (311).

[0143] For example, the injection container valves may be provided in the same number as at least one container.

[0144] For example, the injection vessel valve may be an electronic valve. As an example, the injection vessel valve may be a solenoid valve.

[0145] For example, the pressure control unit (200) may include a syringe pump, an air pressure control module, a vacuum control module, and a valve control module. Here, the syringe pump, the air pressure control module, the vacuum control module, and the valve control module can control the supply of a selected sample from the sample inside the injection container (311) to the microfluidic chip (320) by controlling the pressure inside at least one injection container (311) or by controlling the operating state of the injection container valve.

[0146] However, at least one of the syringe pump, the main force control module, the vacuum control module, and the valve control module in the pressure control unit (200) may be omitted, and the detailed configuration of the pressure control unit (200) described above should be considered merely as an example.

[0147] FIGS. 6a and FIGS. 6b are drawings illustrating an exemplary cartridge (300) according to one embodiment of the present disclosure.

[0148] The cartridge (300) is configured to perform pretreatment of a sample in a single-cell sample pretreatment device (10).

[0149] Specifically, referring to FIG. 6a and FIG. 6b, a cartridge (300) according to one embodiment of the present disclosure may include a sample receiving portion (310), a microfluidic chip (320), and a sample transfer portion (330).

[0150] The sample receiving portion (310) is configured to receive a sample. The sample receiving portion (310) may include at least one injection container (311). Additionally, the sample receiving portion (310) may further include an injection container cover for covering at least one injection container (311).

[0151] At least one injection vessel (311) may include at least one cooling injection vessel (311). At least one cooling injection vessel (311) may be cooled by a cooling unit (600) to be described later.

[0152] For example, referring to FIGS. 6a and 6b, at least one injection container (311) may be provided in a vertical direction. Also, at least one injection container (311) may be guided and positioned by an injection container cover.

[0153] For example, at least one cooling injection container (311) may be spaced apart from the injection container (311) excluding at least one cooling injection container (311). To this end, a first injection container cover covering the injection container (311) excluding the cooling injection container (311) may be spaced apart from a second injection container cover covering the cooling injection container (311).

[0154] For example, referring to FIGS. 6a and FIG. 6b, at least one injection vessel (311) may be 12. And, at least one cooling injection vessel (311) may be 8. However, the number of injection vessels (311) or cooling injection vessels (311) is not limited to the examples described above.

[0155] Referring to FIGS. 6a and 6b, the sample receiving portion (310) may be provided in a predetermined area of ​​the upper surface of the sample transfer portion (330) to be described later. In addition, at least one cooling injection container (311) may come into contact with the cooling portion (600) of the single-cell sample pretreatment device (10) when the cartridge (300) is inserted into the single-cell sample pretreatment device (10). As a result, at least one cooling injection container (311) may be cooled by the cooling portion (600).

[0156] Referring to FIG. 6a, at least one outlet of at least one injection container (311) may be formed at the bottom of each of the injection containers (311). Here, each outlet of at least one injection container (311) may be connected to a sample transport unit (330). For example, each outlet of at least one injection container (311) may be connected to a sample injection channel (331) to be described later.

[0157] The sample transfer unit (330) is configured to transfer a sample inside at least one injection container (311) to the inlet (321) of the microfluidic chip (320). To this end, the sample transfer unit (330) may be connected to the sample receiving unit (310) and the microfluidic chip (320).

[0158] Specifically, the sample transfer unit (330) may include a sample injection channel (331) and a sample transfer valve (332). In some cases, the sample transfer unit (330) may further include a sample transfer unit cover forming an outer shape.

[0159] The sample injection channel (331) is an injection channel connected to each of at least one injection container (311). For example, the sample injection channel (331) may be provided as a tubular member. For example, the sample injection channel (331) may be an injection channel formed inside the sample transport cover.

[0160] Referring to FIGS. 6a and 6b, the sample injection channel (331) can connect the outlet of at least one injection vessel (311) to the sample transfer valve (332).

[0161] The sample transfer valve (332) is a valve for selectively transferring a sample from at least one sample injection channel (331) to the inlet (321) of the microfluidic chip (320). To this end, the sample transfer valve (332) may be connected to at least one sample injection channel (331) and the inlet (321) of the microfluidic chip (320). As an example, the sample transfer valve (332) may be a rotary valve.

[0162] In addition, whether a sample is transferred in at least one sample injection channel (331) can be determined in response to the operating state of the sample transfer valve (332).

[0163] Meanwhile, the pressure control unit (200) described above can be connected to the sample transfer unit (330) when the cartridge (300) is inserted into the housing (100). Here, the pressure control unit (200) can control the operating state of the sample transfer valve (332).

[0164] The pressure control unit (200) can obtain a control signal (e.g., user input) regarding the selection of an injection container (311) containing a sample to be supplied to the microfluidic chip (320) through an input interface or a communication interface.

[0165] And, the pressure control unit (200) can control the injection vessel valve so that the pressure inside the selected injection vessel (311) increases based on a control signal regarding the selection of the injection vessel (311) containing the sample to be supplied to the microfluidic chip (320).

[0166] Additionally, the pressure control unit (200) can control the sample transfer valve (332) so that the sample moves along the sample injection channel (331) connected to the selected injection vessel (311) to the inlet (321) of the microfluidic chip (320).

[0167] For example, if the pressure control unit (200) controls the injection vessel valve to increase the internal pressure of the selected injection vessel (311) based on a control signal, the sample inside the injection vessel (311) can move along the sample injection channel (331) to the sample transfer valve (332). Here, the pressure control unit (200) can control the sample transfer valve (332) so that the sample transfer valve (332) opens to the corresponding sample injection channel (331). Thus, the sample can move from the sample injection channel (331) connected to the selected injection vessel (311) to the microfluidic chip (320) along the sample transfer valve (332).

[0168] Meanwhile, referring to FIGS. 6a and 6b, the sample transfer valve (332) may include a first sample transfer valve (332a) and a second sample transfer valve (332b). Here, the first sample transfer valve (332a) is a valve connected to the sample transfer channel described above, and the second sample transfer valve (332b) is a valve connected from a manual injection channel formed in the housing (100). For example, the manual injection channel may be connected to an injection port (312) of a cartridge (300), and the injection port (312) may be connected to the second sample transfer valve (332b).

[0169] However, the sample transfer valve (332) does not necessarily have to include two sample transfer valves (i.e., the first sample transfer valve (332a) and the second sample transfer valve (332b)), and may be composed of a single sample transfer valve (332). For example, the second sample transfer valve (332b) may be omitted.

[0170] FIGS. 7a and FIGS. 7b are drawings illustrating an exemplary microfluidic chip (320) according to one embodiment of the present disclosure.

[0171] Referring to FIG. 7a and FIG. 7b, a microfluidic chip (320) according to one embodiment of the present disclosure may include a fluid channel (350) formed inside the microfluidic chip (320) to allow a sample to flow.

[0172] Additionally, the microfluidic chip (320) may include an inlet (321) and an outlet (322) connected to a fluid channel (350).

[0173] Specifically, the inlet (321) of the microfluidic chip (320) is an inlet (321) of the microfluidic chip (320) for moving a sample to the microfluidic chip (320), and the outlet (322) of the microfluidic chip (320) is an outlet (322) for discharging a sample from the microfluidic chip (320).

[0174] The inlet (321) of the microfluidic chip (320) can be connected to a sample transfer valve (332). For example, the inlet (321) of the microfluidic chip (320) can be connected to a sample transfer valve (332) along a pipe.

[0175] Additionally, the outlet (322) of the microfluidic chip (320) may be connected to a sample storage unit (400). Here, the sample storage unit (400) is configured to store a sample discharged from the outlet (322) of the microfluidic chip (320).

[0176] For example, the outlet (322) of the microfluidic chip (320) can be connected to the sample storage unit (400) of the single-cell sample pretreatment device (10) by a tube when the cartridge (300) is inserted into the single-cell sample pretreatment device (10).

[0177] Meanwhile, the microfluidic chip (320) may be formed from materials such as silicon, glass, polydimethylsiloxane (PDMS), polymethylmethacrylate (PMMA), and polycarbonate. However, the material of the microfluidic chip (320) is not limited to the examples described above, and various materials may be used within the scope of achieving the purpose of the present disclosure.

[0178] Meanwhile, referring to FIG. 7b, the cross-sectional area of ​​the fluid channel (350) at each of the inlet (321) and outlet (322) of the microfluidic chip (320) may be smaller than the average value of the total cross-sectional area of ​​the fluid channel (350).

[0179] Accordingly, according to the present disclosure, the cross-sectional area of ​​the fluid channel (350) becomes smaller as it moves toward the center along the length of the fluid channel (350), so that observation of the microfluidic chip (320) (e.g., the central region of the microfluidic chip (320)) can be made easier. Additionally, if the cross-sectional area of ​​the fluid channel (350) in the central region of the microfluidic chip (320) is relatively larger than the cross-sectional area in the inlet (321) and outlet (322) regions, the movement speed of the sample in the central region of the microfluidic chip (320) can be relatively slower according to the continuity equation, and more effective pretreatment can be achieved.

[0180] FIG. 8a is a diagram exemplarily illustrating a cross-sectional view of a microfluidic chip (320) according to one embodiment of the present disclosure, and FIG. 8b is a diagram exemplarily illustrating a cross-sectional view of the upper surface of a microfluidic chip (320) according to one embodiment of the present disclosure in an inverted manner.

[0181] Referring to FIGS. 8a and 6b, a microfluidic chip (320) according to one embodiment of the present disclosure may be formed by joining an upper substrate and a lower substrate. However, the microfluidic chip (320) according to the present disclosure does not necessarily have to be formed by joining an upper substrate and a lower substrate, and may be formed integrally from the time of manufacturing.

[0182] Referring to FIG. 8b, the fluid channel (350) may include a main fluid channel (351) and an auxiliary fluid channel (352). Here, the main fluid channel (351) is a fluid channel (350) formed in the center of the upper surface of the fluid channel (350). And, the auxiliary fluid channel (352) is a fluid channel (350) formed on both sides of the main fluid channel (351).

[0183] For example, the auxiliary fluid channel (352) may be an additional gap-shaped fluid channel (350) located at the bottom of the wall of the fluid channel (350) and may be formed to allow capillary action to occur. Here, the sample first flows into the auxiliary fluid channel (352) by means of capillary action, and then, the sample flowing in the auxiliary fluid channel (352) is pulled into the main fluid channel (351), allowing the sample to flow from the inlet (321) toward the outlet (322) inside the fluid channel (350). To this end, the auxiliary fluid channel (352) may be formed lower than the height of the main fluid channel (351).

[0184] FIGS. 9a and 9b are drawings illustrating the fluid transport direction in a fluid channel (350) of a microfluidic chip (320) according to one embodiment of the present disclosure.

[0185] Referring to FIGS. 9a and 9b, a sample can flow within the fluid channel (350) in such a way that the sample first flows along the auxiliary fluid channel (352) by capillary action, and then the sample flowing into the auxiliary fluid channel (352) draws the sample into the main fluid channel (351).

[0186] FIG. 10 is a drawing that exemplarily illustrates a cross-sectional view of the lower surface of a microfluidic chip (320) according to one embodiment of the present disclosure.

[0187] Referring to FIG. 10, the fluid channel (350) may include a plurality of microwells (353) formed on the lower surface of the fluid channel (350).

[0188] Here, a plurality of microwells (353) can be formed to capture a single magnetic bead (M). For example, more than 1,000 microwells (353) can be formed in the lower part of the fluid channel (350).

[0189] FIGS. 11a and FIGS. 11b are drawings illustrating an exemplary microwell (353) according to one embodiment of the present disclosure.

[0190] Referring to FIG. 11a and FIG. 11b, a microwell (353) according to one embodiment of the present disclosure may be formed in a repeating pattern of a plurality of microwells (353). However, the plurality of microwells (353) do not necessarily have to be formed in a repeating pattern and may be formed in various ways.

[0191] Referring to FIG. 11b, one magnetic bead (M) can be placed in one microwell (353). To this end, the cross-sectional area of ​​the microwell (353) in the vertical direction (e.g., the direction of gravity) can be formed to correspond to the size of the magnetic bead (M).

[0192] FIGS. 12a and FIGS. 12b are drawings exemplarily illustrating the flow of a sample in a fluid channel (350) according to one embodiment of the present disclosure.

[0193] Referring to FIG. 12a, a sample (e.g., a cleaning solution) can flow inside a fluid channel (350).

[0194] Referring to FIG. 12b, a sample (e.g., a solution containing magnetic beads (M)) can flow within a fluid channel (350). Here, the magnetic beads (M) in the solution can be captured in a microwell (353). This capture of the magnetic beads (M) can be achieved by gravity, but it can also be achieved by controlling the direction of movement of the magnetic beads (M) by magnetic control by a magnetic control unit (800) described later.

[0195] Referring to Fig. 12b, some of the magnetic beads (M) can capture cells and enter the microwell (353).

[0196] Referring again to FIG. 11b, it can be seen that the magnetic bead (M) in FIG. 12b is captured in the microwell (353), as depicted in FIG. 11b.

[0197] Referring again to FIGS. 6a and 6b, the microfluidic chip (320) may be configured to come into contact with the heating part (700) of the single-cell sample pretreatment device (10) when the cartridge (300) is inserted into the single-cell sample pretreatment device (10).

[0198] The heating unit (700) can heat the microfluid chip (320) for a preset reaction time such that the temperature of the microfluid chip (320) becomes 37 to 42 degrees Celsius. Details regarding the heating unit (700) will be described later in FIG. 16a and below.

[0199] Meanwhile, referring again to FIGS. 6a and 6b, the cartridge (300) may include a microfluidic chip cover (340).

[0200] The microfluidic chip cover (340) is formed integrally with the microfluidic chip (320) and is configured to cover the microfluidic chip (320). Specifically, the microfluidic chip cover (340) may include a housing (100) that covers the microfluidic chip (320) and a coupling part for detachably coupling the microfluidic chip (320) and the sample transfer part (330), but the coupling part may be omitted.

[0201] Specifically, the sample transfer unit (330) and the microfluidic chip cover (340) can be connected as they are detachably coupled. For example, the sample transfer unit (330) and the microfluidic chip cover (340) may be coupled using a snap-fit ​​method, a gasket method, etc. However, the coupling method is not limited to the examples described above, and any mechanical or electronic coupling method that can be employed by a person skilled in the art may be included.

[0202] Additionally, the microfluidic chip cover (340) and the sample transport unit (330) may be formed integrally. For example, the microfluidic chip cover (340) and the sample transport unit (330) may be formed integrally through ultrasonic welding, thermal welding, etc. In this case, the microfluidic chip (320) may also be formed integrally with the sample transport unit (330).

[0203] For example, the microfluidic chip cover (340) may not cover the microfluidic chip (320) in the area corresponding to the central region of the fluid channel (350) (i.e., the area where the cross-sectional area of ​​the fluid channel (350) is larger than the cross-sectional area at the inlet (321) and outlet (322).

[0204] In another example, the microfluidic chip cover (340) may include a window formed in an area corresponding to the central region of the fluid channel (350) (i.e., an area where the cross-sectional area of ​​the fluid channel (350) is larger than the cross-sectional area at the inlet (321) and outlet (322).

[0205] Accordingly, according to the present disclosure, the fluid channel (350) of the microfluidic chip (320) can be observed while preserving the detachable feature of the microfluidic chip (320).

[0206] FIGS. 13a and FIGS. 13b are drawings illustrating an exemplary sample storage unit (400) according to one embodiment of the present disclosure.

[0207] A single-cell sample pretreatment device (10) according to one embodiment of the present disclosure may include a sample storage unit (400).

[0208] The sample storage unit (400) may include at least one storage container (410), a rotating member (420), and a rotating drive module (430).

[0209] At least one storage container (410) is at least one container for storing a sample discharged from the outlet (322) of the microfluidic chip (320). As an example, at least one storage container (410) may include three or more storage containers (410) of different capacities. As an example, at least one storage container (410) may include a reagent tube for storing a sample.

[0210] The rotating member (420) is a member to which at least one storage container (410) is detachably coupled. For example, the rotating member (420) may be a revolver type or a turret type member.

[0211] The rotary drive module (430) is a drive module that drives the rotary member (420). The rotary drive module (430) may include components such as a motor and a motor control unit.

[0212] For example, the rotary drive module (430) can obtain a control signal regarding the selection of a storage container (410) for storing a sample discharged through an input interface or a communication interface.

[0213] And, the rotary drive module (430) can drive the rotary member (420) so that the selected storage container (410) is connected to the discharge port (322) based on a control signal regarding the selection of a storage container (410) for storing the discharged sample.

[0214] For a specific example, the rotary drive module (430) can drive the rotary member (420) based on a control signal regarding the storage container (410) (e.g., a control signal regarding the selection of a 10 ml reagent recovery container) so that the storage container (410) can be connected to the outlet (322) of the microfluidic chip (320) via a tube.

[0215] As an example, the rotary drive module (430) can change the position of the selected storage container (410) by rotating the rotary member (420) with respect to a vertical rotation axis.

[0216] However, the sample storage unit (400) according to the present disclosure does not necessarily have to include a rotation drive module (430), and a method in which the rotation member (420) rotates by manual operation by a user is also possible. In other words, the sample storage unit (400) according to the present disclosure is not limited to the components described above, and some components may be omitted.

[0217] FIGS. 14a, FIGS. 14b and FIGS. 14c are drawings illustrating an optical part (500) according to one embodiment of the present disclosure.

[0218] Referring to FIG. 14a, FIG. 14b and FIG. 14c, a single-cell sample pretreatment device (10) according to one embodiment of the present disclosure may include an optical unit (500).

[0219] The optical unit (500) is configured to photograph a plurality of microwells (353) of the cartridge (300). To this end, the optical unit (500) may include a light source (510), an objective lens (520), and an image sensor (530). However, some of these components may be omitted. Meanwhile, the optical unit (500) may further include a separate aperture, a condenser, and a focal length control module.

[0220] Referring to FIG. 14c, the light source (510) is positioned lower than the cartridge (300) and is configured to irradiate light in a direction toward the cartridge (300).

[0221] Referring to FIG. 14c, the objective lens (520) is a lens through which light emitted from the light source (510) and passed through the cartridge (300) is transmitted. Meanwhile, the objective lens (520) may be provided to be detachable.

[0222] Referring to FIG. 14c, the image sensor (530) is configured to sense light that has passed through the objective lens (520). However, the image sensor (530) may also sense light that has passed through the cartridge (300) directly, rather than light that has passed through the objective lens (520).

[0223] Referring to FIG. 14c, as an example, the optical unit (500) can be configured in a transmission microscope manner.

[0224] As an example, the optical unit (500) can be configured in a reflection microscope manner.

[0225] Referring again to FIG. 11a and FIG. 11b, an image of the fluid channel (350) captured by the optical unit (500) can be seen.

[0226] For example, the optical unit (500) can capture at least one process among the injection, movement, settling, separation, residue removal, and sample recovery of the cell and magnetic bead (M).

[0227] For example, the optical unit (500) can capture at least one of the cell lysis process and the nucleic acid capture process.

[0228] For example, the optical unit (500) can capture the microfluidic chip (320) in real time. The captured image can be stored in the memory (not shown) of the single-cell preprocessing device. Here, the memory stores various programs or data temporarily or indefinitely and transmits the stored information to the processor upon the processor's call. Additionally, the memory can store various information required for the processor's computation, processing, or control operations in an electronic format. Here, the processor may be a processor included in the single-cell sample preprocessing device (10) or a processor separately provided inside the optical unit (500).

[0229] FIGS. 15a, FIGS. 15b and FIGS. 15c are drawings illustrating an exemplary cooling unit (600) according to one embodiment of the present disclosure.

[0230] Referring to FIG. 15a, FIG. 15b and FIG. 15c, a single-cell sample pretreatment device (10) according to one embodiment of the present disclosure may include a cooling unit (600).

[0231] The cooling unit (600) is configured to cool at least one cooling injection container (311) that requires cooling among at least one injection container (311).

[0232] A cooling unit (600) according to one embodiment of the present disclosure may include a first temperature sensor, a Peltier module (610), and a cooling fan (620). However, some of these components may be omitted.

[0233] The first temperature sensor is a sensor that senses the temperature of at least one cooling injection container (311).

[0234] For example, the first temperature sensor may be implemented as at least one of a resistance thermometer (RTD), a thermocouple, a thermistor, a semiconductor temperature sensor, and an infrared temperature sensor.

[0235] The Peltier module (610) is configured to cool at least one cooling injection container (311). To this end, the Peltier module (610) may come into contact with at least one cooling injection container (311). Here, the Peltier module (610) is a device that utilizes the thermoelectric effect, in which heat is absorbed or released at the junction when current flows through two different types of semiconductor materials joined together, and can be used primarily for cooling and temperature control.

[0236] For example, referring to FIG. 15c, the Peltier module (610) may be in contact with at least one cooling injection container (311) on two sides. Here, the contact between the Peltier module (610) and the cooling injection container (311) should be interpreted to include not only direct contact between the surfaces of the two members, but also indirect contact with a second injection container cover placed between them. In other words, the Peltier module (610) may be connected to at least one cooling injection container (311) on two sides.

[0237] Referring to FIG. 6b and FIG. 15c as a specific example, the Peltier module (610) may include a first Peltier member and a second Peltier member. The first Peltier member may be positioned between the first injection container cover and the second injection container cover to cool the second injection container cover. The second Peltier member may be positioned on the side of the second injection container cover to cool the second injection container cover. Meanwhile, the first Peltier member may be positioned spaced apart from the first injection container cover (i.e., without contact). Thus, cooling of the injection container, which does not require cooling, can be prevented due to the first Peltier member.

[0238] The cooling fan (620) is a blower for discharging heat generated by a heat source connected to the Peltier module (610).

[0239] For example, the cooling fan (620) may be a means of blowing air to expel heat generated by the Peltier module (610) to the outside. Specifically, the cooling fan (620) can increase the cooling efficiency of the Peltier module (610) and support stable operation for a long time.

[0240] FIGS. 16a, FIGS. 16b, FIGS. 16c and FIGS. 16d are drawings illustrating an exemplary heating unit (700) according to one embodiment of the present disclosure.

[0241] A single-cell sample pretreatment device (10) according to one embodiment of the present disclosure may include a heating unit (700).

[0242] The heating unit (700) is configured to heat the microfluidic chip (320).

[0243] A heating unit (700) according to one embodiment of the present disclosure may include a second temperature sensor, an air heating module, and a blower module. However, some of these components may be omitted. For example, the air heating module and the blower module may be omitted, and the heating unit (700) may include a conductive heating module (for example, a heating module that heats the microfluidic chip (320) by using heat conduction in contact with the microfluidic chip (320)).

[0244] The second temperature sensor is a sensor that senses the temperature of the microfluidic chip (320). For example, the second temperature sensor may be implemented as at least one of a resistance thermometer (RTD), a thermocouple, a thermistor, a semiconductor temperature sensor, and an infrared temperature sensor.

[0245] An air heating module is a heating module for heating air. For example, the air heating module may be implemented as at least one of an electric heater module, a heat exchanger module, a heat pump module, an infrared heater module, and a gas heater module.

[0246] The blower module is configured to discharge heated air in the direction of the microfluidic chip (320). For example, the blower module may be implemented as at least one of an axial fan module, a centrifugal fan module, a crossflow fan module, a turbo fan module, and a blower fan module.

[0247] Meanwhile, the heating unit (700) can control the temperature of the microfluidic chip (320) within a preset temperature range.

[0248] For example, the heating unit (700) can heat the microfluid chip (320) for a preset reaction time such that the temperature of the microfluid chip (320) becomes 37 to 42 degrees Celsius after the dissolved buffer is transferred from the injection container (311) containing the dissolved buffer to the microfluid chip (320) and a preset dissolution time has elapsed.

[0249] For a specific example, the heating unit (700) can control the temperature of the microfluidic chip (320) to 37 degrees for 30 minutes to support cDNA synthesis proceeding while maintaining the position without recovering the magnetic beads (M) after cell lysis and mRNA capture.

[0250] And, when a sample containing a nuclease (e.g., exonuclease I) is supplied to the microfluidic chip (320), the temperature of the microfluidic chip (320) can be controlled to 37 degrees for 30 minutes to support a reaction taking place at 37 degrees Celsius inside the microfluidic chip (320).

[0251] And, when the reaction of the nuclease enzyme is finished and the washing solution is supplied to the microfluidic chip (320), a washing process can be performed.

[0252] Accordingly, according to the present disclosure, since the temperature of the microfluidic chip (320) can be controlled within a preset temperature range during the process in which a reaction takes place inside the microfluidic chip (320), the efficiency of sample pretreatment can be improved.

[0253] FIGS. 17a and FIGS. 17b are drawings illustrating an exemplary magnetic control unit (800) according to one embodiment of the present disclosure.

[0254] A single-cell sample pretreatment device (10) according to one embodiment of the present disclosure may include a magnetic control unit (800).

[0255] The magnetic force control unit (800) is configured to control the position of the magnet (810). Here, the magnet (810) may include at least one of a permanent magnet, a temporary magnet, an electromagnet, a ferrite magnet, and a neodymium magnet. However, the type of magnet (810) is not limited to the examples described above, and various magnets (810) may be used within the range that can achieve the purpose of the present disclosure.

[0256] The magnetic control unit (800) may include a magnetic driving module (820).

[0257] Specifically, referring to FIG. 17b, the magnetic drive module (820) may include a horizontal movement motor (821), a vertical movement motor (822), and an eccentric disc (830).

[0258] The horizontal movement motor (821) can control the movement of the magnet (810) in the horizontal direction. And, the vertical movement motor (822) can control the movement of the magnet (810) in the vertical direction. To this end, the vertical movement motor (822) can drive the eccentric disc (830). Here, the eccentric disc (830) is connected to the magnet (810), so that the height of the magnet (810) in the vertical direction can be determined according to the rotation of the eccentric disc (830).

[0259] The magnetic control unit (800) can obtain a control signal (e.g., user input) regarding the fixation of the magnetic bead (M) through an input interface or a communication interface.

[0260] And, referring to FIG. 17b, the magnetic control unit (800) can control the magnet (810) to be positioned lower than the microfluidic chip (320) based on a control signal regarding the fixation of the magnetic bead (M). When the magnet (810) is positioned lower than the microfluidic chip (320), the magnetic bead (M) moves to the lower surface of the fluid channel (350), so that the magnetic bead (M) can be captured in the microwell (353).

[0261] Additionally, referring to FIG. 17b, the magnetic control unit (800) can control the magnet (810) to be positioned higher than the microfluidic chip (320) based on a control signal regarding the retrieval of the magnetic bead (M). When the magnet (810) is positioned higher than the microfluidic chip (320), the magnetic bead (M) moves to the upper surface of the fluid channel (350), so that the magnetic bead (M) that was captured in the microwell (353) can move out of the microwell (353).

[0262] At least one of the above-described pressure control unit (200), cartridge (300), sample storage unit (400), optical unit (500), cooling unit (600), heating unit (700), and magnetic force control unit (800) may include a processor. Additionally, at least one of the pressure control unit (200), cartridge (300), sample storage unit (400), optical unit (500), cooling unit (600), heating unit (700), and magnetic force control unit (800) may be controlled by a separate processor included in the single-cell sample pretreatment device (10).

[0263] Specifically, the processor is connected to the configuration of an electronic device including memory as described above, and can control the overall operation of the electronic device by executing at least one instruction stored in the memory as described above. In particular, the processor can be implemented as a single processor as well as as a plurality of processors.

[0264] A processor may be implemented in various ways. For example, one or more processors may include one or more of a CPU (Central Processing Unit), GPU (Graphics Processing Unit), APU (Accelerated Processing Unit), MIC (Many Integrated Core), DSP (Digital Signal Processor), NPU (Neural Processing Unit), hardware accelerator, or machine learning accelerator. One or more processors may control one or any combination of other components of an electronic device and may perform operations or data processing related to communication. One or more processors may execute one or more programs or instructions stored in memory. For example, one or more processors may perform a method according to one embodiment of the present disclosure by executing one or more instructions stored in memory.

[0265] When a method according to one embodiment of the present disclosure includes a plurality of operations, the plurality of operations may be performed by a single processor or by a plurality of processors. For example, when a first operation, a second operation, and a third operation are performed by a method according to one embodiment, the first operation, the second operation, and the third operation may all be performed by a first processor, or the first operation and the second operation may be performed by a first processor (e.g., a general-purpose processor) and the third operation may be performed by a second processor (e.g., an artificial intelligence dedicated processor).

[0266] One or more processors may be implemented as a single-core processor comprising one core, or as one or more multicore processors comprising multiple cores (e.g., homogeneous multicore or heterogeneous multicore). When one or more processors are implemented as multicore processors, each of the multiple cores included in the multicore processor may include internal processor memory such as on-chip memory, and a common cache shared by multiple cores may be included in the multicore processor. Additionally, each of the multiple cores included in the multicore processor (or some of the multiple cores) may independently read and execute program instructions for implementing a method according to one embodiment of the present disclosure, or all (or some) of the multiple cores may be linked together to read and execute program instructions for implementing a method according to one embodiment of the present disclosure.

[0267] When a method according to one embodiment of the present disclosure includes a plurality of operations, the plurality of operations may be performed by one of the plurality of cores included in a multi-core processor, or may be performed by a plurality of cores. For example, when a first operation, a second operation, and a third operation are performed by a method according to one embodiment, the first operation, the second operation, and the third operation may all be performed by a first core included in a multi-core processor, or the first operation and the second operation may be performed by a first core included in a multi-core processor and the third operation may be performed by a second core included in a multi-core processor.

[0268] In the embodiments of the present disclosure, a processor may mean a system-on-chip (SoC) in which one or more processors and other electronic components are integrated, a single-core processor, a multi-core processor, or a core included in a single-core processor or a multi-core processor, wherein the core may be implemented as a CPU, GPU, APU, MIC, DSP, NPU, hardware accelerator, or machine learning accelerator, but the embodiments of the present disclosure are not limited thereto.

[0269] Although preferred embodiments of the present disclosure have been illustrated and described above, the present disclosure is not limited to the specific embodiments described above. It is understood that various modifications can be made by those skilled in the art without departing from the essence of the present disclosure as claimed in the claims, and such modifications should not be understood individually from the technical spirit or perspective of the present disclosure.

[0270] [Explanation of the symbol]

[0271] 10: Single-cell sample pretreatment device

[0272] 100: Housing

[0273] 200: Pressure control unit

[0274] 300: Cartridge

[0275] 400: Sample storage unit

[0276] 500: Optical section

[0277] 600: Cooling section

[0278] 700: Heating part

[0279] 800: Magnetic force control unit

Claims

1. In a single-cell sample pretreatment device, Housing; A sample receiving portion comprising at least one injection vessel for receiving a sample, a microfluidic chip comprising a plurality of microwells, and a sample transfer portion connected to the sample receiving portion and the microfluidic chip and transferring a sample inside the at least one injection vessel to an inlet of the microfluidic chip, and a cartridge inserted into the housing; A pressure control unit disposed inside the housing, connected to the at least one injection vessel, and controlling the pressure inside the at least one injection vessel; and A single-cell sample pretreatment device comprising: a sample storage unit connected to an outlet of the microfluidic chip and storing a sample discharged from the outlet of the microfluidic chip.

2. In Paragraph 1, The above pressure control unit is, Control at least one injection container valve connected to the inlet of each of the above at least one injection container, and A single-cell sample pretreatment device in which, when at least one injection vessel valve is opened, the pressure inside the injection vessel connected to the opened injection vessel valve increases.

3. In Paragraph 2, The above sample transfer unit is, At least one sample injection channel connected to each of the above at least one injection vessel; and A sample transfer valve connected to at least one sample injection channel and an inlet of the microfluidic chip; comprising The above pressure control unit is, Based on a control signal regarding the selection of an injection vessel containing a sample to be supplied to the microfluidic chip, the injection vessel valve is controlled to increase the internal pressure of the selected injection vessel, and A single-cell sample pretreatment device that controls the sample transfer valve so that the sample moves along the sample injection channel connected to the selected injection vessel to the inlet of the microfluidic chip.

4. In Paragraph 1, The above sample storage unit is, At least one storage container for storing a sample discharged from the outlet of the microfluidic chip; A rotating member to which at least one storage container is detachably coupled; and A rotary drive module for driving the above-mentioned rotary member; comprising, The above rotary drive module is, A single-cell sample pretreatment device that drives a rotating member so that the selected storage container is connected to the discharge port based on a control signal regarding the selection of a storage container for storing a discharged sample.

5. In Paragraph 1, The above single-cell sample pretreatment device is, An optical unit for photographing a plurality of microwells of the above cartridge; is included, The above optical unit is, A light source positioned lower than the cartridge and irradiating light in a direction toward the cartridge; An objective lens through which light emitted from the light source and passed through the cartridge is transmitted; and A single-cell sample pretreatment device comprising: an image sensor that senses light transmitted through the objective lens.

6. In Paragraph 1, The above single-cell sample pretreatment device is, A cooling unit for cooling at least one cooling injection container requiring cooling among the at least one injection container mentioned above; comprising The above cooling unit is, A first temperature sensor for sensing the temperature of at least one cooling injection container; A Peltier module for cooling at least one cooling injection vessel; and A single-cell sample pretreatment device including a cooling fan.

7. In Paragraph 6, The above Peltier module is, A single-cell sample pretreatment device in contact with at least one cooling injection vessel on two sides.

8. In Paragraph 3, The above single-cell sample pretreatment device is, A heating unit for heating the above microfluidic chip; comprising, The heating unit above is, A second temperature sensor for sensing the temperature of a microfluidic chip; An air heating module that heats the air; and A single-cell sample pretreatment device comprising: a blower module that discharges the heated air toward the microfluidic chip.

9. In Paragraph 8, The heating unit above is, A single-cell sample pretreatment device that, after the dissolution buffer is transferred from an injection vessel containing the dissolution buffer to the microfluidic chip and a preset dissolution time has elapsed, heats the microfluidic chip for a preset reaction time such that the temperature of the microfluidic chip becomes 37 to 42 degrees Celsius.

10. In Paragraph 1, The above single-cell sample pretreatment device is, It includes a magnetic force control unit that controls the position of the magnet; and The above magnetic force control unit is, Based on a control signal regarding the fixation of the magnetic bead, the magnet is controlled to be positioned lower than the microfluidic chip, and A single-cell sample pretreatment device that controls the magnet to be positioned higher than the microfluidic chip based on a control signal regarding the recovery of magnetic beads.

11. In a cartridge for single-cell sample pretreatment, A sample receiving portion comprising at least one injection container for receiving a sample; A microfluidic chip comprising a plurality of microwells; and A cartridge comprising: a sample receiving portion connected to the sample receiving portion and the microfluidic chip, and a sample transfer portion that transfers a sample inside the at least one injection container to the inlet of the microfluidic chip.

12. In Paragraph 11, The above sample receiving portion is, It is provided in a predetermined area of ​​the upper surface of the sample transfer unit, and The above-mentioned at least one injection vessel is, A cartridge comprising: at least one cooling injection container that contacts a cooling portion of the single-cell sample pretreatment device when the above cartridge is inserted into the single-cell sample pretreatment device.

13. In Paragraph 11, The above microfluidic chip is, A cartridge comprising a fluid channel formed inside the microfluidic chip to allow a sample to flow.

14. In Paragraph 13, The above fluid channel is, A main fluid channel formed at the center of the upper surface of the above fluid channel; and A cartridge comprising auxiliary fluid channels formed on both sides of the main fluid channel. 15.(1) Priming step for treating the fluid channel surface; (2) Cell loading step for positioning cells inside the microwell; (3) A bead loading step in which magnetic beads are placed inside the microwell; (4) A bead washing step to remove magnetic beads that are not located inside the microwell; (5) A lysis step in which cells are lysed using a lysis buffer; (6) A cDNA synthesis step in which the microwell is heated to proceed with cDNA synthesis; (7) A molecule removal step for removing residual molecules by treating with an exonuclease; and (8) A single-cell sample pretreatment method including a bead recovery step.

16. In Paragraph 15, The above (1) priming step is, A step of treating the fluid channel surface using ethanol; A single-cell sample pretreatment method comprising the step of washing fluid channels using a washing buffer.

17. In Paragraph 15, The above (2) cell loading step is, A single-cell sample pretreatment method comprising the step of injecting a fluid containing cells so that the cells are positioned inside the microwell by gravity.

18. In Paragraph 15, The above (5) dissolution step is, A single-cell sample pretreatment method comprising the step of injecting a lysis buffer and reacting for 1 to 5 minutes to lyse the cells.

19. In Paragraph 15, The above (6) cDNA synthesis step is, A single-cell sample pretreatment method performed at a temperature of 37 to 42 degrees for 10 to 60 minutes.

20. In Paragraph 15, The above (7) molecular removal step is, A single-cell sample pretreatment method performed at a temperature of 37 to 42 degrees for 10 to 60 minutes.

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