WO2019049204A1 - Fluid device and use thereof - Google Patents

Fluid device and use thereof Download PDF

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Publication number
WO2019049204A1
WO2019049204A1 PCT/JP2017/031936 JP2017031936W WO2019049204A1 WO 2019049204 A1 WO2019049204 A1 WO 2019049204A1 JP 2017031936 W JP2017031936 W JP 2017031936W WO 2019049204 A1 WO2019049204 A1 WO 2019049204A1
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WO
WIPO (PCT)
Prior art keywords
fluid
flow path
cells
blood cells
flow
Prior art date
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PCT/JP2017/031936
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French (fr)
Japanese (ja)
Inventor
百合香 越智
哲臣 高崎
慶治 三井
Original Assignee
株式会社ニコン
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Publication date
Application filed by 株式会社ニコン filed Critical 株式会社ニコン
Priority to PCT/JP2017/031936 priority Critical patent/WO2019049204A1/en
Priority to JP2019540155A priority patent/JP7020488B2/en
Publication of WO2019049204A1 publication Critical patent/WO2019049204A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/33—Disintegrators
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters

Definitions

  • the present invention relates to fluidic devices and their use. More specifically, the present invention relates to a fluidic device, a method of disrupting cells, and a method of analyzing the number of red blood cells and white blood cells in a blood-derived sample.
  • cells may be broken. Physical methods, chemical methods, and the like exist as methods for disrupting cells.
  • a device or apparatus such as a microbead, a shaker, a centrifuge, or an ultrasonic crusher is used.
  • a worker may come in contact with a sample to be infected with a pathogen or a virus, or may contaminate the environment with an aerosol generated from the sample.
  • Patent No. 3571950 gazette
  • a fluid device includes a circulation channel, a flow channel for flowing a fluid containing cells, and a pump unit provided in the circulation channel and controlling the flow of the fluid or the pressure of the fluid. And an aperture-shaped cell analysis unit provided in the flow path.
  • a method of disrupting cells comprises a flow channel, a pump portion provided in the flow channel, and a compartmental valve for defining a region including the pump portion of the flow channel. Changing the pressure of the fluid by flowing the fluid containing the cells in the flow passage, closing the dividing valve to define a region including the pump portion of the flow passage, and operating the pump portion And c) disrupting the cells.
  • a method of analyzing the number of red blood cells and white blood cells in a blood-derived sample includes a flow path, a pump portion provided in the flow path, and an aperture-shaped cell analysis portion provided in the flow path Flowing the blood-derived sample through the flow channel of the fluidic device, measuring the number of red blood cells and white blood cells in the cell analysis unit, and operating the pump unit to measure the pressure of the blood-derived sample
  • the method comprises the steps of: selectively disrupting the red blood cells by changing; and, after breaking the red blood cells, measuring the number of white blood cells in the cell analysis unit.
  • the present invention comprises a flow path for flowing a fluid containing cells, which comprises a circulation flow path, and a pump unit provided in the circulation flow path for controlling the flow of the fluid or the pressure of the fluid And an aperture-shaped cell analysis unit provided in the flow path.
  • FIG. 1 is a schematic view illustrating the structure of a fluid device 100 according to an embodiment.
  • the fluid device 100 includes a flow channel 110 including a circulation flow channel, a pump unit 130 provided in the circulation flow channel, and an aperture-shaped cell analysis unit 120 provided in the flow channel 110 including the circulation flow channel.
  • the cell analysis unit 120 may be present inside the circulation flow channel, or may be present in a region other than the circulation flow channel in the flow channel 110.
  • the circulation channel means a channel through which the fluid inside can circulate.
  • the circulation flow path may form a closed circuit, or may be configured to be able to form a closed circuit by opening and closing a partition valve 140 described later.
  • the channel 110 is for flowing a fluid L containing cells.
  • the fluid L is not particularly limited, but may be, for example, a blood-derived sample.
  • examples of cells contained in the fluid L include red blood cells, white blood cells and the like.
  • the pump unit 130 By operating the pump unit 130, the flow of the fluid L can be controlled. Also, by operating the pump unit 130, the pressure of the fluid L can be adjusted. For example, by operating the pump unit 130, it is possible to cause the fluid L in a stationary state to flow or to increase the flow rate of the fluid L.
  • the pressure of the fluid L can also be said to be the pressure received by the cells contained in the fluid L. For example, by increasing the flow rate of the fluid L, the pressure of the fluid L can be increased. Alternatively, the pressure of the fluid L can be reduced by reducing the flow rate of the fluid L.
  • Cells can be disrupted using the fluid device of this embodiment. Specifically, cells can be disrupted by changing the pressure of the fluid L flowing through the flow path 110 by the pump unit 130.
  • the cell membrane of a cell can be ruptured by generating a pressure change such as releasing pressure after applying predetermined pressure to the fluid L.
  • a pressure change such as releasing pressure after applying predetermined pressure to the fluid L.
  • only specific cells in the cell mixture can be disrupted. This is considered to be due to the fact that the cell membrane structure is different depending on the cell type, and the resistance to pressure is different.
  • the fluid L contains red blood cells and white blood cells, only red blood cells can be selectively crushed.
  • the fluid device of the present embodiment it is possible to control the scattering of the sample to the outside of the fluid device in order to break up the cells in the fluid device. Therefore, it is possible to reduce the risk of workers being infected with pathogens and viruses and contaminating the environment.
  • the fluidic device of the present embodiment may be a cartridge. That is, the fluid device of the present embodiment is a component that can be freely attached and detached, and may be attached to and detached from the analyzer. Since the fluid device of the present embodiment is a cartridge, it is possible to perform cell disruption and analysis in a closed space within the cartridge, thereby further reducing the risk of infection and contamination.
  • the fluid L inside the circulation flow path provided in the flow path 110 is circulated by operating the pump unit 130 in a state where the partition valves 140a and 140b are closed and the flow path 110 forms a circuit. Can. This makes it possible to disrupt the cells more efficiently.
  • the fluid L flows inside the circulation flow path of the defined volume.
  • “defined” means to distinguish from other areas of the flow path.
  • a part of the flow path 110 may form an introduction flow path 110 a for introducing the fluid L in the area 115 including the pump portion 130, or for discharge for discharging the fluid from the area 115 You may form the flow path 110b.
  • the operation of introducing the fluid L into the region 115 or discharging the fluid L from the region 115 can be easily performed.
  • the introduction channel 110a includes a dividing valve 140a that controls the flow of the fluid L
  • the discharging channel 110b includes a dividing valve 140b that controls the flow of the fluid L.
  • the region 115 including the pump portion 130 is defined from the introduction channel 110a by closing the partition valve 140a and from the discharge channel 110b by closing the partition valve 140b.
  • the fluid device 100 may also include a waste tank 150. By storing the waste liquid in the waste liquid tank 150, the risk of infection or contamination with a germ or virus can be further reduced.
  • the pump unit 130 may be, for example, a valve.
  • the above-mentioned valve is a diaphragm valve provided with a diaphragm member, and the diaphragm member may be formed of an elastomeric material. Then, the flow of the fluid L in the flow path 110 may be controlled by the deformation of the diaphragm member, and the volume of the region 115 including the pump unit 130 may be changed.
  • the deformation direction of the diaphragm member is not particularly limited as long as the flow of the fluid L can be controlled, and may be, for example, a direction perpendicular to the axis of the flow path 110.
  • FIGS. 2A and 2B are cross-sectional views for explaining an example of the structure of the diaphragm valve.
  • 2A shows the diaphragm valve 200 in the open state
  • FIG. 2B shows the diaphragm valve 200 in the closed state.
  • the diaphragm valve 200 includes a first substrate 210, a diaphragm member 230 made of an elastomeric material, and a second substrate 220.
  • the second substrate 220 and the diaphragm member 230 are adhered in close contact with each other. Further, the space between the first substrate 210 and the diaphragm member 230 forms a flow path 110 through which the fluid flows.
  • a through hole 240 is provided in part of the second substrate 220. Further, in the through hole 240, the diaphragm member 230 is exposed.
  • the diaphragm valve 200 is disposed in the flow passage 110 and controls the flow of the fluid L or the pressure of the fluid L inside the flow passage 110.
  • the fluid L can flow inside the flow path 110.
  • FIG. 2B when a fluid for valve control is supplied from the through hole 240 of the diaphragm valve 200 and the inside of the through hole 240 is pressurized, the diaphragm member 230 is deformed and the deformed diaphragm member Part of the substrate 230 is in close contact with the first substrate 210.
  • This state is a closed state of the diaphragm valve 200. As a result, the flow of the fluid L inside the flow path 110 is shut off.
  • a convex portion is formed in the region of the first substrate 210 facing the through hole 240. 211 may be formed.
  • Examples of the fluid for valve control include gases such as N 2 gas and air, and liquids such as water and oil.
  • the fluid for valve control can be supplied, for example, by a tube or the like connected to the through hole 240. Alternatively, opening and closing of the valve may be controlled by mechanical force or electromagnetic force.
  • the elastomer material forming the diaphragm member 230 is not particularly limited as long as it is a material that can be deformed in the axial direction of the through hole 240 according to the pressure change inside the through hole 240, for example, polydimethylsiloxane (PDMS) And silicone-based elastomers such as polymethylphenylsiloxane and polydiphenylsiloxane.
  • PDMS polydimethylsiloxane
  • silicone-based elastomers such as polymethylphenylsiloxane and polydiphenylsiloxane.
  • the structure of the diaphragm valve is not limited to the one described above.
  • the diaphragm valve 300 shown in FIGS. 3A and 3B can also be used.
  • 3A shows the valve 300 in the open state
  • FIG. 3B shows the valve 300 in the closed state.
  • the diaphragm member 230 is not disposed on the entire surface of the second substrate 220, and only around the valve 300.
  • the points that are locally arranged are mainly different.
  • the diaphragm member 230 of the valve 300 is provided with the anchor portion 231, so that even when the diaphragm member 230 is deformed by pressure, it is suppressed that the diaphragm member 230 is damaged and peeled off from the second substrate 220. It is done.
  • pump portion 130 may be comprised of three or more valves.
  • the flow control of the fluid L by the pump unit 130 can be more efficiently performed by three or more valves, that is, by arranging three or more valves.
  • FIGS. 4A to 4D are cross-sectional views for explaining the operation of an example of a pump provided with three diaphragm valves 200a, 200b and 200c.
  • valve 200a is controlled to be closed, and the valves 200b and 200c are kept open. As a result, the flow of the fluid L inside the flow path 110 is blocked by the valve 200a.
  • valve 200a and the valve 200b are controlled to be closed, and the valve 200c is kept open.
  • the fluid L existing around the valve 200 b is pushed away by the deformation of the diaphragm member 230.
  • the valve 200a is in the closed state, the displaced fluid moves in the direction shown by the arrow in FIG. 4C, that is, to the right in FIG. 4C.
  • a flow in the direction indicated by the arrow occurs in the fluid L.
  • the fluid L is pressurized.
  • valves 200b and 200c are controlled to be closed.
  • the fluid L existing around the valve 200 c is pushed away by the deformation of the diaphragm member 230.
  • the valve 200a since the valve 200a is in the closed state, the displaced fluid L moves in the direction shown by the arrow in FIG. 4 (d), that is, to the right in FIG. 4 (d).
  • the valve 200a may be controlled to be open as shown in FIG. 4 (d) or may be kept closed.
  • valves 200a, 200b and 200c are all controlled to be in the open state.
  • the pressurization of the fluid L is released.
  • the fluid L inside the flow path 110 may continue to move to the right toward FIG. 4A due to inertia.
  • the flow of the fluid L inside the flow path 110 or the pressure of the fluid L can be controlled by repeating the above steps.
  • the above process is an example of a control method of a pump provided with three valves, and a control method of a pump provided with three valves is not limited to this.
  • the flow of the fluid L inside the flow passage 110 can be controlled in the reverse direction to that described above by reversing the timing of opening and closing the valve 200a and the valve 200c.
  • the flow of the fluid L or the fluid L can also be adjusted by adjusting the cycle of repeating the operation of FIGS.
  • the pressure of the can be controlled.
  • Division valve Fluid device 100 may further include compartment valve 140.
  • the pressure of the fluid L can be controlled more efficiently by closing the dividing valve 140 (140a, 140b) to define the region 115 including the pump portion 130 of the flow path 110 and operating the pump portion 130.
  • closing the partition valve 140 results in the area 115 including the pump portion 130 being defined. That is, in the fluid device 100, by closing the dividing valve 140 (140a, 140b), the area 115 including the pump portion 130 of the flow path 110 becomes a circulation flow path (loop flow path) and becomes independent from other parts. it can.
  • the flow velocity of the fluid temporarily decreases or stops. Thereafter, when the pump unit 130 is operated, the flow velocity in the region 115 is increased, and the pressure applied to the fluid L is increased.
  • the pump portion 130 is a diaphragm valve
  • closing the diaphragm valve deforms the diaphragm member toward the flow path 110, and the volume in the region 115 is reduced.
  • the pressure of the fluid L in the area 115 is increased.
  • opening the diaphragm valve the diaphragm member is deformed in the direction opposite to the flow path side, and the volume in the region 115 is increased.
  • the pressure of the fluid L decreases.
  • the pressure of the fluid L can be adjusted by changing the volume in the region 115.
  • the dividing valve 140 may be a diaphragm valve including a diaphragm member, and may be similar to the valve constituting the pump unit described above.
  • Compartment valve 140 may be a normally closed valve or a normally open valve.
  • the normally closed valve is a valve that is closed in a steady state and is opened by operating the valve.
  • the normally open valve is a valve that is open in a steady state and is closed by operating the valve.
  • the partition valve 140 When the partition valve 140 is a normally closed valve, at steady state, the partition valve 140 is closed, and by operating the valve, the region 115 defined by the partition valve 140 is released.
  • the dividing valve 140 when the dividing valve 140 is a normally open valve, the dividing valve 140 is open in the steady state and is closed by operating the valve, and a part of the flow path 110 is the dividing valve 140. It becomes an area defined by
  • the fluid device 100 may include one compartment valve 140 or two or more compartment valves. When there are two or more partition valves 140, closing each partition valve 140 defines a region 115 including the pump portion, which can be a temporarily independent space.
  • the cell analysis unit 120 is an area for analyzing cells flowing in the flow channel 110.
  • cell analysis means analyzing predetermined parameters of fluid containing cells.
  • the parameters are not particularly limited, and include, for example, the number of cells in the fluid containing the cells, the size of the cells, and the like.
  • the cell analysis unit has, for example, an aperture shape.
  • the aperture shape is a shape in which the cross-sectional area of the flow channel cross section orthogonal to the flow direction of the fluid in the cell analysis unit 120 is smaller than the cross-sectional area of the flow channel cross section orthogonal to the flow direction of the fluid.
  • the cross-sectional area of the cell analysis unit is smaller than the cross-sectional area of the flow channels of the flow channels 110 before and after the cell analysis unit 120.
  • the diameter of the cell analysis unit 120 is smaller than the diameter of the other region of the flow channel 110. That is, the flow channel cross section of the cell analysis unit having an aperture shape may have a size suitable for analysis of an analyte, or may be a size in which cells to be analyzed pass one by one. Thereby, at least in the cell analysis unit 120, it becomes possible to pass cells one by one. As a result, since cells can be detected for each cell, analysis of cells is facilitated.
  • Cell analysis may be performed, for example, optically or electrically.
  • the analysis items of cells can be set as appropriate. For example, the number of cells may be measured, or the size of cells may be measured.
  • the cross-sectional shape of the flow path and the aperture shape is not limited to a rectangular shape, and may be, for example, a circular shape, or any polygonal shape.
  • the cross section of the flow channel 110 may be a rectangle having a width of 1.5 mm and a height of 0.3 mm.
  • the cross section of the cell analysis unit having an aperture shape may be a rectangle having a width of 30 ⁇ m and a height of 30 ⁇ m. With such a shape and size, for example, white blood cells can pass through a single cell analysis unit that is in the form of flow paths and apertures.
  • the cell analysis unit 120 is present inside the circulation flow channel provided in the flow channel 110. That is, the cell analysis unit 120 is present in the circulation channel.
  • the presence of the cell analysis unit 120 inside the circulation flow channel allows the state of the cells inside the circulation flow channel to be analyzed in real time while the pump unit 130 is operated to break up the cells. As a result, for example, it becomes possible to stop the pump unit 130 when the cell crushing rate reaches an arbitrary value, to continue the cell crushing operation until the cells are completely broken, and the like.
  • FIG. 5 is a schematic view illustrating the structure of the fluid device 500 according to the second embodiment.
  • the fluid device 500 according to the second embodiment is different from the fluid device 500 in that the cell analysis unit 120 exists outside the circuit when the partition valves 140a and 140b are closed to form a circuit in which the circulation channel of the channel 110 is closed.
  • This differs mainly from the fluidic device 100 according to one embodiment. That is, in the fluid device 500, the cell analysis unit 120 is present in a flow channel other than the circulation flow channel.
  • the flow of the fluid L inside the flow path 110 is not influenced by the cell analysis unit 120 because the cell analysis unit 120 is present outside the circuit. Therefore, the degree of freedom in control of the flow of the fluid L by the operation of the pump unit 130 is higher than that of the fluid device 100 according to the first embodiment.
  • the cell analysis unit 120 analyzes the cells flowing in the flow channel 110 before starting the cell disruption or after completing the cell disruption.
  • the cell analysis unit 120 is present in the discharge channel 110b.
  • cells can be analyzed by flowing the fluid L in one passage in the order of the introduction channel 110a, the circulation channel, and the discharge channel 110b.
  • the position of the cell analysis unit 120 is not limited to this, and the cell analysis unit 120 may be present in the introduction channel 110a.
  • cells can be analyzed by flowing the fluid L in the opposite direction again to the introduction channel 110a.
  • FIG. 6 is a schematic view for explaining a modification of the fluid device.
  • the fluid device 600 is mainly different from the fluid device 100 according to the first embodiment in that the circulation channel provided in the flow channel 110 forms two or more closed circuits by controlling the opening and closing of the dividing valve 140. .
  • the flow of the fluid L inside the flow path 110 can be controlled to analyze cells.
  • the cell analysis unit 120 is present inside the circuit.
  • the position of the cell analysis unit 120 is not limited to this, and may be configured to exist outside the circuit.
  • the fluid device 600 includes a first circulation channel and a second circulation channel, and the circuit in which the first circulation channel and the second circulation channel are closed by closing the dividing valve 140. May be formed.
  • the first circulation channel and the second circulation channel in the fluid device 600 share at least a part of the channel, and include a pump unit in the shared channel, and the first circulation
  • the cell analysis unit 120 may be provided in the channel or the second circulation channel.
  • the first circulation channel and the second circulation channel are not shared in the vicinity of both ends of the channel shared by the first circulation channel and the second circulation channel.
  • Each of the flow paths may have a valve.
  • the present invention is a method of disrupting cells, comprising: a flow path, a pump portion provided in the flow path, and a compartment valve for defining a region including the pump portion of the flow path. And allowing the fluid containing the cells to flow in the flow path of the fluidic device, closing the compartment valve to define a region including the pump portion of the flow path, and operating the pump portion Breaking the cells by changing the pressure of the fluid.
  • FIG. 1 is a schematic view illustrating the structure of a fluid device 100 according to an embodiment.
  • the fluid device 100 includes a flow channel 110 including a circulation flow channel, a pump unit 130 provided in the circulation flow channel, and an aperture-shaped cell analysis unit 120 provided in the flow channel 110 including the circulation flow channel. There is.
  • the pump unit 130 may include three or more valves. As described above, control of the flow of the fluid L by the pump unit 130 can be performed more efficiently by using three or more valves, that is, arranging three or more valves.
  • the region 115 of the flow channel 110 forms a closed circuit.
  • the partition valves 140a and 140b of the fluid device 100 are opened, and the partition valves 140c and 140d are closed.
  • the fluid L containing cells is introduced from the introduction channel 110a.
  • the fluid L containing cells may be, for example, a blood-derived sample containing red blood cells and white blood cells.
  • the inside of the flow path 110 is filled with the fluid L containing cells.
  • the excess fluid L passes through the discharge channel 110 b and is stored in the waste liquid tank 150.
  • the flow of the fluid L containing cells may be generated by operating the pump unit 130.
  • the operating condition of the pump unit 130 may be a condition that does not crush cells.
  • the compartment valves 140a and 140b are closed to open 140c.
  • the region 115 of the flow channel 110 forms a closed circuit.
  • the pump unit 130 is operated under the condition of changing the pressure of the fluid L containing cells.
  • the conditions for changing the pressure of the fluid L the driving time of the pump, the driving pressure, the frequency and the like can be mentioned.
  • the flow velocity in the region 115 is increased, and the pressure applied to the fluid L is increased.
  • the pump portion 130 is a diaphragm valve, the diaphragm member is deformed toward the flow passage 110 by closing the diaphragm valve, and the volume in the region 115 is reduced.
  • the pressure of the fluid L in the area 115 is increased.
  • the diaphragm member is deformed in the direction opposite to the flow path side, and the volume in the region 115 is increased. As a result, the pressure of the fluid L decreases. Such pressure changes disrupt the cells.
  • red blood cells in a blood-derived sample containing, for example, red blood cells and white blood cells by adjusting the conditions under which the pressure is changed.
  • the pump unit 130 by operating the pump unit 130 in a state in which the region 115 of the flow channel 110 is closed, the efficiency of cell disruption can be increased.
  • the present invention is a method of measuring the number of red blood cells and white blood cells in a blood-derived sample, comprising: a flow path; a pump unit provided in the flow path; and an aperture provided in the flow path Flowing the blood-derived sample through the flow channel of the fluidic device having the shape of a cell analysis unit, measuring the number of red blood cells and white blood cells in the cell analysis unit, and operating the pump unit to operate the pump unit.
  • a method is provided comprising the steps of: selectively disrupting the red blood cells by changing the pressure of a blood-derived sample; and measuring the number of white blood cells in the cell analysis unit after breaking the red blood cells. It can be said that the method of the present embodiment is a method of blood cell count (blood calculation).
  • the partition valves 140a and 140b of the fluid device 500 are opened, and the partition valves 140c and 140d are closed.
  • a blood-derived sample is introduced from the introduction channel 110a.
  • the introduction of the blood-derived sample may be performed by pressurization or may be performed by operating the pump unit 130.
  • the operating condition of the pump unit 130 be a condition that does not crush cells.
  • the inside of the flow channel 110 is filled with the blood-derived sample.
  • the excess blood-derived sample passes through the cell analysis unit 120 and the discharge channel 110 b and is stored in the waste liquid tank 150.
  • the cell analysis unit 120 measures the number of cells passing through the cell analysis unit 120.
  • red blood cells and white blood cells are not broken. For this reason, the number of cells measured at this stage is the sum of the numbers of red blood cells and white blood cells.
  • the compartment valves 140a and 140b are closed to open 140c.
  • the region 115 of the flow channel 110 forms a closed circuit.
  • the pump unit 130 is operated under conditions to selectively crush the red blood cells in the blood-derived sample to change the pressure of the blood-derived sample, thereby selectively crush the red blood cells.
  • the compartment valve 140b is opened to feed the blood-derived sample. Then, the blood-derived sample in the flow path 110 passes through the cell analysis unit 120 and the discharge flow path 110 b and is stored in the waste liquid tank 150.
  • the cell analysis unit 120 measures the number of cells passing through the cell analysis unit 120.
  • the number of cells measured at this stage is the number of white blood cells.
  • the number of white blood cells in the blood-derived sample can be measured. Also, the number of erythrocytes can be determined more accurately by subtracting the number of leukocytes measured later from the total number of erythrocytes and leukocytes measured first. However, since the number of white blood cells is usually very small relative to the number of red blood cells, the total number of red blood cells and white blood cells initially measured may be used as the number of red blood cells.
  • the conditions for selectively breaking up the red blood cells in the blood-derived sample may be, for example, conditions in which the driving pressure of the pump unit having a diaphragm member is more than 0 kPa, such as 50 to 110 kPa, such as 70 to 110 kPa.
  • the drive frequency of the pump unit having the diaphragm member may be more than 0 Hz, for example, 0.5 to 3.3 Hz, for example, 2 to 3.3 Hz.
  • the operating time of the pump unit may be, for example, 1 to 30 minutes, 5 to 20 minutes, or about 10 minutes.
  • the diameter of the above-mentioned diaphragm member may be, for example, 1 to 3 mm, or 2 to 3 mm.
  • the thickness of the diaphragm member may be, for example, 10 to 2000 ⁇ m, and may be, for example, 100 to 1000 ⁇ m. As the thickness of the diaphragm member is thinner, the required displacement can be obtained at a lower driving pressure.
  • the material of the diaphragm member may be a silicone-based elastomer such as polydimethylsiloxane (PDMS), polymethylphenylsiloxane, polydiphenylsiloxane or the like.
  • erythrocytes can be selectively disrupted.
  • the crushing rate of white blood cells under the above conditions is 2.5% or less.
  • FIGS. 7A to 7C are schematic diagrams illustrating a method of measuring the number of red blood cells and white blood cells in a blood-derived sample using the fluid device 600.
  • FIG. 7A to 7C are schematic diagrams illustrating a method of measuring the number of red blood cells and white blood cells in a blood-derived sample using the fluid device 600.
  • the partition valves 140a2, 140b1, 140c1, 140c2 and 140c5 of the fluid device 600 are opened, and the partition valves 140a1, 140b2, 140c3 and 140c4 are closed.
  • a blood-derived sample is introduced from the introduction channel 110a1.
  • the introduction of the blood-derived sample may be performed by pressurization or may be performed by operating the pump unit 130. In this case, it is preferable that the operating condition of the pump unit 130 be a condition that does not crush cells.
  • the blood-derived sample flows inside the flow channel 110, and the inside of the flow channel 110 is filled with the blood-derived sample.
  • the excess blood-derived sample passes through the discharge channel 110 b 1 and is stored in the waste liquid tank 150.
  • the cell analysis unit 120 measures the number of cells passing through the cell analysis unit 120.
  • red blood cells and white blood cells are not broken. For this reason, the number of cells measured at this stage is the sum of the numbers of red blood cells and white blood cells.
  • the partition valves 140a1, 140b1, 140c3, 140c5 are closed, and the valves 140a2, 140b2, 140c1, 140c2, 140c4 are closed. Open. As a result, the region 115 of the flow channel 110 forms a closed circuit.
  • the pump unit 130 is operated under conditions to selectively crush the red blood cells in the blood-derived sample to change the pressure of the blood-derived sample, thereby selectively crush the red blood cells.
  • the blood-derived sample circulates inside the closed circuit formed by the flow path 110.
  • the blood origin is present in the area including the cell analysis unit 120 of the channel 110.
  • the sample can be drained.
  • the discharge fluid include N 2 gas, gas such as air, buffer, liquid such as oil, and the like.
  • the blood-derived sample passes through the discharge channel 110 b 2 and is stored in the waste liquid tank 150.
  • the discharge fluid is introduced from the introduction channel 110a1.
  • the introduction of the discharge fluid may be performed by pressurization or may be performed by operating the pump unit 130.
  • the operating condition of the pump unit 130 be a condition that does not crush cells.
  • the blood-derived sample in the flow path 110 passes through the cell analysis unit 120 and the discharge flow path 110b2 and is stored in the waste liquid tank 150.
  • the cell analysis unit 120 measures the number of cells passing through the cell analysis unit 120.
  • the number of cells measured at this stage is the number of white blood cells.
  • the device shown in FIG. 8B (hereinafter referred to as “device B”) was the same as device A except that the diameter of each valve was 2 mm.
  • the devices A and B were manufactured for the purpose of evaluating whether or not cells can be disrupted by the operation of the pump unit, and the cell analysis unit was not included.
  • a sample was introduced into each fluid device.
  • the amount of sample introduced was 62.5 ⁇ L for each device.
  • the valve 140 of each device was opened and closed to form a closed circuit of the flow path 110. Thereby, the sample was sealed in the circuit of each device.
  • the pump unit 130 was operated to break up the cells.
  • the operating conditions of the pump unit 130 were set to the conditions shown in Table 1. Moreover, the operating time of the pump part 130 was 10 minutes in all the conditions.
  • Hemolysis rate (%) (absorbance of sample ⁇ absorbance of negative control) / (absorbance of positive control ⁇ absorbance of negative control) ⁇ 100 (1)
  • FIG. 9 is a graph showing the measurement results of the hemolytic rate. As a result, when the valve diameter, the driving pressure and the frequency were increased, the ratio of the erythrocytes to be crushed tended to increase.
  • a sample was introduced into device A.
  • As a sample rabbit blood was used.
  • the amount of sample introduced was 62.5 ⁇ L.
  • the valve 140 of the device A was opened and closed to form a closed circuit of the flow path 110. Thereby, the sample was sealed in the circuit of device A.
  • the pump unit 130 was operated to break up the cells.
  • the operating conditions of the pump unit 130 were set to the conditions shown in Table 2. Moreover, the operating time of the pump part 130 was 10 minutes in all the conditions.
  • the valve 140 of the device A was opened and closed to recover the sample.
  • the hemolysis rate of each collected sample was measured in the same manner as in Experimental Example 2 using a spectrophotometer (UV-1800 / Shimadzu Corporation).
  • the amount of DNA in each sample is subjected to real-time quantitative PCR (Light Cycler 480 System II / Roche Diagnostics). Measured. When white blood cells are destroyed, the amount of DNA in the sample increases. The relative value of the amount of DNA in the recovered sample was calculated when the total amount of DNA contained in the original sample was 100.
  • FIG. 10 is a graph showing the measurement results of the hemolysis rate and the amount of DNA. As a result, it became clear that even if the red blood cells were destroyed by the device A, the white blood cells were hardly destroyed.

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Abstract

A fluid device comprising: a flow path through which a fluid containing cells flows, and which includes a circulation flow path; a pump section which is provided in the circulation flow path, and controls the flow of the fluid or the pressure of the fluid; and a cell analysis section provided in the flow path, wherein the cross-sectional area of the cell analysis section is smaller than the cross-sectional area of the flow path section excluding the cell analysis section.

Description

流体デバイス及びその使用Fluid device and use thereof
　本発明は流体デバイス及びその使用に関する。より具体的には、流体デバイス、細胞を破砕する方法、血液由来試料中の赤血球及び白血球の数を分析する方法に関する。 The present invention relates to fluidic devices and their use. More specifically, the present invention relates to a fluidic device, a method of disrupting cells, and a method of analyzing the number of red blood cells and white blood cells in a blood-derived sample.
　病理学的検査や生物学的な研究において、細胞を破砕する場合がある。細胞を破砕する方法としては、物理的方法、化学的方法等が存在する。細胞を物理的方法で破砕する場合には、例えば、マイクロビーズ、振とう機、遠心機、超音波破砕機等の器具や装置を使用する。しかしながら、細胞を破砕する過程で、作業者が試料に接触して病原菌やウイルスに感染したり、試料から発生したエアロゾルにより周囲を汚染したりする場合がある。 In pathological examination and biological research, cells may be broken. Physical methods, chemical methods, and the like exist as methods for disrupting cells. When cells are to be disrupted by a physical method, for example, a device or apparatus such as a microbead, a shaker, a centrifuge, or an ultrasonic crusher is used. However, in the process of crushing cells, a worker may come in contact with a sample to be infected with a pathogen or a virus, or may contaminate the environment with an aerosol generated from the sample.
　また、細胞を化学的方法で破砕する場合、通常、温度を制御する必要があり、また、検体の希釈が生じる。また、化学処理を停止するための後処理が必要であるため、時間がかかり、目的物が変性してしまう場合がある。 Also, when cells are disrupted by chemical methods, it is usually necessary to control the temperature, and dilution of the sample occurs. In addition, since the post-treatment for stopping the chemical treatment is required, it may take time and the target may be denatured.
　したがって、改良された細胞破砕技術が求められている。 Thus, there is a need for improved cell disruption techniques.
特許第３５７１９５０号公報Patent No. 3571950 gazette
　一実施形態に係る流体デバイスは、循環流路を備える、細胞を含む流体を流すための流路と、前記循環流路に設けられ、前記流体の流れ又は前記流体の圧力を制御するポンプ部と、前記流路に設けられたアパーチャ形状の細胞分析部とを備える。 A fluid device according to one embodiment includes a circulation channel, a flow channel for flowing a fluid containing cells, and a pump unit provided in the circulation channel and controlling the flow of the fluid or the pressure of the fluid. And an aperture-shaped cell analysis unit provided in the flow path.
　一実施形態に係る細胞を破砕する方法は、流路と、前記流路に設けられたポンプ部と、前記流路の前記ポンプ部を含む領域を画定するための区画バルブとを備える流体デバイスの前記流路に、前記細胞を含む流体を流す工程と、前記区画バルブを閉じて前記流路の前記ポンプ部を含む領域を画定する工程と、前記ポンプ部を作動させて前記流体の圧力を変化させることにより前記細胞を破砕する工程とを備える。 A method of disrupting cells according to one embodiment comprises a flow channel, a pump portion provided in the flow channel, and a compartmental valve for defining a region including the pump portion of the flow channel. Changing the pressure of the fluid by flowing the fluid containing the cells in the flow passage, closing the dividing valve to define a region including the pump portion of the flow passage, and operating the pump portion And c) disrupting the cells.
　一実施形態に係る、血液由来試料中の赤血球及び白血球の数を分析する方法は、流路と、前記流路に設けられたポンプ部と、前記流路に設けられたアパーチャ形状の細胞分析部とを備える流体デバイスの前記流路に、前記血液由来試料を流す工程と、前記細胞分析部で赤血球及び白血球の数を測定する工程と、前記ポンプ部を作動させて前記血液由来試料の圧力を変化させることにより前記赤血球を選択的に破砕する工程と、赤血球を破砕した後に、前記細胞分析部で白血球の数を測定する工程とを備える。 According to one embodiment, a method of analyzing the number of red blood cells and white blood cells in a blood-derived sample includes a flow path, a pump portion provided in the flow path, and an aperture-shaped cell analysis portion provided in the flow path Flowing the blood-derived sample through the flow channel of the fluidic device, measuring the number of red blood cells and white blood cells in the cell analysis unit, and operating the pump unit to measure the pressure of the blood-derived sample The method comprises the steps of: selectively disrupting the red blood cells by changing; and, after breaking the red blood cells, measuring the number of white blood cells in the cell analysis unit.
一実施形態に係る流体デバイスの構造を説明する模式図である。It is a schematic diagram explaining the structure of the fluidic device concerning one embodiment. （ａ）及び（ｂ）は、ダイアフラムバルブの構造を説明する断面図である。(A) And (b) is sectional drawing explaining the structure of a diaphragm valve. （ａ）及び（ｂ）は、ダイアフラムバルブの構造を説明する断面図である。(A) And (b) is sectional drawing explaining the structure of a diaphragm valve. （ａ）～（ｄ）は、３個のダイアフラムバルブを備えるポンプの動作を説明する断面図である。(A)-(d) is a sectional view explaining operation of a pump provided with three diaphragm valves. 一実施形態に係る流体デバイスの構造を説明する模式図である。It is a schematic diagram explaining the structure of the fluidic device concerning one embodiment. 一実施形態に係る流体デバイスの構造を説明する模式図である。It is a schematic diagram explaining the structure of the fluidic device concerning one embodiment. （ａ）～（ｃ）は、流体デバイスを用いて血液由来試料中の赤血球及び白血球の数を測定する方法を説明する模式図である。(A) to (c) are schematic diagrams illustrating a method of measuring the number of red blood cells and white blood cells in a blood-derived sample using a fluid device. （ａ）及び（ｂ）は、実験例１で製造した流体デバイスの構造を説明する模式図である。(A) And (b) is a schematic diagram explaining the structure of the fluid device manufactured by Experimental example 1. FIG. 実験例２の結果を示すグラフである。It is a graph which shows the result of example 2 of an experiment. 実験例３の結果を示すグラフである。7 is a graph showing the results of Experimental Example 3.
　以下、場合により図面を参照しつつ、本発明の実施形態について詳細に説明する。なお、図面中、同一又は相当部分には同一又は対応する符号を付し、重複する説明は省略する。なお、各図における寸法比は、説明のため誇張している部分があり、必ずしも実際の寸法比とは一致しない。 Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings as the case may be. In the drawings, the same or corresponding parts will be denoted by the same or corresponding reference symbols, without redundant description. In addition, the dimensional ratio in each figure has the part which exaggerates for description, and does not necessarily correspond with an actual dimensional ratio.
[流体デバイス]
（第１実施形態）
　一実施形態において、本発明は、循環流路を備える、細胞を含む流体を流すための流路と、前記循環流路に設けられ、前記流体の流れ又は前記流体の圧力を制御するポンプ部と、前記流路に設けられたアパーチャ形状の細胞分析部と、を備える、流体デバイスを提供する。 Fluid Device
First Embodiment
In one embodiment, the present invention comprises a flow path for flowing a fluid containing cells, which comprises a circulation flow path, and a pump unit provided in the circulation flow path for controlling the flow of the fluid or the pressure of the fluid And an aperture-shaped cell analysis unit provided in the flow path.
　図１は、一実施形態に係る流体デバイス１００の構造を説明する模式図である。流体デバイス１００は、循環流路を備える流路１１０と、循環流路に設けられたポンプ部１３０と、循環流路を備える流路１１０に設けられたアパーチャ形状の細胞分析部１２０とを備える。後述するように、細胞分析部１２０は、循環流路の内部に存在していてもよく、流路１１０内の循環流路以外の領域に存在していてもよい。 FIG. 1 is a schematic view illustrating the structure of a fluid device 100 according to an embodiment. The fluid device 100 includes a flow channel 110 including a circulation flow channel, a pump unit 130 provided in the circulation flow channel, and an aperture-shaped cell analysis unit 120 provided in the flow channel 110 including the circulation flow channel. As described later, the cell analysis unit 120 may be present inside the circulation flow channel, or may be present in a region other than the circulation flow channel in the flow channel 110.
　本実施形態の流体デバイスにおいて、循環流路とは、内部の流体が循環することができる流路を意味する。循環流路は、閉じた回路を形成していてもよく、後述する区画バルブ１４０を開閉することにより、閉じた回路を形成することができるように構成されていてもよい。 In the fluid device of the present embodiment, the circulation channel means a channel through which the fluid inside can circulate. The circulation flow path may form a closed circuit, or may be configured to be able to form a closed circuit by opening and closing a partition valve 140 described later.
　流路１１０は細胞を含む流体Ｌを流すためのものである。流体Ｌは、特に限定されないが、例えば血液由来試料であってもよい。この場合、流体Ｌに含まれる細胞としては、赤血球、白血球等が挙げられる。ポンプ部１３０を作動させることにより、流体Ｌの流れを制御することができる。また、ポンプ部１３０を作動させることにより、流体Ｌの圧力を調節することができる。例えば、ポンプ部１３０を作動させることにより、静止状態にあった流体Ｌを流動させたり、流体Ｌの流動速度を速めたりすることができる。 The channel 110 is for flowing a fluid L containing cells. The fluid L is not particularly limited, but may be, for example, a blood-derived sample. In this case, examples of cells contained in the fluid L include red blood cells, white blood cells and the like. By operating the pump unit 130, the flow of the fluid L can be controlled. Also, by operating the pump unit 130, the pressure of the fluid L can be adjusted. For example, by operating the pump unit 130, it is possible to cause the fluid L in a stationary state to flow or to increase the flow rate of the fluid L.
　流体Ｌの圧力とは、流体Ｌに含まれる細胞が受ける圧力であるということもできる。例えば、流体Ｌの流動速度を速めることにより、流体Ｌの圧力を上昇させることができる。あるいは、流体Ｌの流動速度を低下させることにより、流体Ｌの圧力を低下させることができる。 The pressure of the fluid L can also be said to be the pressure received by the cells contained in the fluid L. For example, by increasing the flow rate of the fluid L, the pressure of the fluid L can be increased. Alternatively, the pressure of the fluid L can be reduced by reducing the flow rate of the fluid L.
　本実施形態の流体デバイスを用いて細胞を破砕することができる。具体的には、ポンプ部１３０により、流路１１０を流れる流体Ｌの圧力を変化させることにより、細胞を破砕することができる。例えば、流体Ｌに所定の加圧を行った後、加圧を解除する等の圧力変化を発生させることにより、細胞の細胞膜を破裂させることができる。また、圧力変化の条件を制御することにより、細胞混合物中の特定の細胞のみを破砕することができる。これは、細胞の種類によって細胞膜の構造が異なっており、圧力に対する耐性が異なることによると考えられる。実施例において後述するように、例えば、流体Ｌが赤血球及び白血球を含む場合に、赤血球のみを選択的に破砕することができる。 Cells can be disrupted using the fluid device of this embodiment. Specifically, cells can be disrupted by changing the pressure of the fluid L flowing through the flow path 110 by the pump unit 130. For example, the cell membrane of a cell can be ruptured by generating a pressure change such as releasing pressure after applying predetermined pressure to the fluid L. Also, by controlling the conditions of pressure change, only specific cells in the cell mixture can be disrupted. This is considered to be due to the fact that the cell membrane structure is different depending on the cell type, and the resistance to pressure is different. As will be described later in the examples, for example, when the fluid L contains red blood cells and white blood cells, only red blood cells can be selectively crushed.
　本実施形態の流体デバイスにより、従来の細胞破砕装置や試薬を使用しなくても、極めて短時間に細胞を破砕することができる。また、検体も希釈されず、細胞破砕条件が穏やかであるため目的物の変性が生じる恐れが少なく、細胞破砕のために温度を調節する必要もない。細胞の破砕の詳細については後述する。 With the fluid device of the present embodiment, cells can be disrupted in a very short time without using conventional cell disruption devices and reagents. In addition, since the sample is not diluted and the cell disruption conditions are mild, there is little possibility that the target substance will be denatured, and there is no need to control the temperature for cell disruption. Details of cell disruption will be described later.
　また、本実施形態の流体デバイスによれば、流体デバイス内で細胞を破砕するため、試料が流体デバイスの外部に飛散することを制御することができる。このため、作業者が病原菌やウイルスに感染したり、周囲を汚染したりするリスクを低減することができる。 Further, according to the fluid device of the present embodiment, it is possible to control the scattering of the sample to the outside of the fluid device in order to break up the cells in the fluid device. Therefore, it is possible to reduce the risk of workers being infected with pathogens and viruses and contaminating the environment.
　本実施形態の流体デバイスはカートリッジであってもよい。すなわち、本実施形態の流体デバイスは、自由に着脱することができる部品であり、分析装置に着脱するものであってもよい。本実施形態の流体デバイスがカートリッジであることにより、カートリッジ内という閉じられた空間で細胞の破砕及び分析を行うことができるため、感染・汚染のリスクを更に低減することができる。 The fluidic device of the present embodiment may be a cartridge. That is, the fluid device of the present embodiment is a component that can be freely attached and detached, and may be attached to and detached from the analyzer. Since the fluid device of the present embodiment is a cartridge, it is possible to perform cell disruption and analysis in a closed space within the cartridge, thereby further reducing the risk of infection and contamination.
　流体デバイス１００では、区画バルブ１４０ａ及び１４０ｂを閉じて流路１１０が回路を形成した状態で、ポンプ部１３０を作動させることにより、流路１１０が備える循環流路の内部の流体Ｌを循環させることができる。これにより、更に効率よく細胞を破砕することが可能になる。 In the fluid device 100, the fluid L inside the circulation flow path provided in the flow path 110 is circulated by operating the pump unit 130 in a state where the partition valves 140a and 140b are closed and the flow path 110 forms a circuit. Can. This makes it possible to disrupt the cells more efficiently.
　実施例において後述するように、循環流路が閉じた回路を形成した状態で、ポンプ部１３０を作動させることにより、流体Ｌは画定された体積の循環流路の内部を流れることになる。ここで、「画定」とは、流路の他の領域から区別することを意味する。そして、ポンプ部１３０の作動条件を調整して流体Ｌの圧力を変化させると、流体Ｌに含まれる細胞が受ける圧力が変化する。より具体的には、細胞が加圧されたり減圧されたりする。このような圧力変化により、流路１１０における画定された領域１１５の内部で細胞の細胞膜が破裂し、細胞が破砕されると考えられる。 As will be described later in the embodiment, by operating the pump unit 130 with the circulation flow path forming a closed circuit, the fluid L flows inside the circulation flow path of the defined volume. Here, "defined" means to distinguish from other areas of the flow path. When the operating condition of the pump unit 130 is adjusted to change the pressure of the fluid L, the pressure received by the cells contained in the fluid L changes. More specifically, cells are pressurized or depressurized. Such pressure change is considered to cause the cell membrane of the cell to rupture within the defined region 115 in the flow path 110 and the cell to be broken.
　図１に示すように、流路１１０の一部はポンプ部１３０を含む領域１１５に流体Ｌを導入する導入用流路１１０ａを形成していてもよいし、領域１１５から流体を排出する排出用流路１１０ｂを形成していてもよい。これにより、領域１１５に流体Ｌを導入したり、領域１１５から流体Ｌを排出したりする操作を簡便に行うことができる。 As shown in FIG. 1, a part of the flow path 110 may form an introduction flow path 110 a for introducing the fluid L in the area 115 including the pump portion 130, or for discharge for discharging the fluid from the area 115 You may form the flow path 110b. Thus, the operation of introducing the fluid L into the region 115 or discharging the fluid L from the region 115 can be easily performed.
　また、導入用流路１１０ａは、流体Ｌの流れを制御する区画バルブ１４０ａを備えていることが好ましく、排出用流路１１０ｂは、流体Ｌの流れを制御する区画バルブ１４０ｂを備えていることが好ましい。このとき、ポンプ部１３０を含む領域１１５は、区画バルブ１４０ａを閉じることにより導入用流路１１０ａから画定され、区画バルブ１４０ｂを閉じることにより排出用流路１１０ｂから画定される。 Further, it is preferable that the introduction channel 110a includes a dividing valve 140a that controls the flow of the fluid L, and the discharging channel 110b includes a dividing valve 140b that controls the flow of the fluid L. preferable. At this time, the region 115 including the pump portion 130 is defined from the introduction channel 110a by closing the partition valve 140a and from the discharge channel 110b by closing the partition valve 140b.
　また、流体デバイス１００は、廃液タンク１５０を備えていてもよい。廃液を廃液タンク１５０に収容することにより、病原菌やウイルスによる感染や汚染のリスクを更に低減することができる。 The fluid device 100 may also include a waste tank 150. By storing the waste liquid in the waste liquid tank 150, the risk of infection or contamination with a germ or virus can be further reduced.
《ポンプ部》
　ポンプ部１３０は、例えばバルブから構成されていてもよい。また、上記のバルブは、ダイアフラム部材を備えるダイアフラムバルブであり、ダイアフラム部材はエラストマー材料から形成されていてもよい。そして、ダイアフラム部材が変形することで流路１１０における流体Ｌの流れを制御し、ポンプ部１３０を含む領域１１５の体積を変動させるように構成されていてもよい。ここで、ダイアフラム部材の変形方向は、流体Ｌの流れを制御することができれば特に限定されず、例えば流路１１０の軸線に垂直な方向であってもよい。 << pump part >>
The pump unit 130 may be, for example, a valve. Also, the above-mentioned valve is a diaphragm valve provided with a diaphragm member, and the diaphragm member may be formed of an elastomeric material. Then, the flow of the fluid L in the flow path 110 may be controlled by the deformation of the diaphragm member, and the volume of the region 115 including the pump unit 130 may be changed. Here, the deformation direction of the diaphragm member is not particularly limited as long as the flow of the fluid L can be controlled, and may be, for example, a direction perpendicular to the axis of the flow path 110.
　図２（ａ）及び（ｂ）は、ダイアフラムバルブの構造の一例を説明する断面図である。図２（ａ）はダイアフラムバルブ２００の開状態を示し、図２（ｂ）はダイアフラムバルブ２００の閉状態を示す。図２（ａ）及び（ｂ）に示すように、ダイアフラムバルブ２００は、第１の基板２１０と、エラストマー材料からなるダイアフラム部材２３０と、第２の基板２２０とを備えている。第２の基板２２０とダイアフラム部材２３０とは密着した状態で接着されている。また、第１の基板２１０とダイアフラム部材２３０との間の空間は流体が流れる流路１１０を形成している。また、第２の基板２２０の一部には貫通孔２４０が設けられている。また、貫通孔２４０においては、ダイアフラム部材２３０が露出している。 FIGS. 2A and 2B are cross-sectional views for explaining an example of the structure of the diaphragm valve. 2A shows the diaphragm valve 200 in the open state, and FIG. 2B shows the diaphragm valve 200 in the closed state. As shown in FIGS. 2A and 2B, the diaphragm valve 200 includes a first substrate 210, a diaphragm member 230 made of an elastomeric material, and a second substrate 220. The second substrate 220 and the diaphragm member 230 are adhered in close contact with each other. Further, the space between the first substrate 210 and the diaphragm member 230 forms a flow path 110 through which the fluid flows. In addition, a through hole 240 is provided in part of the second substrate 220. Further, in the through hole 240, the diaphragm member 230 is exposed.
　ダイアフラムバルブ２００は、流路１１０に配置されており、流路１１０の内部の流体Ｌの流れ又は流体Ｌの圧力を制御するものである。 The diaphragm valve 200 is disposed in the flow passage 110 and controls the flow of the fluid L or the pressure of the fluid L inside the flow passage 110.
　図２（ａ）に示すダイアフラムバルブ２００の開状態では、流路１１０の内部を流体Ｌが流れることができる。一方、図２（ｂ）に示すように、ダイアフラムバルブ２００の貫通孔２４０からバルブ制御用の流体を供給し、貫通孔２４０の内部を加圧すると、ダイアフラム部材２３０が変形し、変形したダイアフラム部材２３０の一部が第１の基板２１０と密着する。この状態は、ダイアフラムバルブ２００の閉状態である。その結果、流路１１０の内部の流体Ｌの流れが遮断される。 In the open state of the diaphragm valve 200 shown in FIG. 2A, the fluid L can flow inside the flow path 110. On the other hand, as shown in FIG. 2B, when a fluid for valve control is supplied from the through hole 240 of the diaphragm valve 200 and the inside of the through hole 240 is pressurized, the diaphragm member 230 is deformed and the deformed diaphragm member Part of the substrate 230 is in close contact with the first substrate 210. This state is a closed state of the diaphragm valve 200. As a result, the flow of the fluid L inside the flow path 110 is shut off.
　ここで、図２（ａ）及び（ｂ）に示すように、ダイアフラムバルブ２００の閉状態をより強固なものとするために、貫通孔２４０と対向する第１の基板２１０の領域には凸部２１１が形成されていてもよい。 Here, as shown in FIGS. 2A and 2B, in order to make the closed state of the diaphragm valve 200 stronger, a convex portion is formed in the region of the first substrate 210 facing the through hole 240. 211 may be formed.
　バルブ制御用の流体としては、Ｎ２ガス、空気等の気体、水、油等の液体等が挙げられる。バルブ制御用の流体は、例えば、貫通孔２４０に接続されたチューブ等により供給することができる。あるいは、バルブの開閉は、機械的な力や電磁力で制御されてもよい。 Examples of the fluid for valve control include gases such as N 2 gas and air, and liquids such as water and oil. The fluid for valve control can be supplied, for example, by a tube or the like connected to the through hole 240. Alternatively, opening and closing of the valve may be controlled by mechanical force or electromagnetic force.
　また、ダイアフラム部材２３０を形成するエラストマー材料としては、貫通孔２４０の内部の圧力変化に応じて貫通孔２４０の軸線方向に変形可能な材料であれば特に限定されず、例えば、ポリジメチルシロキサン（ＰＤＭＳ）、ポリメチルフェニルシロキサン、ポリジフェニルシロキサン等のシリコーン系エラストマー等が挙げられる。 The elastomer material forming the diaphragm member 230 is not particularly limited as long as it is a material that can be deformed in the axial direction of the through hole 240 according to the pressure change inside the through hole 240, for example, polydimethylsiloxane (PDMS) And silicone-based elastomers such as polymethylphenylsiloxane and polydiphenylsiloxane.
　図２（ｂ）に示す閉状態のダイアフラムバルブ２００において、貫通孔２４０の内部に印加していた圧力を低下させると、変形していたダイアフラム部材２３０が元の形状に戻り、再び図２（ａ）に示す開状態となる。その結果、再び流路１１０の内部を流体Ｌが流れることができるようになる。 In the diaphragm valve 200 in the closed state shown in FIG. 2B, when the pressure applied to the inside of the through hole 240 is reduced, the deformed diaphragm member 230 returns to the original shape, and again FIG. It will be in the open state shown in). As a result, the fluid L can flow again inside the flow path 110.
　ダイアフラムバルブの構造は、上述したものに限られない。例えば、図３（ａ）及び（ｂ）に示すダイアフラムバルブ３００を用いることもできる。図３（ａ）はバルブ３００の開状態を示し、図３（ｂ）はバルブ３００の閉状態を示す。 The structure of the diaphragm valve is not limited to the one described above. For example, the diaphragm valve 300 shown in FIGS. 3A and 3B can also be used. 3A shows the valve 300 in the open state, and FIG. 3B shows the valve 300 in the closed state.
　図３（ａ）及び（ｂ）に示すように、バルブ３００は、上述したバルブ２００と比較すると、ダイアフラム部材２３０が第２の基板２２０の全面に配置されておらず、バルブ３００の周囲のみに局所的に配置されている点が主に異なっている。 As shown in FIGS. 3A and 3B, in the valve 300, compared with the valve 200 described above, the diaphragm member 230 is not disposed on the entire surface of the second substrate 220, and only around the valve 300. The points that are locally arranged are mainly different.
　バルブ３００のダイアフラム部材２３０は、アンカー部２３１を備えていることにより、ダイアフラム部材２３０を加圧により変形させた場合においても、ダイアフラム部材２３０が破損して第２の基板２２０から剥離することが抑制されている。 The diaphragm member 230 of the valve 300 is provided with the anchor portion 231, so that even when the diaphragm member 230 is deformed by pressure, it is suppressed that the diaphragm member 230 is damaged and peeled off from the second substrate 220. It is done.
《３連以上のバルブを含むポンプ部》
　流体デバイス１００のように、ポンプ部１３０は３連以上のバルブから構成されていてもよい。３連以上のバルブにより、すなわちバルブを３個以上並べることにより、ポンプ部１３０による流体Ｌの流れの制御をより効率よく行うことができる。 << Pump part including three or more valves >>
Like fluid device 100, pump portion 130 may be comprised of three or more valves. The flow control of the fluid L by the pump unit 130 can be more efficiently performed by three or more valves, that is, by arranging three or more valves.
　図４（ａ）～（ｄ）を用いて３連バルブの動作について説明する。図４（ａ）～（ｄ）は、３個のダイアフラムバルブ２００ａ、２００ｂ及び２００ｃを備えるポンプの一例の動作を説明する断面図である。 The operation of the triple valve will be described with reference to FIGS. 4 (a) to 4 (d). FIGS. 4A to 4D are cross-sectional views for explaining the operation of an example of a pump provided with three diaphragm valves 200a, 200b and 200c.
　図４（ａ）に示す状態では、３個のバルブ２００ａ、２００ｂ及び２００ｃは、いずれも開状態である。この状態では、流路１１０の内部の流体Ｌの流れは制御されていないため、流体Ｌは図４（ａ）に向かって右側に流れる場合もあるし、左側に流れる場合もあるし、流体の流れが停止（静止）している場合もある。 In the state shown in FIG. 4A, all the three valves 200a, 200b and 200c are in the open state. In this state, since the flow of the fluid L inside the flow passage 110 is not controlled, the fluid L may flow to the right or to the left as shown in FIG. The flow may be stopped (stationary).
　続いて、図４（ｂ）に示すように、バルブ２００ａを閉状態に制御し、バルブ２００ｂ及び２００ｃは開状態のままにする。この結果、流路１１０の内部の流体Ｌの流れはバルブ２００ａによって堰き止められる。 Subsequently, as shown in FIG. 4B, the valve 200a is controlled to be closed, and the valves 200b and 200c are kept open. As a result, the flow of the fluid L inside the flow path 110 is blocked by the valve 200a.
　続いて、図４（ｃ）に示すように、バルブ２００ａ及びバルブ２００ｂを閉状態に制御し、バルブ２００ｃは開状態のままにする。ここで、バルブ２００ｂが開状態から閉状態に変化する過程で、ダイアフラム部材２３０が変形することにより、バルブ２００ｂの周囲に存在していた流体Ｌが押しのけられる。ところが、バルブ２００ａが閉状態にあることから、押しのけられた流体は図４（ｃ）の矢印で示す方向、すなわち、図４（ｃ）に向かって右側に移動する。この結果、流体Ｌに矢印で示す方向の流れが生じる。また、流体Ｌが加圧される。 Subsequently, as shown in FIG. 4C, the valve 200a and the valve 200b are controlled to be closed, and the valve 200c is kept open. Here, in the process of changing the valve 200 b from the open state to the closed state, the fluid L existing around the valve 200 b is pushed away by the deformation of the diaphragm member 230. However, since the valve 200a is in the closed state, the displaced fluid moves in the direction shown by the arrow in FIG. 4C, that is, to the right in FIG. 4C. As a result, a flow in the direction indicated by the arrow occurs in the fluid L. Also, the fluid L is pressurized.
　続いて、図４（ｄ）に示すように、バルブ２００ｂ及び２００ｃを閉状態に制御する。すると、バルブ２００ｃが開状態から閉状態に変化する過程で、ダイアフラム部材２３０が変形することにより、バルブ２００ｃの周囲に存在していた流体Ｌが押しのけられる。ところが、バルブ２００ａが閉状態にあることから、押しのけられた流体Ｌは図４（ｄ）の矢印で示す方向、すなわち、図４（ｄ）に向かって右側に移動する。この結果、流体Ｌに矢印で示す方向の流れが更に加速される。また、流体Ｌが更に加圧される。この時、バルブ２００ａは図４（ｄ）に示すように開状態に制御してもよいし、閉状態のまま維持してもよい。 Subsequently, as shown in FIG. 4 (d), the valves 200b and 200c are controlled to be closed. Then, in the process of the valve 200 c changing from the open state to the closed state, the fluid L existing around the valve 200 c is pushed away by the deformation of the diaphragm member 230. However, since the valve 200a is in the closed state, the displaced fluid L moves in the direction shown by the arrow in FIG. 4 (d), that is, to the right in FIG. 4 (d). As a result, the flow in the direction indicated by the arrows in the fluid L is further accelerated. Also, the fluid L is further pressurized. At this time, the valve 200a may be controlled to be open as shown in FIG. 4 (d) or may be kept closed.
　続いて、再び図４（ａ）に示すように、バルブ２００ａ、２００ｂ及び２００ｃを、いずれも開状態に制御する。この結果、流体Ｌの加圧が解除される。また、この状態において、慣性により、流路１１０の内部の流体Ｌは、図４（ａ）に向かって右側に移動し続けている場合がある。 Subsequently, as shown in FIG. 4A again, the valves 200a, 200b and 200c are all controlled to be in the open state. As a result, the pressurization of the fluid L is released. Further, in this state, the fluid L inside the flow path 110 may continue to move to the right toward FIG. 4A due to inertia.
　更に、以上の工程を繰り返すことにより、流路１１０の内部の流体Ｌの流れ又は流体Ｌの圧力を制御することができる。 Furthermore, the flow of the fluid L inside the flow path 110 or the pressure of the fluid L can be controlled by repeating the above steps.
　以上の工程は、３個のバルブを備えるポンプの制御方法の一例であり、３個のバルブを備えるポンプの制御方法はこれに限られない。例えば、上述したバルブの制御において、バルブ２００ａとバルブ２００ｃの開閉のタイミングを逆にすることにより、流路１１０の内部の流体Ｌの流れを上述したものと逆方向に制御することもできる。また、図４（ａ）～（ｄ）の動作を繰り返す周期、バルブ２００ａ～２００ｃを駆動するバルブ制御用の流体の圧力、バルブの径等を調節することによっても、流体Ｌの流れ又は流体Ｌの圧力を制御することができる。 The above process is an example of a control method of a pump provided with three valves, and a control method of a pump provided with three valves is not limited to this. For example, in the control of the valve described above, the flow of the fluid L inside the flow passage 110 can be controlled in the reverse direction to that described above by reversing the timing of opening and closing the valve 200a and the valve 200c. The flow of the fluid L or the fluid L can also be adjusted by adjusting the cycle of repeating the operation of FIGS. The pressure of the can be controlled.
　また、バルブを４個以上備えるポンプにより、流路１１０の内部の流体Ｌの流れを制御することも可能である。 In addition, it is also possible to control the flow of the fluid L inside the flow passage 110 by a pump having four or more valves.
《区画バルブ》
　流体デバイス１００は、区画バルブ１４０を更に備えていてもよい。区画バルブ１４０（１４０ａ，１４０ｂ）を閉じて流路１１０のポンプ部１３０を含む領域１１５を画定したうえでポンプ部１３０を作動させることにより、より効率よく流体Ｌの圧力を制御することができる。 Division valve
Fluid device 100 may further include compartment valve 140. The pressure of the fluid L can be controlled more efficiently by closing the dividing valve 140 (140a, 140b) to define the region 115 including the pump portion 130 of the flow path 110 and operating the pump portion 130.
　流体デバイス１００では、区画バルブ１４０を閉じることで、ポンプ部１３０を含む領域１１５が画定された領域となる。すなわち、流体デバイス１００では、区画バルブ１４０（１４０ａ，１４０ｂ）を閉じることによって、流路１１０のポンプ部１３０を含む領域１１５が循環流路（ループ流路）となり、その他の部分から独立することができる。 In the fluid device 100, closing the partition valve 140 results in the area 115 including the pump portion 130 being defined. That is, in the fluid device 100, by closing the dividing valve 140 (140a, 140b), the area 115 including the pump portion 130 of the flow path 110 becomes a circulation flow path (loop flow path) and becomes independent from other parts. it can.
　その結果、流路１１０において、一時的に流体の流速が遅くなる、あるいは止まる。その後ポンプ部１３０を作動させると、領域１１５において流速が速くなり、流体Ｌに加えられる圧力が高まる。 As a result, in the flow path 110, the flow velocity of the fluid temporarily decreases or stops. Thereafter, when the pump unit 130 is operated, the flow velocity in the region 115 is increased, and the pressure applied to the fluid L is increased.
　また、例えば、ポンプ部１３０がダイアフラムバルブである場合、ダイアフラムバルブを閉じることでダイアフラム部材が流路１１０側に向かって変形し、領域１１５内の体積が小さくなる。その結果、領域１１５内の流体Ｌの圧力が高まる。一方、ダイアフラムバルブを開けることで、ダイアフラム部材は流路側に反する方向に変形し、領域１１５内の体積が大きくなる。その結果、流体Ｌの圧力が低下する。このようにして、領域１１５内の体積を変動させることで、流体Ｌの圧力を調節することができる。 Also, for example, when the pump portion 130 is a diaphragm valve, closing the diaphragm valve deforms the diaphragm member toward the flow path 110, and the volume in the region 115 is reduced. As a result, the pressure of the fluid L in the area 115 is increased. On the other hand, by opening the diaphragm valve, the diaphragm member is deformed in the direction opposite to the flow path side, and the volume in the region 115 is increased. As a result, the pressure of the fluid L decreases. Thus, the pressure of the fluid L can be adjusted by changing the volume in the region 115.
　区画バルブ１４０は、ダイアフラム部材を備えるダイアフラムバルブであってもよく、上述した、ポンプ部を構成するバルブと同様のものであってもよい。 The dividing valve 140 may be a diaphragm valve including a diaphragm member, and may be similar to the valve constituting the pump unit described above.
　区画バルブ１４０は、ノーマリークローズド・バルブであってもよく、ノーマリーオープン・バルブであってもよい。ノーマリークローズド・バルブは、定常状態において閉状態であり、バルブを作動させることによって開状態となるバルブである。また、ノーマリーオープン・バルブは、定常状態において開状態であり、バルブを作動させることによって閉状態となるバルブである。 Compartment valve 140 may be a normally closed valve or a normally open valve. The normally closed valve is a valve that is closed in a steady state and is opened by operating the valve. In addition, the normally open valve is a valve that is open in a steady state and is closed by operating the valve.
　区画バルブ１４０がノーマリークローズド・バルブである場合、定常状態において区画バルブ１４０は閉状態であり、バルブを作動させることによって、区画バルブ１４０により画定された領域１１５が解放される。 When the partition valve 140 is a normally closed valve, at steady state, the partition valve 140 is closed, and by operating the valve, the region 115 defined by the partition valve 140 is released.
　また、区画バルブ１４０がノーマリーオープン・バルブである場合、定常状態において区画バルブ１４０は開状態であり、バルブを作動させることによって閉状態となり、流路１１０の一部の領域１１５が区画バルブ１４０により画定された領域となる。 Also, when the dividing valve 140 is a normally open valve, the dividing valve 140 is open in the steady state and is closed by operating the valve, and a part of the flow path 110 is the dividing valve 140. It becomes an area defined by
　また、流体デバイス１００は、区画バルブ１４０を１個備えていてもよく、２個以上備えていてもよい。区画バルブ１４０が２個以上ある場合には、各区画バルブ１４０を閉じることによってポンプ部を含む領域１１５を画定し、一時的に独立した空間とすることができる。 In addition, the fluid device 100 may include one compartment valve 140 or two or more compartment valves. When there are two or more partition valves 140, closing each partition valve 140 defines a region 115 including the pump portion, which can be a temporarily independent space.
《細胞分析部》
　細胞分析部１２０は、流路１１０を流れる細胞を分析する領域である。ここで、細胞分析とは、細胞を含む流体の所定のパラメータを分析することを意味する。パラメータとしては特に限定されず、例えば、細胞を含む流体中の細胞数、細胞の大きさ等が挙げられる。細胞分析部は、例えばアパーチャ形状である。アパーチャ形状とは、細胞分析部１２０について流体の進行方向と直交する流路断面の断面積が、流路１１０について流体の進行方向と直交する流路断面の断面積よりも小さい形状である。例えば、細胞分析部１２０の前後の流路１１０の流路の断面積よりも、細胞分析部の断面積は小さい。例えば流路が円環状である場合、細胞分析部１２０の径は、流路１１０の他の領域の径と比較して小さい。すなわち、アパーチャ形状である細胞分析部の流路断面は、分析対象物の分析に適したサイズであってもよく、分析対象である細胞が一つずつ通過するサイズとしてもよい。これにより、少なくとも細胞分析部１２０では、細胞を１個ずつ通過させることが可能になる。その結果、１細胞ごとに細胞を検出することができるため、細胞の分析が容易になる。細胞の分析は、例えば、光学的に行ってもよいし、電気的に行ってもよい。細胞の分析項目は適宜設定することができる。例えば、細胞の数を測定してもよいし、細胞の大きさを測定してもよい。 << Cell Analysis Division >>
The cell analysis unit 120 is an area for analyzing cells flowing in the flow channel 110. Here, cell analysis means analyzing predetermined parameters of fluid containing cells. The parameters are not particularly limited, and include, for example, the number of cells in the fluid containing the cells, the size of the cells, and the like. The cell analysis unit has, for example, an aperture shape. The aperture shape is a shape in which the cross-sectional area of the flow channel cross section orthogonal to the flow direction of the fluid in the cell analysis unit 120 is smaller than the cross-sectional area of the flow channel cross section orthogonal to the flow direction of the fluid. For example, the cross-sectional area of the cell analysis unit is smaller than the cross-sectional area of the flow channels of the flow channels 110 before and after the cell analysis unit 120. For example, when the flow channel is annular, the diameter of the cell analysis unit 120 is smaller than the diameter of the other region of the flow channel 110. That is, the flow channel cross section of the cell analysis unit having an aperture shape may have a size suitable for analysis of an analyte, or may be a size in which cells to be analyzed pass one by one. Thereby, at least in the cell analysis unit 120, it becomes possible to pass cells one by one. As a result, since cells can be detected for each cell, analysis of cells is facilitated. Cell analysis may be performed, for example, optically or electrically. The analysis items of cells can be set as appropriate. For example, the number of cells may be measured, or the size of cells may be measured.
　流路及びアパーチャ形状の断面形状は矩形に限られず、例えば円形であってもよく、任意の多角形であってもよい。例えば、流路１１０の断面は、幅１．５ｍｍ、高さ０．３ｍｍの矩形であってもよい。また、アパーチャ形状である細胞分析部の断面は、幅３０μｍ、高さ３０μｍの矩形であってもよい。流路及びアパーチャ形状である細胞分析部が、このような形状及び大きさであると、例えば白血球が単一で通過することができる。 The cross-sectional shape of the flow path and the aperture shape is not limited to a rectangular shape, and may be, for example, a circular shape, or any polygonal shape. For example, the cross section of the flow channel 110 may be a rectangle having a width of 1.5 mm and a height of 0.3 mm. In addition, the cross section of the cell analysis unit having an aperture shape may be a rectangle having a width of 30 μm and a height of 30 μm. With such a shape and size, for example, white blood cells can pass through a single cell analysis unit that is in the form of flow paths and apertures.
《細胞分析部の位置》
　流体デバイス１００では、細胞分析部１２０が、流路１１０が備える循環流路の内部に存在している。すなわち、細胞分析部１２０が循環流路に存在している。細胞分析部１２０が循環流路の内部に存在することにより、ポンプ部１３０を作動させて細胞を破砕している最中に循環流路の内部の細胞の状態をリアルタイムで分析することができる。この結果、例えば、細胞破砕率が任意の値に達した時点でポンプ部１３０を停止させることや、細胞が完全に破砕されるまで細胞破砕操作を継続すること等が可能になる。 Position of cell analysis section
In the fluid device 100, the cell analysis unit 120 is present inside the circulation flow channel provided in the flow channel 110. That is, the cell analysis unit 120 is present in the circulation channel. The presence of the cell analysis unit 120 inside the circulation flow channel allows the state of the cells inside the circulation flow channel to be analyzed in real time while the pump unit 130 is operated to break up the cells. As a result, for example, it becomes possible to stop the pump unit 130 when the cell crushing rate reaches an arbitrary value, to continue the cell crushing operation until the cells are completely broken, and the like.
（第２実施形態）
　図５は、第２実施形態に係る流体デバイス５００の構造を説明する模式図である。第２実施形態の流体デバイス５００は、区画バルブ１４０ａ及び１４０ｂを閉じて流路１１０が備える循環流路が閉じた回路を形成した場合に細胞分析部１２０が回路の外部に存在する点において、第１実施形態に係る流体デバイス１００と主に異なる。すなわち、流体デバイス５００では、細胞分析部１２０が循環流路以外の流路に存在している。 Second Embodiment
FIG. 5 is a schematic view illustrating the structure of the fluid device 500 according to the second embodiment. The fluid device 500 according to the second embodiment is different from the fluid device 500 in that the cell analysis unit 120 exists outside the circuit when the partition valves 140a and 140b are closed to form a circuit in which the circulation channel of the channel 110 is closed. This differs mainly from the fluidic device 100 according to one embodiment. That is, in the fluid device 500, the cell analysis unit 120 is present in a flow channel other than the circulation flow channel.
　第２実施形態の流体デバイス５００では、細胞分析部１２０が回路の外部に存在することから、流路１１０の内部の流体Ｌの流れが細胞分析部１２０の影響を受けない。このため、第１実施形態に係る流体デバイス１００よりも、ポンプ部１３０の作動による流体Ｌの流れの制御の自由度が高い。 In the fluid device 500 of the second embodiment, the flow of the fluid L inside the flow path 110 is not influenced by the cell analysis unit 120 because the cell analysis unit 120 is present outside the circuit. Therefore, the degree of freedom in control of the flow of the fluid L by the operation of the pump unit 130 is higher than that of the fluid device 100 according to the first embodiment.
　第２実施形態の流体デバイス５００では、例えば、細胞の破砕を開始する前や、細胞の破砕を完了した後に、細胞分析部１２０で流路１１０を流れる細胞を分析することになる。 In the fluid device 500 according to the second embodiment, for example, the cell analysis unit 120 analyzes the cells flowing in the flow channel 110 before starting the cell disruption or after completing the cell disruption.
　流体デバイス５００では、細胞分析部１２０が排出用流路１１０ｂに存在している。この場合、流体Ｌを、導入用流路１１０ａ、循環流路、排出用流路１１０ｂの順に一方通行で流すことにより細胞を分析することができる。しかしながら、細胞分析部１２０の位置はこれに限られず、細胞分析部１２０は導入用流路１１０ａに存在していてもよい。この場合においても、流体Ｌを、導入用流路１１０ａ、循環流路の順に送液した後、再度導入用流路１１０ａに逆方向に流すことにより、細胞を分析することができる。 In the fluid device 500, the cell analysis unit 120 is present in the discharge channel 110b. In this case, cells can be analyzed by flowing the fluid L in one passage in the order of the introduction channel 110a, the circulation channel, and the discharge channel 110b. However, the position of the cell analysis unit 120 is not limited to this, and the cell analysis unit 120 may be present in the introduction channel 110a. Also in this case, after the fluid L is sent in the order of the introduction channel 110a and the circulation channel, cells can be analyzed by flowing the fluid L in the opposite direction again to the introduction channel 110a.
（変形例）
　図６は、流体デバイスの変形例を説明する模式図である。流体デバイス６００は、区画バルブ１４０の開閉を制御することにより流路１１０が備える循環流路が２個以上の閉じた回路を形成する点において、第１実施形態に係る流体デバイス１００と主に異なる。流体デバイス６００では、バルブ１４０の開閉を制御することにより、流路１１０の内部の流体Ｌの流れを制御し、細胞を分析することができる。 (Modification)
FIG. 6 is a schematic view for explaining a modification of the fluid device. The fluid device 600 is mainly different from the fluid device 100 according to the first embodiment in that the circulation channel provided in the flow channel 110 forms two or more closed circuits by controlling the opening and closing of the dividing valve 140. . In the fluid device 600, by controlling the opening and closing of the valve 140, the flow of the fluid L inside the flow path 110 can be controlled to analyze cells.
　流体デバイス６００では、区画バルブ１４０ａ１，１４０ａ２，１４０ｂ１，１４０ｂ２を閉じて流路１１０が回路を形成した場合に、細胞分析部１２０は回路の内部に存在している。しかしながら、細胞分析部１２０の位置はこれに限られず、回路の外部に存在する構成としてもよい。 In the fluid device 600, when the partition valves 140a1, 140a2, 140b1, and 140b2 are closed and the flow path 110 forms a circuit, the cell analysis unit 120 is present inside the circuit. However, the position of the cell analysis unit 120 is not limited to this, and may be configured to exist outside the circuit.
　図６に示すように、流体デバイス６００は、第１循環流路と第２循環流路とを含み、区画バルブ１４０を閉じることにより、第１循環流路及び第２循環流路が閉じた回路を形成する構成であってもよい。 As shown in FIG. 6, the fluid device 600 includes a first circulation channel and a second circulation channel, and the circuit in which the first circulation channel and the second circulation channel are closed by closing the dividing valve 140. May be formed.
　また、図６に示すように、流体デバイス６００における第１循環流路と第２循環流路とは、少なくとも一部の流路を共有し、共有する流路にポンプ部を備え、第１循環流路又は第２循環流路に細胞分析部１２０を備えていてもよい。 Further, as shown in FIG. 6, the first circulation channel and the second circulation channel in the fluid device 600 share at least a part of the channel, and include a pump unit in the shared channel, and the first circulation The cell analysis unit 120 may be provided in the channel or the second circulation channel.
　また、図６に示すように、流体デバイス６００は、第１循環流路と第２循環流路が共有する流路の両端の近傍における、第１循環流路及び第２循環流路が共有しない流路のそれぞれにバルブを有していてもよい。 Further, as shown in FIG. 6, in the fluid device 600, the first circulation channel and the second circulation channel are not shared in the vicinity of both ends of the channel shared by the first circulation channel and the second circulation channel. Each of the flow paths may have a valve.
［細胞を破砕する方法］
　一実施形態において、本発明は、細胞を破砕する方法であって、流路と、前記流路に設けられたポンプ部と、前記流路の前記ポンプ部を含む領域を画定するための区画バルブと、を備える流体デバイスの前記流路に、前記細胞を含む流体を流す工程と、前記区画バルブを閉じて前記流路の前記ポンプ部を含む領域を画定する工程と、前記ポンプ部を作動させて前記流体の圧力を変化させることにより前記細胞を破砕する工程と、を備える方法を提供する。 [How to disrupt cells]
In one embodiment, the present invention is a method of disrupting cells, comprising: a flow path, a pump portion provided in the flow path, and a compartment valve for defining a region including the pump portion of the flow path. And allowing the fluid containing the cells to flow in the flow path of the fluidic device, closing the compartment valve to define a region including the pump portion of the flow path, and operating the pump portion Breaking the cells by changing the pressure of the fluid.
　ここで、一例として、図１に示す流体デバイス１００を用いて細胞を破砕する方法について説明する。図１は、一実施形態に係る流体デバイス１００の構造を説明する模式図である。流体デバイス１００は、循環流路を備える流路１１０と、循環流路に設けられたポンプ部１３０と、循環流路を備える流路１１０に設けられたアパーチャ形状の細胞分析部１２０とを備えている。 Here, as an example, a method of disrupting cells using the fluid device 100 shown in FIG. 1 will be described. FIG. 1 is a schematic view illustrating the structure of a fluid device 100 according to an embodiment. The fluid device 100 includes a flow channel 110 including a circulation flow channel, a pump unit 130 provided in the circulation flow channel, and an aperture-shaped cell analysis unit 120 provided in the flow channel 110 including the circulation flow channel. There is.
　ここで、ポンプ部１３０は３連以上のバルブを含んでいてもよい。上述したように、３連以上のバルブにより、すなわちバルブを３個以上並べることにより、ポンプ部１３０による流体Ｌの流れの制御をより効率よく行うことができる。 Here, the pump unit 130 may include three or more valves. As described above, control of the flow of the fluid L by the pump unit 130 can be performed more efficiently by using three or more valves, that is, arranging three or more valves.
　流体デバイス１００では、区画バルブ１４０ａ，１４０ｂを閉じることにより、流路１１０の領域１１５が閉じた回路を形成する。 In the fluid device 100, by closing the partition valves 140a and 140b, the region 115 of the flow channel 110 forms a closed circuit.
　まず、流体デバイス１００の区画バルブ１４０ａ，１４０ｂを開放し、区画バルブ１４０ｃ，１４０ｄを閉じる。この状態で、導入用流路１１０ａから細胞を含む流体Ｌを導入する。細胞を含む流体Ｌは、例えば、赤血球及び白血球を含む血液由来試料であってもよい。その結果、流路１１０の内部が細胞を含む流体Ｌで満たされる。余剰の流体Ｌは排出用流路１１０ｂを通過して廃液タンク１５０に収容される。ここで、細胞を含む流体Ｌの流れは、ポンプ部１３０を作動させることによって生じさせてもよい。この場合、ポンプ部１３０の作動条件は、細胞を破砕しない条件としてもよい。 First, the partition valves 140a and 140b of the fluid device 100 are opened, and the partition valves 140c and 140d are closed. In this state, the fluid L containing cells is introduced from the introduction channel 110a. The fluid L containing cells may be, for example, a blood-derived sample containing red blood cells and white blood cells. As a result, the inside of the flow path 110 is filled with the fluid L containing cells. The excess fluid L passes through the discharge channel 110 b and is stored in the waste liquid tank 150. Here, the flow of the fluid L containing cells may be generated by operating the pump unit 130. In this case, the operating condition of the pump unit 130 may be a condition that does not crush cells.
　続いて、流路１１０の内部が細胞を含む流体Ｌで満たされた後、区画バルブ１４０ａ，１４０ｂを閉じ、１４０ｃを開放する。その結果、流路１１０の領域１１５が閉じた回路を形成する。 Subsequently, after the inside of the flow path 110 is filled with the fluid L containing cells, the compartment valves 140a and 140b are closed to open 140c. As a result, the region 115 of the flow channel 110 forms a closed circuit.
　続いて、細胞を含む流体Ｌの圧力を変化させる条件でポンプ部１３０を作動させる。ここで、流体Ｌの圧力を変化させる条件としては、ポンプの駆動時間、駆動圧、周波数等が挙げられる。その結果、領域１１５において流速が速くなり、流体Ｌに加えられる圧力が高まる。また、ポンプ部１３０がダイアフラムバルブである場合、ダイアフラムバルブを閉じることでダイアフラム部材が流路１１０側に向かって変形し、領域１１５内の体積が小さくなる。その結果、領域１１５内の流体Ｌの圧力が高まる。一方、ダイアフラムバルブを開けることで、ダイアフラム部材は流路側に反する方向に変形し、領域１１５内の体積が大きくなる。その結果、流体Ｌの圧力が低下する。このような圧力変化により、細胞が破砕される。 Subsequently, the pump unit 130 is operated under the condition of changing the pressure of the fluid L containing cells. Here, as the conditions for changing the pressure of the fluid L, the driving time of the pump, the driving pressure, the frequency and the like can be mentioned. As a result, the flow velocity in the region 115 is increased, and the pressure applied to the fluid L is increased. When the pump portion 130 is a diaphragm valve, the diaphragm member is deformed toward the flow passage 110 by closing the diaphragm valve, and the volume in the region 115 is reduced. As a result, the pressure of the fluid L in the area 115 is increased. On the other hand, by opening the diaphragm valve, the diaphragm member is deformed in the direction opposite to the flow path side, and the volume in the region 115 is increased. As a result, the pressure of the fluid L decreases. Such pressure changes disrupt the cells.
　ここで、圧力を変化させる条件を調整することにより、例えば、赤血球及び白血球を含む血液由来試料中の赤血球のみを選択的に破砕することもできる。また、流路１１０の領域１１５が閉じた回路を形成した状態でポンプ部１３０を作動させることにより、細胞を破砕する効率を高めることができる。 Here, it is also possible to selectively disrupt only red blood cells in a blood-derived sample containing, for example, red blood cells and white blood cells by adjusting the conditions under which the pressure is changed. In addition, by operating the pump unit 130 in a state in which the region 115 of the flow channel 110 is closed, the efficiency of cell disruption can be increased.
［血液由来試料中の赤血球及び白血球の数を測定する方法］
　一実施形態において、本発明は、血液由来試料中の赤血球及び白血球の数を測定する方法であって、流路と、前記流路に設けられたポンプ部と、前記流路に設けられたアパーチャ形状の細胞分析部と、を備える流体デバイスの前記流路に、前記血液由来試料を流す工程と、前記細胞分析部で赤血球及び白血球の数を測定する工程と、前記ポンプ部を作動させて前記血液由来試料の圧力を変化させることにより前記赤血球を選択的に破砕する工程と、赤血球を破砕した後に、前記細胞分析部で白血球の数を測定する工程と、を備える方法を提供する。本実施形態の方法は、血球算定（血算）を行う方法であるともいえる。 [Method for measuring the number of red blood cells and white blood cells in a blood-derived sample]
In one embodiment, the present invention is a method of measuring the number of red blood cells and white blood cells in a blood-derived sample, comprising: a flow path; a pump unit provided in the flow path; and an aperture provided in the flow path Flowing the blood-derived sample through the flow channel of the fluidic device having the shape of a cell analysis unit, measuring the number of red blood cells and white blood cells in the cell analysis unit, and operating the pump unit to operate the pump unit. A method is provided comprising the steps of: selectively disrupting the red blood cells by changing the pressure of a blood-derived sample; and measuring the number of white blood cells in the cell analysis unit after breaking the red blood cells. It can be said that the method of the present embodiment is a method of blood cell count (blood calculation).
　ここで、一例として、図５に示す流体デバイス５００を用いて血液由来試料中の赤血球及び白血球の数を測定する方法について説明する。 Here, as an example, a method of measuring the number of red blood cells and white blood cells in a blood-derived sample using the fluid device 500 shown in FIG. 5 will be described.
　まず、流体デバイス５００の区画バルブ１４０ａ，１４０ｂを開放し、区画バルブ１４０ｃ，１４０ｄを閉じる。この状態で、導入用流路１１０ａから血液由来試料を導入する。血液由来試料の導入は加圧により行ってもよいし、ポンプ部１３０を作動させることにより行ってもよい。この場合、ポンプ部１３０の作動条件は、細胞を破砕しない条件であることが好ましい。 First, the partition valves 140a and 140b of the fluid device 500 are opened, and the partition valves 140c and 140d are closed. In this state, a blood-derived sample is introduced from the introduction channel 110a. The introduction of the blood-derived sample may be performed by pressurization or may be performed by operating the pump unit 130. In this case, it is preferable that the operating condition of the pump unit 130 be a condition that does not crush cells.
　その結果、流路１１０の内部が血液由来試料で満たされる。余剰の血液由来試料は細胞分析部１２０及び排出用流路１１０ｂを通過して廃液タンク１５０に収容される。ここで、細胞分析部１２０で、細胞分析部１２０を通過する細胞の数を測定する。この時、細胞分析部１２０を通過する血液由来試料は、赤血球及び白血球が破砕されていない。このため、この段階で測定した細胞の数は、赤血球及び白血球の数の合計である。 As a result, the inside of the flow channel 110 is filled with the blood-derived sample. The excess blood-derived sample passes through the cell analysis unit 120 and the discharge channel 110 b and is stored in the waste liquid tank 150. Here, the cell analysis unit 120 measures the number of cells passing through the cell analysis unit 120. At this time, in the blood-derived sample that passes through the cell analysis unit 120, red blood cells and white blood cells are not broken. For this reason, the number of cells measured at this stage is the sum of the numbers of red blood cells and white blood cells.
　続いて、流路１１０の内部が血液由来試料で満たされた後、区画バルブ１４０ａ，１４０ｂを閉じ、１４０ｃを開放する。その結果、流路１１０の領域１１５が閉じた回路を形成する。 Subsequently, after the inside of the flow path 110 is filled with the blood-derived sample, the compartment valves 140a and 140b are closed to open 140c. As a result, the region 115 of the flow channel 110 forms a closed circuit.
　続いて、血液由来試料中の赤血球を選択的に破砕する条件でポンプ部１３０を作動させて、血液由来試料の圧力を変化させることにより、赤血球を選択的に破砕する。 Subsequently, the pump unit 130 is operated under conditions to selectively crush the red blood cells in the blood-derived sample to change the pressure of the blood-derived sample, thereby selectively crush the red blood cells.
　続いて、区画バルブ１４０ｂを開放し、血液由来試料を送液する。すると、流路１１０の内部の血液由来試料は細胞分析部１２０及び排出用流路１１０ｂを通過して廃液タンク１５０に収容される。 Subsequently, the compartment valve 140b is opened to feed the blood-derived sample. Then, the blood-derived sample in the flow path 110 passes through the cell analysis unit 120 and the discharge flow path 110 b and is stored in the waste liquid tank 150.
　この時、細胞分析部１２０で、細胞分析部１２０を通過する細胞の数を測定する。ここで、細胞分析部１２０を通過する血液由来試料は、赤血球のみが選択的に破砕されており、白血球は破砕されていない。このため、この段階で測定した細胞の数は、白血球の数である。 At this time, the cell analysis unit 120 measures the number of cells passing through the cell analysis unit 120. Here, in the blood-derived sample that passes through the cell analysis unit 120, only red blood cells are selectively broken, and white blood cells are not broken. Therefore, the number of cells measured at this stage is the number of white blood cells.
　以上の工程により、血液由来試料中の白血球の数を測定することができる。また、最初に測定した赤血球及び白血球の合計の数から、後で測定した白血球のみの数を差し引くことにより、赤血球の数をより正確に求めることができる。しかしながら、赤血球の数に対し、白血球の数は非常に少ない場合が通常であるため、最初に測定した赤血球及び白血球の合計の数を赤血球の数として用いてもよい。 Through the above steps, the number of white blood cells in the blood-derived sample can be measured. Also, the number of erythrocytes can be determined more accurately by subtracting the number of leukocytes measured later from the total number of erythrocytes and leukocytes measured first. However, since the number of white blood cells is usually very small relative to the number of red blood cells, the total number of red blood cells and white blood cells initially measured may be used as the number of red blood cells.
　血液由来試料中の赤血球を選択的に破砕する条件は、例えば、ダイアフラム部材を有するポンプ部の駆動圧力が、０ｋＰａ超１１０ｋＰａ、例えば５０～１１０ｋＰａ、例えば７０～１１０ｋＰａである条件であってもよい。また、ダイアフラム部材を有するポンプ部の駆動周波数が、０Ｈｚ超３．３Ｈｚ、例えば０．５～３．３Ｈｚ、例えば２～３．３Ｈｚである条件であってもよい。また、ポンプ部の作動時間は、例えば１～３０分間であってもよく、５～２０分間であってもよく、約１０分間であってもよい。 The conditions for selectively breaking up the red blood cells in the blood-derived sample may be, for example, conditions in which the driving pressure of the pump unit having a diaphragm member is more than 0 kPa, such as 50 to 110 kPa, such as 70 to 110 kPa. In addition, the drive frequency of the pump unit having the diaphragm member may be more than 0 Hz, for example, 0.5 to 3.3 Hz, for example, 2 to 3.3 Hz. The operating time of the pump unit may be, for example, 1 to 30 minutes, 5 to 20 minutes, or about 10 minutes.
　また、上記のダイアフラム部材の直径は、例えば１～３ｍｍであってもよく、２～３ｍｍであってもよい。また、ダイアフラム部材の厚さは、例えば１０～２０００μｍであってもよく、例えば１００～１０００μｍであってもよい。ダイアフラム部材の厚さは、薄いほど低駆動圧で必要変位が得られる。また、ダイアフラム部材の材質は、ポリジメチルシロキサン（ＰＤＭＳ）、ポリメチルフェニルシロキサン、ポリジフェニルシロキサン等のシリコーン系エラストマー等であってもよい。 In addition, the diameter of the above-mentioned diaphragm member may be, for example, 1 to 3 mm, or 2 to 3 mm. The thickness of the diaphragm member may be, for example, 10 to 2000 μm, and may be, for example, 100 to 1000 μm. As the thickness of the diaphragm member is thinner, the required displacement can be obtained at a lower driving pressure. Further, the material of the diaphragm member may be a silicone-based elastomer such as polydimethylsiloxane (PDMS), polymethylphenylsiloxane, polydiphenylsiloxane or the like.
　上記の条件では、赤血球を選択的に破砕することができる。上記の条件における白血球の破砕率は２．５％以下である。 Under the above conditions, erythrocytes can be selectively disrupted. The crushing rate of white blood cells under the above conditions is 2.5% or less.
　続いて、別の例として、図６に示す流体デバイス６００を用いて血液由来試料中の赤血球及び白血球の数を測定する方法について説明する。図７（ａ）～（ｃ）は、流体デバイス６００を用いて血液由来試料中の赤血球及び白血球の数を測定する方法を説明する模式図である。 Subsequently, as another example, a method of measuring the number of red blood cells and white blood cells in a blood-derived sample using the fluid device 600 shown in FIG. 6 will be described. FIGS. 7A to 7C are schematic diagrams illustrating a method of measuring the number of red blood cells and white blood cells in a blood-derived sample using the fluid device 600. FIG.
　まず、図７（ａ）に示すように、流体デバイス６００の区画バルブ１４０ａ２，１４０ｂ１，１４０ｃ１，１４０ｃ２，１４０ｃ５を開放し、区画バルブ１４０ａ１，１４０ｂ２，１４０ｃ３，１４０ｃ４を閉じる。続いて、導入用流路１１０ａ１から血液由来試料を導入する。血液由来試料の導入は加圧により行ってもよいし、ポンプ部１３０を作動させることにより行ってもよい。この場合、ポンプ部１３０の作動条件は、細胞を破砕しない条件であることが好ましい。 First, as shown in FIG. 7A, the partition valves 140a2, 140b1, 140c1, 140c2 and 140c5 of the fluid device 600 are opened, and the partition valves 140a1, 140b2, 140c3 and 140c4 are closed. Subsequently, a blood-derived sample is introduced from the introduction channel 110a1. The introduction of the blood-derived sample may be performed by pressurization or may be performed by operating the pump unit 130. In this case, it is preferable that the operating condition of the pump unit 130 be a condition that does not crush cells.
　その結果、図７（ａ）の矢印で示すように、流路１１０の内部を血液由来試料が流れ、流路１１０の内部が血液由来試料で満たされる。余剰の血液由来試料は排出用流路１１０ｂ１を通過して廃液タンク１５０に収容される。ここで、細胞分析部１２０で、細胞分析部１２０を通過する細胞の数を測定する。この時、細胞分析部１２０を通過する血液由来試料は、赤血球及び白血球が破砕されていない。このため、この段階で測定した細胞の数は、赤血球及び白血球の数の合計である。 As a result, as shown by the arrow in FIG. 7A, the blood-derived sample flows inside the flow channel 110, and the inside of the flow channel 110 is filled with the blood-derived sample. The excess blood-derived sample passes through the discharge channel 110 b 1 and is stored in the waste liquid tank 150. Here, the cell analysis unit 120 measures the number of cells passing through the cell analysis unit 120. At this time, in the blood-derived sample that passes through the cell analysis unit 120, red blood cells and white blood cells are not broken. For this reason, the number of cells measured at this stage is the sum of the numbers of red blood cells and white blood cells.
　続いて、流路１１０の内部が血液由来試料で満たされた後、図７（ｂ）に示すように、区画バルブ１４０ａ１，１４０ｂ１，１４０ｃ３，１４０ｃ５を閉じ、１４０ａ２，１４０ｂ２，１４０ｃ１，１４０ｃ２，１４０ｃ４を開放する。その結果、流路１１０の領域１１５が閉じた回路を形成する。 Subsequently, after the inside of the flow path 110 is filled with the blood-derived sample, as shown in FIG. 7B, the partition valves 140a1, 140b1, 140c3, 140c5 are closed, and the valves 140a2, 140b2, 140c1, 140c2, 140c4 are closed. Open. As a result, the region 115 of the flow channel 110 forms a closed circuit.
　続いて、血液由来試料中の赤血球を選択的に破砕する条件でポンプ部１３０を作動させて、血液由来試料の圧力を変化させることにより、赤血球を選択的に破砕する。この間、図７（ｂ）の矢印で示すように、血液由来試料は、流路１１０により形成された、閉じた回路の内部を循環することになる。 Subsequently, the pump unit 130 is operated under conditions to selectively crush the red blood cells in the blood-derived sample to change the pressure of the blood-derived sample, thereby selectively crush the red blood cells. During this time, as shown by the arrow in FIG. 7 (b), the blood-derived sample circulates inside the closed circuit formed by the flow path 110.
　また、図７（ｂ）の矢印で示すように、導入用流路１１０ａ２から、排出用流体を導入することにより、流路１１０の細胞分析部１２０を含む領域の内部に存在していた血液由来試料を排出することができる。排出用流体としては、Ｎ２ガス、空気等の気体、バッファー、油等の液体等が挙げられる。血液由来試料は、排出用流路１１０ｂ２を通過して廃液タンク１５０に収容される。 Further, as shown by the arrows in FIG. 7B, by introducing the discharge fluid from the introduction channel 110a2, the blood origin is present in the area including the cell analysis unit 120 of the channel 110. The sample can be drained. Examples of the discharge fluid include N 2 gas, gas such as air, buffer, liquid such as oil, and the like. The blood-derived sample passes through the discharge channel 110 b 2 and is stored in the waste liquid tank 150.
　続いて、図７（ｃ）に示すように、区画バルブ１４０ａ２，１４０ｂ１，１４０ｃ２，１４０ｃ５を閉じ、１４０ａ１，１４０ｂ２，１４０ｃ１，１４０ｃ３，１４０ｃ４を開放する。 Subsequently, as shown in FIG. 7C, the dividing valves 140a2, 140b1, 140c2, and 140c5 are closed, and the valves 140a1, 140b2, 140c1, 140c3, and 140c4 are opened.
　続いて、この状態で、導入用流路１１０ａ１から排出用流体を導入する。排出用流体の導入は加圧により行ってもよいし、ポンプ部１３０を作動させることにより行ってもよい。この場合、ポンプ部１３０の作動条件は、細胞を破砕しない条件であることが好ましい。 Subsequently, in this state, the discharge fluid is introduced from the introduction channel 110a1. The introduction of the discharge fluid may be performed by pressurization or may be performed by operating the pump unit 130. In this case, it is preferable that the operating condition of the pump unit 130 be a condition that does not crush cells.
　すると、図７（ｃ）の矢印で示すように、流路１１０の内部の血液由来試料は細胞分析部１２０及び排出用流路１１０ｂ２を通過して廃液タンク１５０に収容される。この時、細胞分析部１２０で、細胞分析部１２０を通過する細胞の数を測定する。ここで、細胞分析部１２０を通過する血液由来試料は、赤血球のみが選択的に破砕されており、白血球は破砕されていない。このため、この段階で測定した細胞の数は、白血球の数である。以上の工程により、血液由来試料中の白血球の数を測定することができる。 Then, as shown by the arrow in FIG. 7C, the blood-derived sample in the flow path 110 passes through the cell analysis unit 120 and the discharge flow path 110b2 and is stored in the waste liquid tank 150. At this time, the cell analysis unit 120 measures the number of cells passing through the cell analysis unit 120. Here, in the blood-derived sample that passes through the cell analysis unit 120, only red blood cells are selectively broken, and white blood cells are not broken. Therefore, the number of cells measured at this stage is the number of white blood cells. Through the above steps, the number of white blood cells in the blood-derived sample can be measured.
　次に実施例を示して本実施形態を説明するが、本発明は以下の実施例に限定されるものではない。 Next, the present embodiment will be described by way of examples, but the present invention is not limited to the following examples.
［実験例１］
（流体デバイスの製造） [Experimental Example 1]
(Manufacturing of fluid devices)
　また、図８（ｂ）に示すデバイス（以下、「デバイスＢ」という。）は、各バルブの直径が２ｍｍである以外はデバイスＡと同様であった。なお、デバイスＡ及びＢは、ポンプ部の作動により細胞を破砕できるか否かを評価することを目的として製造したものであり、細胞分析部は有していないものであった。 The device shown in FIG. 8B (hereinafter referred to as “device B”) was the same as device A except that the diameter of each valve was 2 mm. The devices A and B were manufactured for the purpose of evaluating whether or not cells can be disrupted by the operation of the pump unit, and the cell analysis unit was not included.
［実験例２］
（赤血球の破砕の検討）
　実験例１で製造したデバイスＡ及びＢを使用して、細胞の破砕を検討した。試料としては、ウサギの血液を用いた。 [Experimental Example 2]
(Examination of erythrocyte disruption)
Cell disruption was examined using devices A and B manufactured in Experimental Example 1. Rabbit blood was used as a sample.
　まず、各流体デバイスに試料を導入した。試料の導入量は、各デバイスにつき６２．５μＬずつであった。続いて、各デバイスのバルブ１４０を開閉し、流路１１０の閉じた回路を形成した。これにより、試料が各デバイスの回路内に密閉された。 First, a sample was introduced into each fluid device. The amount of sample introduced was 62.5 μL for each device. Subsequently, the valve 140 of each device was opened and closed to form a closed circuit of the flow path 110. Thereby, the sample was sealed in the circuit of each device.
　続いて、ポンプ部１３０を作動させ、細胞の破砕を行った。ポンプ部１３０の作動条件は、表１に示す各条件に設定した。また、ポンプ部１３０の作動時間は全ての条件において１０分間とした。 Subsequently, the pump unit 130 was operated to break up the cells. The operating conditions of the pump unit 130 were set to the conditions shown in Table 1. Moreover, the operating time of the pump part 130 was 10 minutes in all the conditions.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　続いて、各デバイスのバルブ１４０を開閉し、各デバイスから試料を回収した。続いて、回収した各試料の５７６ｎｍにおける吸光度を分光光度計（ＵＶ－１８００／島津製作所)を使用して測定した。陰性対照として生理食塩液の吸光度を測定した。また、陽性対照として生理食塩水の代わりに注射用水（大塚製薬）を添加して完全溶血させた試料の吸光度を測定した。各試料の溶血率は、下記式（１）を用いて算出した。
　溶血率（％）＝（試料の吸光度－陰性対照の吸光度）／（陽性対照の吸光度－陰性対照の吸光度）×１００　…（１） Subsequently, the valve 140 of each device was opened and closed, and the sample was collected from each device. Subsequently, the absorbance at 576 nm of each collected sample was measured using a spectrophotometer (UV-1800 / Shimadzu Corporation). Absorbance of physiological saline was measured as a negative control. In addition, as a positive control, water for injection (Otsuka Pharmaceutical) was added instead of physiological saline to measure the absorbance of a sample completely hemolyzed. The hemolysis rate of each sample was calculated using the following formula (1).
Hemolysis rate (%) = (absorbance of sample−absorbance of negative control) / (absorbance of positive control−absorbance of negative control) × 100 (1)
　図９は、溶血率の測定結果を示すグラフである。その結果、バルブの直径や駆動圧、周波数を高くすると、破砕される赤血球の割合が増加する傾向が認められた。 FIG. 9 is a graph showing the measurement results of the hemolytic rate. As a result, when the valve diameter, the driving pressure and the frequency were increased, the ratio of the erythrocytes to be crushed tended to increase.
［実験例３］
（細胞破砕処理が白血球に及ぼす影響の検討）
　実験例２と同様にして、ウサギの血液中の赤血球を破砕し、白血球に与える影響を検討した。 [Experimental Example 3]
(Study on the effect of cell disruption treatment on leukocytes)
In the same manner as in Experimental Example 2, erythrocytes in the blood of rabbits were crushed, and the influence on the leukocytes was examined.
　まず、デバイスＡに試料を導入した。試料としては、ウサギの血液を使用した。試料の導入量は６２．５μＬであった。続いて、デバイスＡのバルブ１４０を開閉し、流路１１０の閉じた回路を形成した。これにより、試料がデバイスＡの回路内に密閉された。 First, a sample was introduced into device A. As a sample, rabbit blood was used. The amount of sample introduced was 62.5 μL. Subsequently, the valve 140 of the device A was opened and closed to form a closed circuit of the flow path 110. Thereby, the sample was sealed in the circuit of device A.
　続いて、ポンプ部１３０を作動させ、細胞の破砕を行った。ポンプ部１３０の作動条件は、表２に示す各条件に設定した。また、ポンプ部１３０の作動時間は全ての条件において１０分間とした。 Subsequently, the pump unit 130 was operated to break up the cells. The operating conditions of the pump unit 130 were set to the conditions shown in Table 2. Moreover, the operating time of the pump part 130 was 10 minutes in all the conditions.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　続いて、デバイスＡのバルブ１４０を開閉し、試料を回収した。続いて、回収した各試料の溶血率を、実験例２と同様にして分光光度計（ＵＶ－１８００／島津製作所)を使用して測定した。また、ポアサイズ１．２μｍのフィルターを通過させることにより試料中の細胞を除去した後、各試料中のＤＮＡ量をリアルタイム定量ＰＣＲ（ライトサイクラー４８０システムＩＩ／ロシュ・ダイアグノスティックス社）を使用して測定した。白血球が破壊された場合、試料中のＤＮＡ量が増加する。元の試料中に含まれる全ＤＮＡ量を１００とした際の回収試料におけるＤＮＡ量の相対値を算出した。 Subsequently, the valve 140 of the device A was opened and closed to recover the sample. Subsequently, the hemolysis rate of each collected sample was measured in the same manner as in Experimental Example 2 using a spectrophotometer (UV-1800 / Shimadzu Corporation). In addition, after removing cells in the sample by passing through a filter with a pore size of 1.2 μm, the amount of DNA in each sample is subjected to real-time quantitative PCR (Light Cycler 480 System II / Roche Diagnostics). Measured. When white blood cells are destroyed, the amount of DNA in the sample increases. The relative value of the amount of DNA in the recovered sample was calculated when the total amount of DNA contained in the original sample was 100.
　図１０は、溶血率及びＤＮＡ量の測定結果を示すグラフである。その結果、デバイスＡで赤血球を破壊しても、白血球はほとんど破壊されていないことが明らかとなった。 FIG. 10 is a graph showing the measurement results of the hemolysis rate and the amount of DNA. As a result, it became clear that even if the red blood cells were destroyed by the device A, the white blood cells were hardly destroyed.
１００，５００，６００…流体デバイス、１１０…流路、１１０ａ，１１０ａ１，１１０ａ２…導入用流路、１１０ｂ，１１０ｂ１，１１０ｂ２…排出用流路、１１５…領域、１２０…細胞分析部、１３０，Ｐ…ポンプ部、１４０，１４０ａ，１４０ａ１，１４０ａ２，１４０ｂ，１４０ｂ１，１４０ｂ２，１４０ｃ，１４０ｃ１，１４０ｃ２，１４０ｃ３，１４０ｃ４，１４０ｃ５，１４０ｄ…区画バルブ、１５０…廃液タンク、２００，２００ａ，２００ｂ，２００ｃ，３００…ダイアフラムバルブ（バルブ）、２１０…第１の基板、２１１，２１１ａ，２１１ｂ，２１１ｃ…凸部、２２０…第２の基板、２３０…ダイアフラム部材、２３１…アンカー部、２４０…貫通孔、Ｌ…流体。 100, 500, 600: fluid device, 110: flow path, 110a, 110a1, 110a2: introduction flow path, 110b, 110b1, 110b2: discharge flow path, 115: area, 120: cell analysis unit, 130, P: Pump parts 140, 140a, 140a1, 140a2, 140b, 140b1, 140b2, 140c1, 140c2, 140c3, 140c4, 140c5, 140d ... division valves, 150 ... waste liquid tanks, 200, 200a, 200b, 200c, 300 ... diaphragms Valve (valve) 210: first substrate 211, 211a, 211b, 211c: convex portion 220: second substrate 230: diaphragm member 231: anchor portion 240: through hole L: fluid.

Claims (19)

  1. 　循環流路を備える、細胞を含む流体を流すための流路と、
    　前記循環流路に設けられ、前記流体の流れ又は前記流体の圧力を制御するポンプ部と、
    　前記流路に設けられた細胞分析部と、
    　を備え、
    　前記細胞分析部の断面積は、前記細胞分析部以外の流路の断面積と比べて小さい、流体デバイス。     A flow path for flowing a fluid containing cells, comprising a circulation flow path;
    A pump unit provided in the circulation flow path for controlling the flow of the fluid or the pressure of the fluid;
    A cell analysis unit provided in the flow path;
    Equipped with
    The fluid device, wherein the cross-sectional area of the cell analysis unit is smaller than the cross-sectional area of the flow path other than the cell analysis unit.
  2. 　前記ポンプ部を含む領域を画定するための区画バルブを更に備える、請求項１に記載の流体デバイス。 The fluidic device of claim 1, further comprising a compartment valve for defining an area including the pump portion.
  3. 　前記区画バルブは前記循環流路以外の流路に設けられ、前記区画バルブを閉じることにより、前記循環流路が閉じた回路を形成する、請求項２に記載の流体デバイス。 The fluid device according to claim 2, wherein the dividing valve is provided in a flow channel other than the circulation flow channel, and closing the dividing valve forms a closed circuit of the circulation flow channel.
  4. 　前記細胞分析部が前記循環流路に存在する、請求項１～３のいずれか一項に記載の流体デバイス。 The fluid device according to any one of claims 1 to 3, wherein the cell analysis unit is present in the circulation channel.
  5. 　前記細胞分析部が前記循環流路以外の流路に存在する、請求項１～３のいずれか一項に記載の流体デバイス。 The fluid device according to any one of claims 1 to 3, wherein the cell analysis unit is present in a flow channel other than the circulation channel.
  6. 　前記ポンプ部は３連以上のバルブを含む、請求項１～５のいずれか一項に記載の流体デバイス。 The fluid device according to any one of claims 1 to 5, wherein the pump unit includes three or more valves.
  7. 　前記ポンプ部はダイアフラム部材を有し、
    　前記ダイアフラム部材が変形することで前記流路における流体の流れを制御し、前記ポンプ部を含む領域の体積を変動させる、請求項２～６のいずれか一項に記載の流体デバイス。     The pump portion has a diaphragm member,
    The fluid device according to any one of claims 2 to 6, wherein the deformation of the diaphragm member controls the flow of fluid in the flow passage, and the volume of the region including the pump portion is varied.
  8. 　前記流体が血液由来試料であり、前記細胞が赤血球及び白血球を含む、請求項１～７のいずれか一項に記載の流体デバイス。 The fluid device according to any one of the preceding claims, wherein the fluid is a blood-derived sample and the cells comprise red blood cells and white blood cells.
  9. 　前記循環流路は、第１循環流路と第２循環流路とを含み、
    　前記第１循環流路と前記第２循環流路とは、少なくとも一部の流路を共有する、請求項３～８のいずれか一項に記載の流体デバイス。     The circulation channel includes a first circulation channel and a second circulation channel,
    The fluid device according to any one of claims 3 to 8, wherein the first circulation channel and the second circulation channel share at least a part of a channel.
  10. 　前記共有する流路に、前記ポンプ部を備え、
    　前記第１循環流路又は前記第２循環流路に前記細胞分析部を備える、請求項９に記載の流体デバイス。     The pump unit is provided in the shared flow path,
    The fluid device according to claim 9, wherein the cell analysis unit is provided in the first circulation channel or the second circulation channel.
  11. 　前記第１循環流路と第２循環流路とが共有する流路の両端の近傍における、前記第１循環流路及び前記第２循環流路が共有しない流路のそれぞれにバルブを有する、請求項９又は１０に記載の流体デバイス。 In the vicinity of both ends of the flow path shared by the first circulation flow path and the second circulation flow path, valves are provided in each of the flow paths not shared by the first circulation flow path and the second circulation flow path. Item 11. The fluid device according to item 9 or 10.
  12. 　請求項１～１１のいずれか一項に記載の流体デバイスを備え、
    　前記ポンプ部が、前記流体の流れ又は前記流体の圧力を、（ｉ）試料中の赤血球も白血球も破砕せずに試料を循環させられる程度、及び（ｉｉ）試料中の赤血球のみ破砕し、白血球は破砕せずに試料を循環させられる程度、に制御可能に構成されている、血液算定用流体デバイス。     A fluidic device according to any one of the preceding claims,
    To the extent that the pump unit circulates the flow of the fluid or the pressure of the fluid (i) without breaking the red blood cells or white blood cells in the sample, and (ii) only red blood cells in the sample, white blood cells A fluid device for calculating blood that is configured to be controllable to the extent that the sample can be circulated without breaking.
  13. 　細胞を破砕する方法であって、
    　流路と、前記流路に設けられたポンプ部と、前記流路の前記ポンプ部を含む領域を画定するための区画バルブと、を備える流体デバイスの前記流路に、前記細胞を含む流体を流す工程と、
    　前記区画バルブを閉じて前記流路の前記ポンプ部を含む領域を画定する工程と、
    　前記ポンプ部を作動させて前記流体の圧力を変化させることにより前記細胞を破砕する工程と、
    　を備える方法。     A method of disrupting cells,
    A fluid containing a cell is contained in the flow path of a fluid device including a flow path, a pump portion provided in the flow path, and a dividing valve for defining a region including the pump portion of the flow path. Flowing step,
    Closing the partition valve to define an area including the pump portion of the flow path;
    Crushing the cells by operating the pump unit to change the pressure of the fluid;
    How to provide.
  14. 　前記流体デバイスは、前記流路に設けられたアパーチャ形状の細胞分析部を更に備えており、
    　前記細胞分析部において、前記流体中の細胞を分析する工程を更に備える、請求項１３に記載の方法。     The fluid device further includes an aperture-shaped cell analysis unit provided in the flow path,
    The method according to claim 13, further comprising the step of analyzing cells in the fluid in the cell analysis unit.
  15. 　前記区画バルブを閉じることにより、前記流路が閉じた回路を形成する、請求項１３又は１４に記載の方法。 15. A method according to claim 13 or 14, wherein closing the partition valve causes the flow path to form a closed circuit.
  16. 　前記ポンプ部は３連以上のバルブを含む、請求項１３～１５のいずれか一項に記載の方法。 The method according to any one of claims 13 to 15, wherein the pump part comprises three or more valves.
  17. 　前記流体は複数種類の細胞を含み、細胞を破砕する前記工程において、少なくとも１種類の細胞を破砕する条件で前記ポンプ部を作動させる、請求項１３～１６のいずれか一項に記載の方法。 The method according to any one of claims 13 to 16, wherein the fluid contains a plurality of types of cells, and in the step of disrupting the cells, the pump unit is operated under conditions for disrupting at least one type of cells.
  18. 　前記流体が血液由来試料であり、前記細胞が赤血球及び白血球を含む、請求項１３～１７のいずれか一項に記載の方法。 The method according to any one of claims 13 to 17, wherein the fluid is a blood-derived sample and the cells comprise red blood cells and white blood cells.
  19. 　血液由来試料中の赤血球及び白血球の数を測定する方法であって、
    　流路と、前記流路に設けられたポンプ部と、前記流路に設けられたアパーチャ形状の細胞分析部と、を備える流体デバイスの前記流路に、前記血液由来試料を流す工程と、
    　前記細胞分析部で赤血球及び白血球の数を測定する工程と、
    　前記ポンプ部を作動させて前記血液由来試料の圧力を変化させることにより前記赤血球を選択的に破砕する工程と、
    　赤血球を破砕した後に、前記細胞分析部で白血球の数を測定する工程と、
    　を備える方法。     A method of measuring the number of red blood cells and white blood cells in a blood-derived sample, comprising:
    Flowing the blood-derived sample through the flow path of the fluidic device comprising a flow path, a pump portion provided in the flow path, and an aperture-shaped cell analysis portion provided in the flow path;
    Measuring the number of red blood cells and white blood cells in the cell analysis unit;
    Selectively crushing the red blood cells by operating the pump unit to change the pressure of the blood-derived sample;
    Measuring the number of white blood cells in the cell analysis unit after crushing the red blood cells;
    How to provide.
PCT/JP2017/031936 2017-09-05 2017-09-05 Fluid device and use thereof WO2019049204A1 (en)

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Citations (3)

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JP2008136475A (en) * 2006-11-10 2008-06-19 Univ Waseda Cell catching device and cell catching method using the same
JP2014132860A (en) * 2013-01-10 2014-07-24 Aquatech Co Ltd Micropump unit
WO2016013395A1 (en) * 2014-07-22 2016-01-28 株式会社日立ハイテクノロジーズ Cell dispersion measurement mechanism, and cell subculture system utilizing same

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Publication number Priority date Publication date Assignee Title
US20070105206A1 (en) 2005-10-19 2007-05-10 Chang Lu Fluidic device

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008136475A (en) * 2006-11-10 2008-06-19 Univ Waseda Cell catching device and cell catching method using the same
JP2014132860A (en) * 2013-01-10 2014-07-24 Aquatech Co Ltd Micropump unit
WO2016013395A1 (en) * 2014-07-22 2016-01-28 株式会社日立ハイテクノロジーズ Cell dispersion measurement mechanism, and cell subculture system utilizing same

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