EP3300805B1 - Mikrovorrichtung zum abfangen von partikeln und verfahren zum erfassen, konzentrieren oder abtrennen von partikeln damit - Google Patents

Mikrovorrichtung zum abfangen von partikeln und verfahren zum erfassen, konzentrieren oder abtrennen von partikeln damit Download PDF

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Publication number
EP3300805B1
EP3300805B1 EP17194232.9A EP17194232A EP3300805B1 EP 3300805 B1 EP3300805 B1 EP 3300805B1 EP 17194232 A EP17194232 A EP 17194232A EP 3300805 B1 EP3300805 B1 EP 3300805B1
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EP
European Patent Office
Prior art keywords
flow channel
channel chamber
less
particles
enlarged portion
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EP17194232.9A
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English (en)
French (fr)
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EP3300805A1 (de
Inventor
Teruo Fujii
Soo Hyeon Kim
Hiroshi Ito
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Arkray Inc
University of Tokyo NUC
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Arkray Inc
University of Tokyo NUC
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C5/00—Separating dispersed particles from liquids by electrostatic effect
    • B03C5/02—Separators
    • B03C5/022—Non-uniform field separators
    • B03C5/026—Non-uniform field separators using open-gradient differential dielectric separation, i.e. using electrodes of special shapes for non-uniform field creation, e.g. Fluid Integrated Circuit [FIC]

Definitions

  • the present disclosure relates to a microdevice for capturing particles in a sample, a method for capturing particles in a sample, and a method for concentrating particles using the same.
  • the microdevice, the capturing method, and the concentrating method of the present disclosure can be used to capture, or concentrate cells in a sample.
  • JP 2012-34641A discloses a microchamber array apparatus that is capable of capturing an object to be examined, with use of dielectrophoresis (DEP) and disrupting an object to be examined, with use of cell-disruption (electroporation, EP) in a single well.
  • JP 2008-249513A discloses an apparatus for dividing, with use of dielectrophoresis, liquid containing microparticles into a concentrated liquid having a high concentration of microparticles and a diluted liquid having a low concentration of microparticles.
  • US 2009/0148937 A1 discloses a cell sorting apparatus comprising a cage-like electrode.
  • US 2009/0148937 A1 discloses a cell sorting apparatus comprising a cage-like electrode.
  • the cell sorting apparatus comprises an inlet, an outlet, and a channel connecting the inlet and outlet.
  • the channel comprises a channel widening, in which both the height and the width of the channel are enlarged.
  • the cage-like electrode is discussed as being a dielectrophoretically acting electrode arrangement.
  • US 2009/0148937 A1 also refers to " A 3-D microelectrode system for handling and caging single cells and particles" by T. Muller et al, published in Biosensors and Bioelectronics, vol. 14, no. 3, 1 March 1999 . This article discloses that the electrode arrangement may be disposed on the bottom of the channel.
  • US 2006/121555 A1 discloses a flow cell for collecting and concentrating a sample dispersed in a flowing medium.
  • the device comprises a body defining an inlet, an outlet, and opposing bottom and top walls extending at least partially therebetween and defining an expansion cavity.
  • the cavity includes a collection wall extending from a downstream region of the bottom wall.
  • the device also comprises a travelling wave grid disposed along the bottom wall and adapted to transport particulates proximate to the grid, to the collection wall. In the expansion region, the height of the cavity increases by reducing the height of the bottom wall.
  • US 2009/0155877 A1 discloses a biochip for lysing and/or cell separation wherein a microfluidic channel is formed between two electrodes.
  • WO 2016/062878 A1 discloses a microfluidic system having a capture area.
  • CTCs circulating tumor cells
  • immune cells circulating tumor cells
  • CTCs are cells that separate from primary tumor tissues or metastatic tumor tissues and invade blood, and thus it has been reported that the number of CTCs in blood relates to the possibility of cancer metastasis and prognosis. Thus, there is demand to accurately analyze these cells.
  • rare cells such as the above-described CTCs are present in a sample in an extremely small amount, such as about several cells. Therefore, for the convenience of use for analysis, rare cells need to be collected in a state in which particles included in the sample are concentrated.
  • loss of cells caused by centrifugation which is a common concentrating method, is a very significant problem.
  • centrifugation is used to concentrate the sample, the degree of loss that occurs is high between workers, experiments, or the like, and thus centrifugation is problematic for reproducibility.
  • the present disclosure relates to a device and a method with which particles such as rare cells in a sample can be captured precisely, and preferably relates to a device and a method with which particles such as rare cells in a sample can be concentrated.
  • the present disclosure relates to a microdevice for capturing particles according to the independent claim 1, the microdevice comprising: an inlet; an outlet; and a flow channel comprising a flow channel chamber that connects the inlet and the outlet, wherein the flow channel chamber has an enlarged portion in which a cross-sectional area of the flow channel enlarges in a height direction with respect to a bottom surface of the flow channel chamber by increasing the height of an upper surface of the flow channel chamber and a portion upstream of the enlarged portion, wherein the bottom surface of the flow channel chamber is a flat surface, and wherein the flow channel chamber is provided with an electric field generation means which is disposed on the bottom surface of the flow channel chamber at least in the enlarged portion or the vicinity of the enlarged portion.
  • the present disclosure relates to a method for capturing particles in a sample in a flow channel chamber of a microdevice, the microdevice being the above-described microdevice for capturing particles, the method including causing the electric field generation means of the microdevice to generate an electric field, and introducing the sample into the flow channel chamber from the inlet of the microdevice.
  • the present disclosure relates to a method for concentrating, separating, observing, or collecting particles in a sample, the method including capturing particles in a sample with the method for capturing particles of the present disclosure.
  • the present disclosure it is possible to precisely capture particles such as rare cells in a sample. Also, in at least preferred embodiments of the present disclosure, it is possible to capture or collect particles such as rare cells in a sample with a high reproducibility while reducing loss. Also, in at least preferred embodiments of the present disclosure, it is possible to concentrate, separate, observe, or collect particles such as rare cells in a sample.
  • the present disclosure is based on new findings found by the inventors that when a non-uniform electric field is generated in a flow channel chamber having an enlarged portion in which a cross-sectional area of a flow channel enlarges from the upstream side toward the downstream side and a cell liquid is introduced into the flow channel chamber from the upstream side of the flow channel chamber in this state, cells that flow at a reduced flow speed in the enlarged portion can be more easily captured with a dielectrophoretic force and cells can be concentrated.
  • the present disclosure is based on new findings found by the inventors that the efficiency of concentrating cells can be increased by utilizing the dielectrophoretic force of cells and the flow channel chamber having the enlarged portion in which the cross-sectional area of the flow channel enlarges.
  • a distribution of flow velocities occurs in the flow channel such that the flow velocity is lower on the wall surface sides (for example, the upper surface side and the bottom surface side) of the flow channel compared to the central portion of the flow channel, and thus, by reducing the height of the flow channel in front of the enlarged portion, a state is created in which particles are physically close to the bottom surface side such that particles reach the enlarged portion or the vicinity thereof in a state in which the particles are close to the bottom surface side on which the flow velocity is low. Furthermore, because the dielectrophoretic force has a stronger effect the closer the distance to the electric field generation means is, a strong dielectrophoretic force is applied to particles that are close to the bottom surface by installing the electric field generation means on the bottom surface of the flow channel.
  • the present disclosure relates to a microdevice (the microdevice of the present disclosure) for capturing particles in a sample through dielectrophoresis.
  • the microdevice of the present disclosure includes an inlet, an outlet, and a flow channel comprising a flow channel chamber that connects the inlet and the outlet, in which the flow channel chamber has an enlarged portion in which the cross-sectional area of the flow channel enlarges from the inlet toward the outlet , and the flow channel chamber is provided with an electric field generation means disposed at least in the enlarged portion or the vicinity of the enlarged portion.
  • the microdevice of the present disclosure in one or more embodiments, it is possible to precisely capture particles such as rare cells in a sample. Also, according to the microdevice of the present disclosure, in one or more embodiments, it is possible to efficiently concentrate particles. In one or more embodiments, the microdevice of the present disclosure enables observation, analysis, or collection of captured or concentrated particles.
  • the flow channel chamber in the microdevice of the present disclosure connects the inlet and the outlet, and is capable of ejecting, from the outlet, a sample that is introduced from the inlet. Also, particles captured in the flow channel chamber can be collected from the flow channel chamber by introducing a collection liquid from the inlet or the outlet.
  • the flow channel chamber in the microdevice of the present disclosure has an enlarged portion in which the cross-sectional area of the flow channel enlarges from the inlet toward the outlet.
  • the flow velocity of a sample (the speed of particles) can be rapidly reduced in a state in which particles in the sample that is introduced into the flow channel chamber are close to the electric field generation means (for example, electrodes), and a dielectrophoresis force generated by the electric field generation means in the flow channel chamber can be applied to particles whose speed is reduced.
  • the electric field generation means for example, electrodes
  • the cross-sectional area of the flow channel enlarges in a height direction or, in one or more embodiments, in both the height direction and the width direction with respect to the bottom surface of the flow channel chamber. Enlargement of the flow channel in the height direction is achieved by increasing the height of the upper surface of the flow channel chamber.
  • enlargement of the flow channel in the height direction may be achieved by enlarging the flow channel in the height direction at 90 degrees (in the perpendicular direction to a direction in which the sample flows) or at approximately 90 degrees, enlarging the flow channel in a linear manner, in a gradual manner, or in a curved manner in the height direction from the inlet toward the outlet, or enlarging the flow channel with a combination of these manners.
  • gradual enlargement includes stepwise (including single step) enlargement.
  • enlargement of the flow channel in the width direction can be achieved by increasing the width of the flow channel chamber.
  • enlargement of the flow channel in the width direction may be achieved by enlarging the width of the flow channel at 180 degrees (in the horizontal direction with respect to a direction in which the sample flows) or at approximately 180 degrees, enlarging the width in a linear manner, in a gradual manner, or in a curved manner in the width direction from the inlet toward the outlet, or enlarging the width with a combination of these manners.
  • gradual enlargement includes stepwise (including single step) enlargement.
  • enlarged portion when the enlarged portion enlarges in a linear manner or in a curved manner in the height direction and/or the width direction from the inlet toward the outlet, a region extending from a portion at which the enlargement starts to a portion having the maximum height and/or a portion having the maximum width is referred to as "enlarged portion".
  • the flow channel enlarges in the height direction.
  • an example of the enlarged portion includes a portion in which the cross-sectional area of the flow channel that is orthogonal to a straight line direction between the inlet and the outlet (the direction in which the sample flows) enlarges, too.
  • the cross-sectional area of the flow channel refers to the area of the cross section of the flow channel chamber in a direction that is orthogonal to the direction in which the sample flows. In one or more embodiments, enlargement of the cross-sectional area is achieved by the flow channel cross-sectional area of the flow channel chamber being larger than the flow channel cross-sectional area that is located in front of the enlarged portion.
  • the flow channel cross-sectional area need only be determined as appropriate in accordance with the captured particles, the sample, the flow velocity, and the like.
  • the flow channel cross-sectional area of flow channel chamber is 1.5 times or more, 2 times or more, 2.5 times or more, 3 times or more, 3.5 times or more, 4 times or more, 4.5 times or more, 5 times or more, 5.5 times or more, or 6 times or more the flow channel cross-sectional area that is located in front of the enlarged portion.
  • a flow channel cross-sectional area ratio between the enlarged portion and the portion that is located in front of the enlarged portion is 1.5 or more, 2 or more, 2.5 or more, 3 or more, 3.5 or more, 4 or more, 4.5 or more, 5 or more, 5.5 or more, or 6 or more, and/or 10 or less, 9 or less, 8 or less, or 7 or less.
  • the flow channel cross-sectional area of the enlarged portion refers to the flow channel cross-sectional area having the maximum flow channel cross-sectional area in the enlarged portion.
  • the flow channel cross-sectional area of a portion that is located in front of the enlarged portion refers to the flow channel cross-sectional area that is located on the upstream side of the enlarged portion and just before the flow channel cross-sectional area changes (enlarges).
  • the enlarged portion enlarges in the height direction with respect to the bottom surface of the flow channel chamber.
  • a ratio (He/Hb) between a height (He) of the enlarged portion and a height (Hb) of the portion that is located in front of the enlarged portion (enlargement change point) need only be determined as appropriate in accordance with the captured particles, the sample, the flow velocity, and the like, and in one or more embodiments, the ratio (He/Hb) is 1.5 or more, and from the viewpoint of reducing the flow velocity and further increasing the capture ratio in the enlarged portion, the ratio (He/Hb) is preferably 1.5 or more, 2 or more, 2.5 or more, 3 or more, 3.5 or more, 4 or more, 4.5 or more, 5 or more, 5.5 or more, or 6 or more.
  • the upper limit of the above-described ratio (He/Hb) is 10 or less, 9 or less, 8 or less, or 7 or less.
  • the height (He) of the enlarged portion refers to the height of a portion of the flow channel chamber having the maximum height in the enlarged portion.
  • the height (Hb) of the portion that is located in front of the enlarged portion refers to the height of the flow channel chamber that is located on the upstream side of the enlarged portion and just before the flow channel cross-sectional area increases.
  • the height (He) of the enlarged portion may be 100 ⁇ m or more, and from the viewpoint of reducing the flow velocity in the enlarged portion, in one or more embodiments, the height (He) is 100 pm or more, 200 pm or more, 300 ⁇ m or more, 400 ⁇ m or more, 500 pm or more, or 600 ⁇ m or more. In one or more embodiments, the height of the enlarged portion is 1000 ⁇ m or less, 900 ⁇ m or less, 800 ⁇ m or less, or 700 ⁇ m or less.
  • the height (Hb) of the portion that is located in front of the enlarged portion is 200 ⁇ m or less, 150 ⁇ m or less, 100 ⁇ m or less, 50 ⁇ m or less, or 40 ⁇ m or less. In one or more embodiments, the height (Hb) of the portion that is located in front of the enlarged portion is 20 ⁇ m or more or 30 ⁇ m or more.
  • the width of the flow channel chamber in this embodiment is 0.05 mm or more, 0.1 mm or more, or 0.5 mm or more, and from the viewpoint of concentrating particles, the width is 50 mm or less, 40 mm or less, 30 mm or less, 20 mm or less, 10 mm or less, 9 mm or less, 8 mm or less, 7 mm or less, 6 mm or less, 5 mm or less, 4 mm or less, 3 mm or less, 2 mm or less, or 1 mm or less.
  • the width of the flow channel chamber refers to the length of the flow channel in a direction that is orthogonal to the direction in which the sample flows.
  • the height (He) of the enlarged portion, the height (Hb) of the portion that is located in front of the enlarged portion, the width of the flow channel chamber, and the like need only be determined as appropriate in accordance with the captured particles, the sample, the flow velocity, and the like.
  • a ratio (We/ Wb) between a width (We) of the enlarged portion and a width (Wb) of the portion that is located in front of the enlarged portion is 1.5 or more, and from the viewpoint of reducing the flow velocity and further increasing the capture ratio in the enlarged potion, the ratio (We/Wb) is preferably 1.5 or more, 2 or more, 2.5 or more, 3 or more, 3.5 or more, 4 or more, 4.5 or more, 5 or more, 5.5 or more, or 6 or more. Also, in one or more embodiments, the upper limit of the above-described ratio (We/Wb) is 10 or less, 9 or less, 8 or less, or 7 or less.
  • the width (We) of the enlarged portion refers to the width of a portion of the flow channel chamber having the maximum width in the enlarged portion.
  • the width (Wb) of the portion that is located in front of the enlarged portion refers to the width of the flow channel chamber that is located on the upstream side of the enlarged portion and just before the flow channel cross-sectional area increases.
  • the width (We) of the enlarged portion may be 0.075 mm or more, and from the viewpoint of reducing the flow velocity and further increasing the capture ratio in the enlarged portion, in one or more embodiments, the width (We) is 0.1 mm or more, 0.2 mm or more, 0.3 mm or more, 0.4 mm or more, 0.5 mm or more, 1 mm or more, 2 mm or more, 3 mm or more, 4 mm or more, 5 mm or more, 6 mm or more, 7 mm or more, 8 mm or more, 9 mm or more, or 10 mm or more.
  • the width (We) of the enlarged portion is 500 mm or less, 400 mm or less, 300 mm or less, 200 mm or less, 100 mm or less, 90 mm or less, 80 mm or less, 70 mm or less, 60 mm or less, 50 mm or less, 40 mm or less, 30 mm or less, or 20 mm or less.
  • the width (Wb) of the portion that is located in front of the enlarged portion is 50 mm or less, 40 mm or less, 30 mm or less, 20 mm or less, 10 mm or les, 9 mm or less, 8 mm or less, 7 mm or less, 6 mm or less, 5 mm or less, 4 mm or less, 3 mm or less, 2 mm or less, or 1 mm or less.
  • the width (Wb) of the portion that is located in front of the enlarged portion is 0.05 mm or more, 0.1 mm or more, or 0.5 mm or more.
  • the height of the flow channel chamber from the inlet side to the enlargement change point is 200 ⁇ m or less, 150 ⁇ m or less, 100 ⁇ m or less, 50 ⁇ m or less, 40 um or less, 30 ⁇ m or less, or 20 ⁇ m or less. In one or more embodiments, the height of the flow channel chamber is 20 ⁇ m or more or 30 ⁇ m or more.
  • the length of the flow channel chamber is 0.05 mm or more, 0.1 mm or more, 0.5 mm or more, or 1 mm or more, and from the viewpoint of concentrating particles, the length is 100 mm or less, 50 mm or less, 40 mm or less, 30 mm or less, 20 mm or less, 10 mm or less, 9 mm or less, 8 mm or less, 7 mm or less, 6 mm or less, 5 mm or less, 4 mm or less, 3 mm or less, or 2 mm or less.
  • the length of the flow channel chamber refers to the length of the flow channel in the direction in which the sample flows.
  • the volume (capacity) of the flow channel chamber is 10 pl or more, 100 pl or more, 1 nl or more, 10 nl or more, 0.1 ⁇ l or more, 0.2 ⁇ l or more, 0.3 ⁇ l or more, 0.4 ⁇ l or more, 0.5 ⁇ l or more, 0.6 ⁇ l or more, 0.7 ⁇ l or more, 0.8 ⁇ l or more, 0.9 ⁇ l or more, or 1 ⁇ l or more, and/or 10 ml or less, 5 ml or less, 1 ml or less, 0.5 ml or less, 0.3 ml or less, 0.1 ml or less, 90 ⁇ l or less, 80 ⁇ l or less, 70 ⁇ l or less, 60 ⁇ l or less, 50 ⁇ l or less, 40 ⁇ l or less, 30 ⁇ l or less, 20 ⁇ l or less, or 10 ⁇ l or less.
  • the width (We) of the enlarged portion, the width (Wb) of the portion that is located in front of the enlarged portion, the length of the flow channel chamber, the volume of the flow channel chamber, and the like need only be determined as appropriate in accordance with the captured particles, the sample, the flow velocity, and the like.
  • the bottom surface of the flow channel chamber is a flat surface.
  • the flow channel chamber is provided with an electric field generation means for causing an electric field.
  • the microdevice of the present disclosure is capable of generating a non-uniform electric field by applying an electric field to the electric field generation means disposed in the flow channel chamber and causing dielectrophoresis. From the viewpoint of further increasing the particle capture ratio, it is sufficient that the electric field generation means is disposed at least in the enlarged portion or the vicinity thereof. In one or more embodiments, disposing the electric field generation means in the enlarged portion or the vicinity thereof is achieved by disposing the electric field generation means at a position that faces the enlarged portion when the enlarged portion has a shape such that one wall surface enlarges upward.
  • the electric field generation means is disposed on the bottom surface of the flow channel chamber. From the viewpoint of further increasing the particle capture ratio by reducing the flow velocity in the enlarged portion in a state in which particles are close to the bottom surface of the flow channel chamber, the electric field generation means is disposed at least on the bottom surface of the flow channel chamber that faces the enlarged portion.
  • an example of the electric field generation means is a counter electrode for dielectrophoresis.
  • the bottom surface of the flow channel chamber is provided with a counter electrode for dielectrophoresis.
  • the microdevice of the present disclosure may be capable of generating a non-uniform electric field by applying an electric field to the counter electrode disposed on the bottom surface of the flow channel chamber, and causing dielectrophoresis.
  • the electrodes need only be disposed at least near the enlarged portion, and from the viewpoint of further increasing the particle capture ratio, it is preferable that the electrodes are disposed on the entire bottom surface of the flow channel chamber from the upstream side to the downstream side. In one or more embodiments, the electrodes are preferably disposed on the bottom surface of the inner wall surface of the flow channel chamber.
  • the electrodes there is no particular limitation on the form of the electrodes, and in one or more embodiments, an example of the electrode is a comb electrode (interdigital electrode).
  • the comb electrodes are preferably disposed such that longitudinal directions of electrode fingers of the comb electrodes are orthogonal to the straight line direction (the direction in which the sample flows) between the inlet and the outlet.
  • the width of electrodes is 0.1 ⁇ m or more, 0.5 ⁇ m or more, 1 ⁇ m or more, 2 ⁇ m or more, 3 ⁇ m or more, 4 ⁇ m or more, 5 ⁇ m or more, 6 ⁇ m or more, 7 ⁇ m or more, 8 ⁇ m or more, 9 ⁇ m or more, or 10 pm or more, and/or 5000 ⁇ m or less, 1000 pm or less, 900 ⁇ m or less, 800 pm or less, 700 ⁇ m or less, 600 ⁇ m or less, 500 ⁇ m or less, 400 ⁇ m or less, 300 ⁇ m or less, 200 ⁇ m or less, or 100 ⁇ m or less.
  • the widths of electrodes may be equal to or different from each other. In the present disclosure, the width of electrodes refers to the length of electrodes in the direction in which the sample flows.
  • the gap between electrodes is 1 ⁇ m or more, 2 ⁇ m or more, 3 ⁇ m or more, 4 ⁇ m or more, 5 ⁇ m or more, 6 ⁇ m or more, 7 ⁇ m or more, 8 ⁇ m or more, 9 ⁇ m or more, or 10 ⁇ m or more, and/or 1000 ⁇ m or less, 900 ⁇ m or less, 800 ⁇ m or less, 700 ⁇ m or less, 600 ⁇ m or less, 500 ⁇ m or less, 400 ⁇ m or less, 300 ⁇ m or less, 200 ⁇ m or less, or 100 ⁇ m or less.
  • the gap between electrodes refers to an interval (distance) between electrodes that are adjacent in the direction in which the sample flows.
  • the thickness of the electrode is 0.1 nm or more, 0.5 nm or more, 1 nm or more, 2 nm or more, 3 nm or more, 4 nm or more, 5 nm or more, 6 nm or more, 7 nm or more, 8 nm or more, 9 nm or more, or 10 nm or more, and/or 1000 nm or less, 900 nm or less, 800 nm or less, 700 nm or less, 600 nm or less, or 500 nm or less.
  • the length of each electrode finger can be determined as appropriate in accordance with the width of the flow channel chamber. In one or more embodiments, the length of each electrode finger is 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, or 70% or more, and/or 100% or less, 95% or less, 90% or less, or 85% or less of the width of the flow channel chamber. From the viewpoint of further increasing the particle capture ratio, in one or more embodiments, the electrode fingers are preferably disposed entirely across the width of the flow channel chamber.
  • examples of the material of the electrode include indium tin oxide (ITO), titanium, chromium, gold, platinum, ZnO (zinc oxide), fluorine-doped tin oxide (FTO), silver, copper, and conductive materials (conductive polymer and the like).
  • the electrodes are preferably transparent to enable the captured particles to be easily observed or analyzed.
  • positions at which the inlet and the outlet are formed are side surfaces, an upper surface, or a lower surface of the microdevice.
  • the material of the microdevice includes glass, molten silica, and resins such as plastic.
  • the plastic include polymethyl methacrylate (PMMA), polycarbonate, polystyrene, polytetrafluoroethylene (PTFE), polyether ether ketone (PEEK), and silicone.
  • the microdevice is preferably transparent to enable the captured particles to be easily observed or analyzed.
  • the microdevice of the present disclosure can be manufactured by forming electrodes on a substrate, and joining the substrate provided with the electrodes and a substrate provided with a flow channel chamber including an enlarged portion in which the cross-sectional area of the flow channel enlarges, for example.
  • the present disclosure relates to a method for manufacturing a microdevice including forming electrodes on a substrate and joining the substrate provided with the electrodes and a substrate provided with a flow channel chamber including an enlarged portion in which the cross-sectional area of the flow channel enlarges from the upstream side toward the downstream side.
  • the substrates are joined so as to cover the electrodes formed on the substrate provided with the flow channel chamber.
  • electrodes can be formed with a conventionally known method.
  • examples of the formation method include photolithography technology and printing technology such as screen printing, gravure printing, and flexographic printing.
  • a flow channel can be formed with a conventionally known method.
  • examples of the formation method include cutting technology and casting technology.
  • the present disclosure relates to a method for capturing particles in a sample (a capturing method of the present disclosure) including generating, in a flow channel chamber having an enlarged portion in which the cross-sectional area of a flow channel enlarges (optionally from the upstream side toward the downstream side), an electric field for applying an dielectrophoretic force to particles, and introducing a sample containing particles into the flow channel chamber from the upstream side of the flow channel chamber.
  • the time at which the electric field starts to be generated and the time at which the sample starts to be introduced into the inlet may be the same, or the electric field may start to be generated after the sample is introduced and before the sample reaches the enlarged portion.
  • the capturing method of the present disclosure because the sample containing particles is introduced from the upstream side of the flow channel chamber into the flow channel chamber having the enlarged portion in which the cross-sectional area of the flow channel enlarges from the upstream side toward the downstream side, it is possible to precisely capture particles and easily concentrate particles.
  • the capturing method of the present disclosure is performed using the microdevice of the present disclosure.
  • the present disclosure relates to a method for capturing particles in a sample in a flow channel chamber of a microdevice, in which the microdevice is the microdevice of the present disclosure, and the method for capturing particles includes causing the electric field generation means of the microdevice to generate an electric field, and introducing the sample into the flow channel chamber from the inlet of the microdevice.
  • the electric field need only be generated in a portion corresponding to at least the enlarged portion.
  • the capturing method of the present disclosure includes generating an electric field for applying a dielectrophoretic force to particles on at least part of a bottom surface of the flow channel chamber or the entire bottom surface, and from the viewpoint of further increasing the capture ratio, the capturing method includes generating an electric field for applying a dielectrophoretic force to particles on the bottom surface of the flow channel chamber in the enlarged portion, or generating an electric field for applying a dielectrophoretic force to particles on the entire bottom surface of the flow channel chamber.
  • the flow rate of the sample may be 1 ⁇ L/min or more, 2 ⁇ L/min or more, 3 ⁇ L/min or more, 4 ⁇ L/min or more, 5 ⁇ L/min or more, 6 ⁇ L/min or more, 7 ⁇ L/ min or more, 8 ⁇ L/min or more, 9 ⁇ L/min or more, or 10 ⁇ L/min or more.
  • the flow rate may be 1000 ⁇ L/min or less, 900 ⁇ L/min or less, 800 ⁇ L/min or less, 700 ⁇ L/min or less, 600 ⁇ L/min or less, 500 ⁇ L/min or less, 400 ⁇ L/min or less, 300 ⁇ L/min or less, 200 ⁇ L/min or less, or 100 ⁇ L/min or less.
  • the amount of the sample introduced into the flow channel chamber preferably exceeds the capacity of the flow channel chamber (i.e. such that the sample may overflow from the outlet).
  • the capturing method of the present disclosure includes introducing the sample in an amount exceeding the capacity of the flow channel chamber (i.e. such that the sample may overflow from the outlet).
  • the electric field can be generated by applying an alternating voltage to electrodes that are disposed on the bottom surface of the flow channel chamber.
  • the applied voltage is 0.1 V or more, 0.5 V or more, 1 V or more, 2 V or more, 3 V or more, 4 V or more, 5 V or more, 6 V or more, 7 V or more, 8 V or more, 9 V or more, or 10 V or more, and in one or more embodiments, the applied voltage is 100 V or less, 90 V or less, 80 V or less, 70 V or less, 60 V or less, 50 V or less, 40 V or less, or 30 V or less.
  • the applied frequency need only be a frequency with which particles can be captured on the electrodes, and in one or more embodiments, the applied frequency is 1 kHz or more, 5 kHz or more, 10 kHz or more, 50 kHz or more, 100 kHz or more, 200 kHz or more, 300 kHz or more, 400 kHz or more, 500 kHz or more, 600 kHz or more, 700 kHz or more, 800 kHz or more, 900 kHz or more, or 1 MH or more, and/or 100 MHz or less, 90 MHz or less, 80 MHz or less, 70 MHz or less, 60 MHz or less, 50 MHz or less, 40 MHz or less, 30 MHz or less, 20 MHz or less, or 10 MHz or less.
  • the sample includes particles and a solvent (liquid) that suspends or disperses the particles.
  • an example of the particles is cells.
  • an example of the cells is rare cells such as CTCs.
  • examples of the rare cells include human colon cancer cells, human stomach cancer cells, human large bowel cancer cells, human lung cancer cells.
  • the solvent in which particles are suspended or dispersed is desired to have an electrical conductivity (conductance) that is as low as possible.
  • the solvent preferably contains an electrolyte in a small amount.
  • the solvent is preferably a non-electrolytic isotonic solution such as a sucrose isotonic solution.
  • the present disclosure relates to a method for concentrating particles in a sample that includes capturing particles in a sample with the capturing method of the present disclosure.
  • the concentrating method of the present disclosure may include introducing a collection liquid into a flow channel chamber that captured particles, and collecting the particles captured in the flow channel chamber from the flow channel chamber.
  • the present disclosure relates to a method for collecting particles in a sample that includes capturing particles, in a flow channel chamber, in a sample with the capturing method of the present disclosure, introducing a collection liquid in the flow channel chamber, and collecting the particles captured in the flow channel chamber from the flow channel chamber.
  • the present disclosure relates to a method for analyzing particles in a sample that includes capturing particles in a sample with the capturing method of the present disclosure.
  • the present disclosure relates to a method for observing or analyzing particles that includes capturing, in the flow channel chamber, particles in a sample with the capturing method of the present disclosure, and observing or analyzing the particles captured in the flow channel chamber.
  • particles can be observed through microscopy or the like.
  • particles can be analyzed with the microdevice of the present disclosure, and for example, after the particles are captured in the flow channel chamber, the particles can be analyzed in the flow channel chamber.
  • the microdevice and the capturing method of the present disclosure in one or more embodiments, it is possible to capture, in different capture regions, particles that have different balances between the dielectrophoretic force received from the electric field and the resistance received from the liquid flow. That is, according to the microdevice and the capturing method of the present disclosure, in one or more embodiments, if the sample contains a plurality of types of particles (for example, if the sample contains two or more types of particles having different balances between the dielectrophoretic force received from the electric field and the resistance received from liquid flow), these particles can be captured in different capture regions.
  • the present disclosure relates to a method for separating particles in a sample, and the method for separating particles includes generating an electric field for applying a dielectrophoretic force to particles, in a flow channel chamber having an enlarged portion in which the cross-sectional area of a flow channel enlarges from the upstream side toward the downstream side, and introducing the sample containing particles into the flow channel chamber from the upstream side of the flow channel chamber.
  • the separation method of the present disclosure in one or more embodiments, it is possible to capture particles having different balances between the dielectrophoretic force received from the electric field and resistance received from liquid flow in different capture regions.
  • the separation method of the present disclosure in one or more embodiments, if the sample contains CTCs and white blood cells, white blood cells that receive a small resistance from liquid flow can be captured on the upstream side of the enlarged portion where there is a high cross-sectional flow velocity, and CTCs that receive a larger resistance from liquid flow than white blood cells can be captured in the vicinity of the enlarged portion where there is a low cross-sectional flow velocity.
  • a plurality of types of particles included in a sample can be separated.
  • the present disclosure relates to a method for separating particles in a sample that includes generating an electric field for applying a dielectrophoretic force to particles, at least in the enlarged portion or the vicinity of the enlarged portion in a flow channel chamber having an enlarged portion in which the cross-sectional area of a flow channel enlarges from the upstream side toward the downstream side, introducing the sample containing particles into the flow channel chamber from the upstream side of the flow channel chamber, and separating a plurality of types of particles included in the sample.
  • FIG. 1 is a schematic diagram of one embodiment of the microdevice of the present disclosure.
  • (A) is a top view of a microdevice 1
  • (B) is a cross-sectional view taken along a direction I-I in (A)
  • (C) is a cross-sectional view taken along a direction II-II in (B).
  • the microdevice 1 has an inlet 10, a flow channel chamber 11, an outlet 12, and comb electrodes 13.
  • the inlet 10 and the outlet 12 are formed on an upper surface of the microdevice 1, and are continuous with the flow channel chamber 11 formed in a longitudinal direction along a bottom surface of the microdevice 1.
  • a portion of the flow channel chamber 11 that is in contact with the inlet 10 may be provided with a tapered portion which is widening from the inlet toward to the enlarged portion 14.
  • a configuration in which no tapered portion is provided may be adopted.
  • the flow channel chamber 11 has an enlarged portion 14 in which the cross-sectional area of a flow channel enlarges in the height direction.
  • the enlarged portion 14 is formed in an approximately central portion of the flow channel chamber 11.
  • a ratio (He/Hb) between a height (He) of the enlarged portion 14 and a height (Hb) at an enlargement change point is approximately 3.
  • a height (Hu) of the flow channel chamber 11 on the upstream side of the enlarged portion 14 is equal to the height (Hb) at the enlargement change point. That is, in the microdevice 1 in FIG. 1 , the height from the most upstream portion of the flow channel chamber 11 to the enlargement change point is approximately constant.
  • the height (He) of the enlarged portion 14 is equal to a height (Hd) of the flow channel chamber 11 on the downstream side of the enlarged portion 14. That is, in the microdevice 1 in FIG. 1 , the height from the enlarged portion 14 to the most downstream portion of the flow channel chamber 11 is approximately constant.
  • the height of the flow channel (the cross-sectional area of the flow channel) enlarges rapidly.
  • the height (Hb) at the enlargement change point is preferably as low as possible in order to reduce the distance to the electrodes as much as possible when the flow velocity of cells rapidly decreases due to the cells reaching the enlarged portion 14, and as a result, to further increase the cell capture ratio.
  • the height (He) of the enlarged portion 14 can be determined as appropriate in accordance with the height (Hb) at the enlargement change point.
  • the comb electrodes 13 are formed on the upper surface of a substrate that constitutes the bottom surface of the flow channel chamber 11.
  • a microdevice shown in FIG. 1 was produced with a procedure below.
  • the flow chamber of the microdevice has a tapered portion which is extending in width and which is formed on the inlet side, and an enlarged portion in which the cross-sectional area of a flow channel enlarges in the height direction and which is formed on an approximately central portion of the flow channel chamber.
  • the height (Hb) of the portion that is located in front of the enlarged portion (on the upstream side (the inlet side) of the enlarged portion) was approximately 50 pm
  • the height (He) of the enlarged portion was approximately 100 ⁇ m
  • the volume of the flow channel chamber was approximately 4 ⁇ l.
  • SNU-1 cells that were stained with Celltracker green were added to a dispersion liquid (a buffer for dielectrophoresis) below, and the liquid containing these cells was fed to a microdevice under conditions below, and the cells were captured. After the end of liquid feeding, the number of cells that were captured in the flow channel chamber was measured using a microscope, and the capture ratio was obtained by dividing the value by the number of added cells. The results are shown in Table 1 below.
  • Example 1 Comp. Ex. 1 Comp. Ex. 2 Enlarged portion present not present not present Height ( ⁇ m) 50 ⁇ 100 100 50 Width (mm) 7 7 7 Length (mm) 10 10 10 Volume ( ⁇ L) 3.9 5.3 2.6 Capture ratio (%) 95 41 42
  • the device of Example 1 having the enlarged portion captured cells at a capture ratio that was higher than that of the devices of Comparative Examples 1 and 2 having no enlarged portion.
  • the volume of the flow channel chamber was 3.9 ⁇ l, and thus the cells were significantly concentrated by introducing a cell liquid into the device of Example 1 such that the volume of the cell liquid was reduced to 1/50 or less that of the cell liquid before the introduction (treatment) (50-fold concentrated).
  • Example 1 Although the experiment was performed with a treatment liquid amount of 200 ⁇ l in Example 1, it was possible to similarly perform treatment at a high capture ratio with a treatment liquid amount of 1 ml or more, and the cells were concentrated 250-fold or more with this method.
  • Example 2 An experiment was performed similarly to Example 1 except that SNU-1 cells that were stained with Celltracker green and were treated, fixed, and underwent membrane permeabilization treatment under treatment conditions below using paraformaldehyde (PFA) and Tween20 were used. The results are shown in Table 2 below and FIG. 2 .
  • PFA paraformaldehyde
  • Example 2 Comp. Ex. 3
  • Comp. Ex. 4 Enlarged portion present not present not present Height ( ⁇ m) 50 ⁇ 100 100 50 Width (mm) 7 7 7 Length (mm) 10 10 10 Volume ( ⁇ L) 3.9 5.3 2.6 Capture ratio (%) 83 53 4
  • the device of the Example 2 having the enlarged portion captured cells at a capture ratio that was higher than that of the devices of Comparative Examples 3 and 4 having no enlarged portion.
  • the volume of the flow channel chamber was 3.9 pL, and thus the volume of the cell liquid was reduced to 1/50 or less that of the cell liquid before the introduction (treatment) by introducing the cell liquid into the device of Example 2, and as a result, the cells were easily concentrated significantly.
  • FIG. 2 is an image showing the distribution of the captured cells in Example 2 and Comparative Example 3.
  • FIG. 2(a) is the image of Example 2
  • FIG. 2(b) is the image of Comparative Example 3.
  • FIGS. 2(c) and 2(d) show enlarged views of the region surrounded with white broken lines in FIGS. 2(a) and 2(b) .
  • White dots in FIGS. 2(a) to 2(d) show the captured cells.
  • locations at which the cells were captured were distributed.
  • FIGS. 2(a) and 2(c) in the device of Example 2, many cells were captured in the central portion of the device, that is, near the enlarged portion. That is, with the device of Example 2, cells were captured locally and the captured cells were observed easily.
  • Example 3 Capture ratio Height ( ⁇ m) 50 ⁇ 100 50 ⁇ 300 Flow rate ( ⁇ L/min) 50 108% 116% 100 82% 98% 200 - 77%
  • Example 2 1 mL of cell liquid containing cells that were fixed and underwent membrane permeabilization treatment in Example 2 was introduced into a microcentrifuge tube and centrifuged for 5 minutes at 200 ⁇ g so as to collect the cells. The number of collected cells was measured and their collection ratio was obtained. As a result, the collection ratio was 22%.
  • Example 1 Similarly to Example 1, 1 ml of cell liquid that was the same as that of Comparative Example 5 was concentrated with the microdevice of the present disclosure. As a result, the ratio of collecting cells that were collected in the device was 98% (treatment flow rate: 50 ⁇ L/min).
  • Example 2 Similarly to Example 1, 1 mL of cell liquid containing SW620 cells (human colon cancer cells) that were stained with Celltracker green was concentrated with the microdevice of the present disclosure (treatment flow rate: 20 ⁇ L/min). Next, 10 ⁇ l of PBS (-) was fed with a pipette from the outlet of the microdevice, and the cells that were captured in the microdevice were collected from the microdevice. The number of collected cells was measured using a microscope, and the collection ratio was obtained by dividing the value by the number of cells (rough number) in the cell liquid before liquid feeding. The results are shown in Table 4 below.
  • Example 4 1 ml of cell liquid containing SW620 cells that were treated under the conditions below was concentrated with the microdevice of the present disclosure, and the number of cells that were captured in the device was measured using a microscope.
  • 20 ⁇ l of PBS (-) was fed with a pipette from the outlet of the microdevice, and the cells that were captured in the microdevice were collected from the microdevice.
  • the number of collected cells was measured using a microscope, and the collection ratio was obtained by dividing the value by the number of cells (rough number) in the cell liquid before liquid feeding. The results are shown in Table 4 below.
  • microdevice of the present disclosure made it possible to easily collect cells while suppressing the loss of cells with a small amount of a collection liquid. That is, according to the microdevice of the present disclosure, cells were easily concentrated.
  • Example 1 a cell liquid obtained by mixing SW620 cells that were treated under the conditions below and white blood cells that were treated under the conditions below was concentrated with the microdevice of the present disclosure (treatment flow rate: 20 ⁇ L/min).
  • FIG. 3 is an image showing the distribution of the captured cells in Example 7.
  • the situation of the distribution of captured cells is schematically shown by surrounding cancer cells with triangles and white blood cells with circles.
  • the region of the upstream side of the enlarged portion i.e. the region including the tapered portion
  • vicinity of the enlarged portion is shown by surrounding with broken line.
  • white blood cells were captured on the upstream side (the inlet side) of the enlarged portion (in a region surrounded with long chain lines in FIG. 3 ), and many cancer cells were captured in the vicinity of the enlarged portion (in a region surrounded with broken line in FIG. 3 ).
  • Example 7 it was suggested that in a case where a sample would contain a plurality of cells, the cells would be captured utilizing a difference in balance between a resistance of cells received from liquid flow and a dielectrophoretic force of cells received from an electric field such that positions at which those cells were captured were separated from each other.

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Claims (10)

  1. Mikrovorrichtung (1) zum Fangen von Teilchen, wobei die Mikrovorrichtung umfasst:
    einen Einlass (10);
    einen Auslass (12); und
    einen Strömungskanal, der eine Strömungskanalkammer (11) umfasst, die den Einlass (10) und den Auslass (12) verbindet, wobei die Strömungskanalkammer (11) eine Bodenoberfläche und eine obere Oberfläche aufweist,
    wobei die Bodenoberfläche der Strömungskanalkammer (11) eine flache Oberfläche ist,
    wobei die Strömungskanalkammer (11) einen verbreiterten Abschnitt (14) und einen Abschnitt stromaufwärts des verbreiterten Abschnitts aufweist, wobei sich eine Querschnittfläche des verbreiterten Strömungskanals in dem verbreiterten Abschnitt (14) in einer Höhenrichtung bezüglich der Bodenoberfläche der Strömungskanalkammer (11) durch Erhöhen der Höhe (He) der oberen Oberfläche der Strömungskanalkammer verbreitert,
    und wobei die Strömungskanalkammer (11) mit einem Mittel (13) zum Erzeugen eines elektrischen Feldes versehen ist, um eine dielektrophoretische Kraft an Teilchen auf mindestens einem Teil der Bodenoberfläche der Strömungskanalkammer anzulegen, wobei das Mittel (13) zum Erzeugen eines elektrischen Feldes auf der Bodenoberfläche der Strömungskanalkammer (11) mindestens in dem verbreiterten Abschnitt (14) oder in der Nähe des verbreiterten Abschnitts (14) angeordnet ist.
  2. Mikrovorrichtung nach Anspruch 1,
    wobei sich die Querschnittfläche des Strömungskanals in dem verbreiterten Abschnitt (14) auch auf eine schrittweise Art in eine Breitenrichtung in Bezug zu der Bodenoberfläche der Strömungskanalkammer (11) verbreitert.
  3. Mikrovorrichtung nach Anspruch 1 oder 2,
    wobei das Mittel (13) zum Erzeugen eines elektrischen Feldes eine Gegenelektrode zur Dielektrophorese umfasst und bevorzugt eine Kammelektrode umfasst.
  4. Mikrovorrichtung nach einem der Ansprüche 1 bis 3,
    wobei das Mittel (13) zum Erzeugen eines elektrischen Feldes dazu konfiguriert ist, ein nicht einheitliches elektrisches Feld zu erzeugen.
  5. Verfahren zum Fangen von Teilchen in einer Probe in einer Strömungskanalkammer (11) in
    einer Mikrovorrichtung (1),
    wobei die Mikrovorrichtung (1) eine Mikrovorrichtung nach einem der Ansprüche 1 bis 4 ist, wobei das Verfahren zum Fangen von Teilchen umfasst:
    Bewirken, dass das Mittel (13) zum Erzeugen eines elektrischen Feldes der Mikrovorrichtung (1) ein elektrisches Feld erzeugt; und
    Einführen der Probe in die Strömungskanalkammer (11) von dem Einlass (10) der Mikrovorrichtung her.
  6. Verfahren nach Anspruch 5,
    das das Einführen der Probe in einer Menge, die eine Kapazität der Strömungskanalkammer (11) überschreitet, umfasst.
  7. Verfahren zum Konzentrieren einer Probe, das umfasst:
    Fangen von Teilchen in der Probe in der Strömungskanalkammer (11) mit dem Verfahren nach Anspruch 5 oder 6;
    Einführen einer Sammelflüssigkeit in die Strömungskanalkammer (11); und
    Sammeln der Teilchen, die in der Strömungskanalkammer (11) gefangen sind, aus der Strömungskanalkammer (11).
  8. Verfahren nach einem der Ansprüche 5 bis 7,
    wobei das erzeugte elektrische Feld ein nicht einheitliches elektrisches Feld ist.
  9. Verfahren nach einem der Ansprüche 5 bis 8,
    wobei das elektrische Feld durch Anlegen einer Wechselspannung an das Mittel (13) zum Erzeugen eines elektrischen Feldes erzeugt wird.
  10. Verfahren nach einem der Ansprüche 5 bis 9,
    wobei die Probe aus Blut besteht, und/oder die Teilchen Zellen sind, und bevorzugt zirkulierende Tumorzellen und/oder Immunzellen sind.
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