Patent Grant
11305281
This application claims the benefit under 35 U.S.C. § 371 as a U.S. National Stage Entry of International Application No. PCT/JP2018/005148, filed in the Japanese Patent Office as a Receiving Office on Feb. 15, 2018, which claims priority to Japanese Patent Application Number JP2017-027583, filed in the Japanese Patent Office on Feb. 17, 2017, each of which is hereby incorporated by reference in its entirety.
The present technology relates to a microchip and a microparticle fractionating device. More particularly, the present technology relates to a microchip having a structure suitable for fractionating microparticles and a microparticle fractionating device including the microchip.
Various devices have been developed so far to fractionate microparticles. For example, in microparticle fractionating systems used in flow cytometers, laminar flows constituted of sample liquids containing cells and sheath liquids are discharged from orifices formed in flow cells or microchips. Predetermined vibrations are exerted on such a laminar flow to form droplets when the laminar flow is discharged. A direction of movement of each of the formed droplets is electrically controlled depending on whether the formed droplets contain or do not contain target microparticles so that the target microparticles can be fractionated.
Techniques for fractionating target microparticles in microchips without forming droplets as described above have also been developed. For example, the following Patent Literature 1 discloses “a microchip including a sample liquid introduction flow path through which a sample liquid containing microparticles flows, at least one pair of sheath liquid introduction flow paths which join the sample liquid introduction flow path from both sides thereof and introduce a sheath liquid around the sample liquid, a joining flow path which communicates with the sample liquid introduction flow path and the sheath liquid introduction flow path and in which liquids flowing through the flow paths join and flow, a negative pressure suction unit which communicates with the joining flow path and suctions and pulls microparticles to be collected, and at least one pair of disposal flow paths which are provided on both sides of the negative pressure suction unit and communicate with the joining flow path” (claim 1). In such a microchip, target microparticles are collected by the negative pressure suction unit through suction.
Also, the following Patent Literature 2 discloses “a method for fractionating microparticles including a procedure of fractionating microparticles in a liquid flowing through a main flow path into a branch flow path communicating with the main flow path by generating a negative pressure in the branch flow path, in which, in the procedure, a flow of a liquid leading from the branch flow path side to the main flow path side is formed in a communication port of the main flow path and the branch flow path” (claim 1). In such a fractionating method, entering of non-target particles or a sample liquid containing such non-target particles and a sheath liquid during a non-fractionating operation is suppressed due to the flow of the liquid leading to the main flow path side. Furthermore, Patent Literature 2 also discloses a microchip for fractionating microparticles capable of carrying out the method for fractionating microparticles (claim 9).
PTL 1: JP 2012-127922A
PTL 2: JP 2014-036604A
In each of the microchips disclosed in the above-described Patent Literatures 1 and 2, when a liquid flowing through the main flow path does not contain target microparticles, such a liquid flows to, for example, the disposal flow path and such a liquid is guided into a predetermined flow path so that the target microparticles are collected only when the liquid flowing through the main flow path contains the target microparticles. In this way, each of the microchips has a section in which the flow of such a liquid changes, that is, a branch section configured to fractionate the target microparticles, depending on whether the liquid flowing through the main flow path contains the target microparticles.
A purpose of the present technology is to fractionate microparticles at a higher speed in a microchip having a branch section as described above.
The inventors of the present technology found that the above-described purpose can be attained by adopting a specific structure in the branch section.
That is, an embodiment of the present technology provides a microchip including: a main flow path through which a liquid containing microparticles flows; and a branch flow path that branches from the main flow path. A cross-sectional area of a portion of the main flow path is substantially constant up to a branch start position or decreases toward the branch start position, and a radius of curvature R of a side wall that connects a side wall of the main flow path and a side wall of the branch flow path is 0.5 mm or less and more than 0 mm.
In the microchip, an orifice section which is coaxial with the main flow path is provided; and the side wall that connects the side wall of the main flow path and the side wall of the branch flow path is between 0 μm and 300 μm from an orifice inlet to the orifice section.
In the microchip, the cross-sectional area of the portion of the main flow path leading up to the branch start position is substantially constant, or decreases, so that the radius of curvature R of the side wall begins the branching of the branch flow path from the main flow path.
In the microchip, a maximum cross-sectional area of the branch flow path is not more than half of the cross-sectional area of the main flow path at the branch start position.
In the microchip, the branch flow path branches from the main flow path in a Y shape.
In the microchip, a cross-sectional area of the branch flow path continuously decreases in an advancing direction of a flow of a liquid from a start position of the branch flow path.
In the microchip, a cross-sectional area of the branch flow path continuously decreases from a start position of the branch flow path to a predetermined position in an advancing direction of a flow of a liquid and continuously increases from the predetermined position in an advancing direction of a flow of a liquid.
In the microchip, a side wall of the branch flow path connected to the orifice section is bent toward an axis of the main flow path.
In the microchip, an orifice section which is coaxial with the main flow path is provided; and an angle of a side wall of the branch flow path connected to the orifice section with respect to an axis of the main flow path is 115 to 160 degrees.
In the microchip, an orifice section which is coaxial with the main flow path is provide; and an angle of a side wall of the branch flow path connected to the orifice section with respect to an axis of the main flow path is 125 to 160 degrees.
In the microchip, an orifice section which is coaxial with the main flow path is provided; and a pressure chamber communicates with the orifice section and a pressure in the pressure chamber is reduced or increased.
In the microchip, microparticles are guided into the orifice section by reducing a pressure in the pressure chamber or microparticles are advanced to the branch flow path by increasing a pressure in the pressure chamber.
In the microchip, an orifice section which is coaxial with the main flow path is provided; and a flow path configured to form a flow of a liquid leading from the orifice section to the main flow path is provided in the orifice section, such that microparticles are advanced to the branch flow path due to the formed flow.
In the microchip, the cross-sectional area of the portion of the main flow path leading up to the branch start position is constant up to the branch start position; and the cross-sectional area of a second portion of the main flow path increases as it extends from the branch start position along the side wall.
An embodiment of the present technology provides a microparticle sorting device able to receive a microchip, the microchip including: a main flow path through which a liquid containing microparticles flows; and a branch flow path that branches from the main flow path. A cross-sectional area of a portion of the main flow path is substantially constant up to a branch start position or decreases toward the branch start position, and a radius of curvature R of a side wall that connects a side wall of the main flow path and a side wall of the branch flow path is 0.5 mm or less.
The microparticle sorting device may include: a light irradiation unit configured to irradiate microparticles flowing through the main flow path with light; a detection unit configured to detect scattered light and/or fluorescence emitted from the microparticles; and a control unit configured to control an advancing direction of the microparticles flowing through the main flow path on the basis of data detected by the detection unit.
In the microparticle sorting device, the control unit may control a pressure in a pressure chamber communicating with the orifice section.
In the microparticle sorting device, the control unit may control a flow of a liquid in a flow path provided to form a flow of a liquid leading from the orifice section to the main flow path.
According to the present technology, microparticles can be fractionated at a higher speed. Note that effects attained by the present technology are not necessarily limited to the effects described herein, but may be any of the effects described in the present specification.
Hereinafter, preferred embodiments for carrying out the present technology will be described. Note that the embodiments which will be described below illustrate representative embodiments of the present technology and thus the scope of the present technology is not narrowly interpreted. A description will be provided in the following order.
1. Description of Related Technology
2. First Embodiment (Microchip)
(1) Configuration of Microchip
(2) Configuration of Branch Section
3. Second Embodiment (Microparticle Fractionating Device)
4. Examples
A technique for fractionating target microparticles in a microchip will be described below with reference to
As illustrated in
A sheath liquid flowing through the sheath liquid flow path 504 joins with a sample liquid flowing from both sides of the sample liquid flow path 502 and a laminar flow in which the sample liquid is surrounded by the sheath liquid is formed. The laminar flow flows toward a branch section 507 through a main flow path 505.
The microchip 500 includes the branch section 507. The laminar flow flowing through the main flow path 505 and reaching the branch section 507 flows to a branch flow path 508 in the branch section 507. Furthermore, only when particles to be collected flowing and reaching the branch section 507, a flow to a particle fractionating flow path 509 is formed in the branch section 507 and the particles are collected. When the particles are suctioned into the particle fractionating flow path 509, the sample liquid constituting the laminar flow or the sample liquid and the sheath liquid constituting the laminar flow can also flow to the a particle fractionating flow path. In this way, the microparticles are fractionated in the branch section 507.
When particles are collected in a microchip having such a flow path structure, a flow flowing from the main flow path 505 to the particle fractionating flow path 509 via the orifice section 601 (hereinafter also referred to as a “flow at a time of particle collection”) is formed. Such a flow is not form except when particles are collected. For example, a flow from the orifice section 601 to the main flow path 505 is formed by a sheath liquid introduced from the gate flow inlet 602, except when particles are collected.
After the flow at the time of particle collection is formed, a flow from the orifice section 601 to the main flow path 505 is formed again and thus a flow in the branch section 507 changes. Moreover, when other particles to be collected approach the vicinity of the orifice section after the flow in the branch section 507 is settled, a flow at a time of particle collection is formed again.
When the flow at the time of particle collection is formed again before the flow in the branch section 507 is settled, possibility of microparticles to be collected not being collected and/or possibility of microparticles not to be collected entering the particle fractionating flow path 509 increases. For this reason, in order to speed up the fractionating of microparticles, it is necessary to shorten a time from formation of a flow at a time of particle collection to stabilization of a flow in the branch section (hereinafter also referred to as a “continuous fractionable interval”).
When the inventors of the present technology measured continuous fractionable intervals in microchips disclosed in Patent Literatures 1 and 2, the continuous fractionable intervals were at most about 200 μs. Thus, the inventors of the present technology examined a way to further shorten a continuous fractionable interval.
In the above-described examination, attention was paid to a structure of the branch section 507. In a flow path having the branch section 507, generally, a cross-sectional area of a main flow path thereof increases toward the branch section to minimize pressure loss in the branch flow path. Furthermore, a cross-sectional area of a flow path after branching may be equal to or larger than the cross-sectional area of the main flow path thereof. Such a flow path structure (hereinafter also referred to as a “general flow path structure”) is generally adopted.
An example of the branch section having such a general flow path structure is as illustrated in
The inventors of the present technology found that stagnation points (portions A and B in
Also, the inventors of the present technology found that, particularly, after target microparticles are collected, microparticles stay at the stagnation point (B in
The inventors of the present technology also found that a core flow spreads in the vicinity of the stagnation points. In other words, it is also found that microparticles can flow through a portion deviating from a center of a main flow path and/or that a flow of a sample liquid can spread. When the core flow spreads, it is necessary to guide a larger amount of fluid into a predetermined flow path to fractionate target microparticles. As a result, fractionating efficiency is lowered. For this reason, it is necessary to suppress the spread of the core flow.
The inventors of the present technology found the above-described problems in the above-described branch section. According to the present technology, at least one of the above-described problems can be solved.
A microchip 100 according to an embodiment of the present technology may include a flow path structure as illustrated in
A sheath liquid flowing through the sheath liquid flow path 104 joins with a sample liquid flowing from both sides of a sample liquid flow path 102, and a laminar flow in which the sample liquid is surrounded by the sheath liquid or a laminar flow in which the sample liquid is sandwiched between the sheath liquids is formed. Such laminar flows flow toward a branch section 107 though a main flow path 105.
The main flow path 105 may include a detection region 106. In the detection region 106, particles in a sample liquid can be irradiated with light. It can be determined whether such particles are to be collected on the basis of fluorescence and/or scattered light generated due to the irradiation of the light.
The microchip 100 includes the branch section 107. The laminar flow flowing through the main flow path 105 and reaching the branch section 107 flows to a branch flow path 108 in the branch section 107. Furthermore, only when particles to be collected flowing and reaching the branch section 107, a flow to a particle fractionating flow path 109 is formed in the branch section 107. The particles to be collected are suctioned into the particle fractionating flow path 109 using a flow to a particle fractionating flow path 109. When the particles are suctioned into the particle fractionating flow path 109, the sample liquid constituting the laminar flow or the sample liquid and the sheath liquid constituting the laminar flow can also flow to the the particle fractionating flow path 109.
Note that, in
The orifice section 201 may include a gate flow inlet 202. A sheath liquid or a sample liquid can be introduced into the orifice section through the gate flow inlet 202. A flow from the orifice section 201 toward the main flow path 105 is formed using the sheath liquid or the sample liquid introduced from the gate flow inlet 202. The entering of particles not to be collected into the particle fractionating flow path 109 can be prevented due to such a flow. The particles not to be collected flow to the branch flow path 108.
Note that the gate flow inlet 202 may not include an orifice section. In this case, for example, a flow from the particle fractionating flow path 109 toward a main flow path thereof may be formed due to a change in pressure in the particle fractionating flow path 109 or such a pressure can be maintained so that a flow from the main flow path 105 to the particle fractionating flow path 109 is not formed. As a result, the entering of microparticles into the particle fractionating flow path 109 can be prevented. The particles not to be collected flow to the branch flow path 108.
In the microchip according to an embodiment of the present technology, the branch section 107 has a specific structure. In other words, in the microchip according to an embodiment of the present technology, (a) a cross-sectional area of a main flow path is constant up to a branch start position or continuously decreases toward such a branch start position and (b) a minimum cross-sectional area of a branch flow path is equal to or less than half of the cross-sectional area of the main flow path at the branch start position. The characteristics of (a) and (b) above will be described below with reference to
Regarding Characteristic of (a)
Regarding Characteristic of (b)
In
Also, in the microchip according to an embodiment of the present technology, a minimum cross-sectional area of a branch flow path may be, for example, ⅕ or more, preferably ¼ or more, and more preferably ⅓ or more of a cross-sectional area of the main flow path at the branch start position. The clogging of a liquid can be suppressed due to a lower limit of such a minimum cross-sectional area.
In the present technology, effects capable of fractionating microparticles at a higher speed are attained due to the characteristics of (a) and (b) above.
Also, in the microchip according to an embodiment of the present technology, a side wall configured to connect a side wall of the main flow path and a side wall of the branch flow path can be at between 0 μm and 300 μm from the orifice inlet. The microparticles can be fractionated at a high speed due to such a characteristic. Such a characteristic will be described below with reference to
In
In an embodiment of the present technology, the end of the connection side wall on the main flow path side thereof may be within 300 μm from the orifice inlet. In other words, the branch start position may be within 300 μm from the orifice inlet. Note that, in the present technology, the orifice inlet is indicated by reference numeral 404 in
The end of the connection side wall on the main flow path side is within 300 μm from the orifice inlet so that transition in a direction of the side wall from the side wall of the main flow path to the side wall of the branch flow path starts immediately before an orifice. Microparticles can be fractionated at a higher speed by causing the transition to start immediately before the orifice.
The distance 1 may be preferably 280 μm or less, more preferably 250 μm or less, and even more preferably 200 μm or less.
Also, in an embodiment of the present technology, the end of the connection side wall on the main flow path side may be within 300 μm from the orifice inlet and an end of the connection side wall on a branch flow path side may be at a distance of 0 μm or more from the orifice inlet. This means that, in
The end of the connection side wall on the main flow path is within 300 μm from the orifice inlet and the end of the connection side wall on the branch flow path side is at the distance of 0 μm or more from the orifice inlet so that the transition in the direction of the side wall from the side wall of the main flow path to the side wall of the branch flow path starts and ends immediately before the orifice. Such transition starts and ends immediately before the orifice so that microparticles can be fractionated at a higher speed.
The distance 2 may be preferably 10 μm or more, more preferably 20 μm or more, and even more preferably 30 μm or more.
In an embodiment of the present technology, the connection side wall may be a curved surface or a flat surface and may be preferably a curved surface. When the connection side wall is a curved surface, a radius of curvature R of the connection side wall may be 0.5 mm or less, more preferably 0.4 mm or less, and even more preferably 0.3 mm or less. The radius of curvature R may be more than 0 mm. In some embodiments of the present technology, the radius of curvature R may be 0.5 mm or less and more than 0 mm. With such a radius of curvature, microparticles can be fractionated at a higher speed. When the connection side wall is a flat surface, the flat surface may be constituted of a plurality of flat surfaces or may be constituted of one flat surface such that transition is made stepwise from a direction of the side wall of the main flow path to a direction of the side wall of the branch flow path.
In an embodiment of the present technology, the maximum cross-sectional area of the branch flow path may be not more than half of the cross-sectional area of the main flow path at the branch start position. The maximum cross-sectional area refers to a maximum cross-sectional area of the branch flow path on the orifice from a position of the branch flow path having the minimum cross-sectional area. For example, in
Also, the maximum cross-sectional area may be, for example, ⅕ or more, preferably ¼ or more, and more preferably ⅓ or more of the cross-sectional area of the main flow path at the branch start position. With such a lower limit of the minimum cross-sectional area, the clogging of a liquid can be suppressed.
In an embodiment of the present technology, the branch flow path may have a structure in which a cross-sectional area of the branch flow path from the start position of the branch flow path continuously decreases in an advancing direction of a flow of a liquid. Here, the start position of the branch flow path is the same as a position of the end of the connection side wall on the branch flow path side. The decrease in the cross-sectional area of the branch flow path may continue to a predetermined position of the branch flow path. Although the predetermined position may be appropriately set by a person of ordinary skill in the art so that effects which will be described below are attained, for example, the predetermined position may be within 1000 μm and preferably 300 μm from the start position of the branch flow path. Furthermore, in an embodiment of the present technology, the branch flow path may have a structure in which the branch flow path continuously decreases from the branch start position to the predetermined position thereof in an advancing direction of a flow of a liquid and continuously increase from the predetermined position thereof in an advancing direction of a flow of a liquid. In other words, the microchip according to an embodiment of the present technology may have a structure in which a cross-sectional area of the branch flow path continuously decreases from a position in which the cross-sectional area of the branch flow path is maximized in an advancing direction of a flow of a liquid, the cross-sectional area of the branch flow path is maximized, and then a cross-sectional area of a flow path continuously increases in the advancing direction of the flow of the liquid again (in the present specification, a branch flow path in the branch flow path which is on a branch flow path end side from a position in which a cross-sectional area of the branch flow path is minimized is also referred to as a “flow path after branching.”). With such a structure, an effect of preventing stagnation points from occurring in the branch flow path and/or an effect of increasing a flow rate in the vicinity of a wall surface of the branch flow path can be attained. As a result, the fractionating of particles can be speeded up.
In an embodiment of the present technology, the branch flow path may branch from the main flow path to one or two or more branch flow paths. In the case of branching into one branch flow path, for example, a main flow path and an orifice section are coaxial with each other and a branch flow path may branch from its axis. A main flow path may branch into two branch flow paths. The two branch flow paths preferably branch in a Y shape. A flow of a liquid in a branch flow path can be smoother by branching a flow path into a Y shape. The orifice section may be provided on a center of the Y-shaped branched branch flow path. Furthermore, a main flow path may branch into three branch flow paths or four branch flow paths.
In an embodiment of the present technology, the cross-sectional area of the flow path after branching may continuously increase from the position in which the cross-sectional area of the branch flow path is minimized in a direction in which the liquid flows. Thus, pressure loss is reduced so that a high flow rate at a low pressure is possible. The cross-sectional area of the flow path after branching may continuously increase to a predetermined position. Although the predetermined position may be appropriately set by a person of ordinary skill in the art, for example, as illustrated in
In an embodiment of the present technology, a side wall of the branch flow path connected to the orifice section may be bent toward the axis of the main flow path. In the branch flow path, a maximum flow rate section is located on the particle fractionating flow path side rather than a center of a flow path in some cases. In other words, a difference may occur between a flow rate in the branch flow path on the particle fractionating flow path side and a flow rate on an opposite side thereof. As a result, stagnation may occur in the branch flow path. The side wall of the branch flow path is bent toward the axis of the main flow path so that the difference between the flow rate in the branch flow path on the particle fractionating flow path side and the flow rate on the opposite side thereof decreases, that is, the imbalance between the flow rates is eliminated, and thus occurrence of stagnation in the branch flow path can be suppressed. The flow rate in the branch flow path can be improved and microparticles can be fractionated at a higher speed by suppressing the occurrence of stagnation.
In an embodiment of the present technology, an angle of the side wall of the branch flow path connected to the orifice section with respect to the axis of the main flow path may be preferably 115 to 160 degrees and more preferably 125 to 160 degrees. Such an angle is 115 degrees or more so that occurrence of stagnation in the vicinity of the orifice section inlet can be prevented. Furthermore, such an angle is 125 degrees or more so that collisions of particles to the orifice section inlet can be suppressed. Such an angle is 160 degrees or less so that a microchip can be more easily manufactured.
In the microchip according to an embodiment of the present technology, a pressure chamber may communicate with the orifice section. The pressure chamber may be included in the particle fractionating flow path or the particle fractionating flow path itself may function as a pressure chamber. A pressure in such a pressure chamber can be reduced or increased. A pressure in the pressure chamber is reduced so that microparticles are guided into the orifice section or a pressure in the pressure chamber is increased so that entering of microparticles into the orifice section is prevented. Only particles to be collected can be fractionated by adjusting a pressure in the pressure chamber as described above.
Also, in the microchip according to an embodiment of the present technology, a flow path configured to form a flow (in the present specification, also referred to as a “gate flow”) of a liquid leading from the orifice section to the main flow path may be provided in the orifice section and the liquid can advance to the particle fractionating flow path of microparticles due to the formed flow. Entering of particles not to be collected into the orifice section can be prevented by such a gate flow. In an embodiment of the present technology, the gate flow may be introduced into the orifice section at all times while particles are being fractionated and a pressure in the pressure chamber can be reduced only when microparticles are guided into the particle fractionating flow path. A flow stronger than a flow from the orifice section to the main flow path caused by the gate flow is formed from the main flow path to the particle fractionating flow path due to a decrease in the pressure, and thus target particles can be fractionated in the particle fractionating flow path.
In an embodiment of the present technology, microparticles may be appropriately selected by a person of ordinary skill in the art. In an embodiment of the present technology, microparticles may include biological microparticles such as cells, microorganisms, and liposomes, synthetic particles such as latex particles, gel particles, and industrial particles, and the like. The biological microparticles may include chromosomes, liposomes, mitochondria, organelles (cell organelles), and the like constituting various cells. The cells may include animal cells (hemocyte cells and the like) and plant cells. The microorganisms may include bacteria such as Escherichia coli, viruses such as tobacco mosaic virus, and fungi such as yeast. In addition, the biological microparticles may also include biological macromolecules such as nucleic acids, proteins, and complexes thereof. The synthetic particles may be, for example, particles made of organic or inorganic polymeric materials, metals, or the like. The organic polymeric materials may include polystyrene, styrene, divinylbenzene, polymethyl methacrylate, and the like. The inorganic polymeric materials may include glass, silica, magnetic materials, and the like. The metals may include gold colloid, aluminum, and the like. A shape of microparticles may be generally a spherical shape or a substantially spherical shape, or a non-spherical shape. A size and mass of microparticles may be appropriately selected by a person of ordinary skill in the art depending on a size of a flow path of a microchip. On the other hand, the size of the flow path of the microchip can also be appropriately selected depending on a size and mass of microparticles. In an embodiment of the present technology, a chemical or biological label, for example, a fluorescence dye or the like is attached to microparticles as necessary. Such microparticles can be more easily detected using such a label. A label to be attached can be appropriately selected by a person of ordinary skill in the art.
The microchip according to an embodiment of the present technology may be manufactured by a known method in the related art. The microchip according to an embodiment of the present technology can be manufactured, for example, by bonding two substrates in which flow paths are formed as described above. Flow paths may be formed on both of the two substrates or may be formed only in one of the substrates. In order to easily adjust a position when substrates are bonded, it is desirable that flow paths are formed only in one of the substrates.
As materials used to form the microchip according to an embodiment of the present technology, known materials in the related art may be used. Examples of the materials include polycarbonate, cycloolefin polymers, polypropylene, polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), polyethylene, polystyrene, glass, and silicone, but the present technology is not limited thereto. Particularly, polymer materials such as polycarbonate, cycloolefin polymers, and polypropylene are particularly preferable because the polymer materials have excellent processability and can manufacture a microchip inexpensively using a forming device.
A microparticle fractionating device (also referred to herein as a microparticle sorting device) according to an embodiment of the present technology includes a main flow path through which a liquid containing microparticles flows, a branch flow path configured to branch from the main flow path, an orifice section which is coaxial with the main flow path, and a microchip in which a cross-sectional area of the main flow path is constant up to a branch start position or continuously decreases toward the branch start position and a minimum cross-sectional area of the branch flow path is half or less of the cross-sectional area of the main flow path at the branch start position. Since such a microchip has been described in 2. First embodiment described above, a description of the microchip will be omitted.
The microparticle fractionating device according to an embodiment of the present technology may include a light irradiation unit configured to irradiate microparticles flowing through the main flow path with light, a detection unit configured to detect scattered light and/or fluorescence emitted from the microparticles, and a control unit configured to control an advancing direction of microparticles flowing through the main flow path on the basis of data detected by the detection unit. The light irradiation unit, the detection unit, and the control unit will be described below.
In an embodiment of the present technology, the light irradiation unit irradiates microparticles flowing through the main flow path with light (excitation light). The light irradiation unit may include a light source configured to emit excitation light and objective lenses configured to collect the excitation light to microparticles flowing through the main flow path. The light source may be appropriately selected from laser diodes, second harmonic generation (SHG) lasers, solid lasers, gas lasers, high brightness light-emitting diodes (LEDs), and the like in accordance with the purpose of analysis. The light irradiation unit may include other optical elements as necessary in addition to the light source and the objective lenses.
In an embodiment of the present technology, the detection unit detects scattered light and/or fluorescence emitted from the microparticles by light irradiation using the light irradiation unit. The detection unit may include condenser lenses configured to collect fluorescence and/or scattered light emitted from microparticles and a detector. Photo-multiplier tubes (PMTs), photodiodes, charge-coupled devices (CCDs), complementary metal-oxide-semiconductors (CMOSs), and the like are used as such a detector, but the present technology is not limited thereto. The detection unit may include other optical elements as necessary in addition to the condenser lenses and the detector.
Fluorescence detected by the detection unit may be fluorescence generated from fluorescence generated from microparticles themselves, substances labeled on microparticles, for example, fluorescence substances, and the like, but the present technology is not limited thereto. Scattered light detected by the detection unit may be forward scattered light, side scattered light, Rayleigh scattering, and/or Mie scattering, but the present technology is not limited thereto.
In an embodiment of the present technology, the control unit controls whether microparticles flowing through the main flow path are advanced to the branch flow path or suctioned into the particle fractionating flow path on the basis of data detected by the detection unit. Fluorescence and scattered light detected by the detection unit can be converted into electric signals. In other words, the microparticle fractionating device according to an embodiment of the present technology may include an electric signal conversion unit. The electric signal conversion unit may be included in the control unit and may not be included in the control unit. The control unit may receive such electric signals and determine optical characteristics of microparticles on the basis of the electric signals. When microparticles are to be collected, the control unit may change a flow in a flow path on the basis of the result of the determination so that the microparticles advance to a microparticle fractionating flow path through the orifice section. The change in the flow can be made, for example, by reducing a pressure in the pressure chamber. Furthermore, after microparticles are collected, the control unit may change the flow in the flow path again. Such a change of the flow again can be made by increasing a pressure in the pressure chamber. In other words, the control unit may control a pressure in the pressure chamber communicating with the orifice section on the basis of data detected by the detection unit. Furthermore, the control unit may control a flow of a liquid in a flow path provided to form a flow of the liquid leading from the orifice section to the main flow path. The control unit may have the same function as that of a drive unit disclosed in JP 2014-036604A.
A flow of a liquid in a microchip (hereinafter referred to as a “microchip of Comparative Example 1”) including a branch section with a flow path structure in which a cross-sectional area of a main flow path increases toward a branch section and a cross-sectional area of a flow path after branching is the cross-sectional area or more of the main flow path is simulated using COMSOL Multiphysics v5.1. The branch section is as illustrated in
Analysis conditions in the above-described simulation are as follows.
Flow rate of sheath liquid: 5 ml/minute
Flow rate of sample liquid: 0.25 ml/minute
Flow rate of gate flow: 0.3 ml/minute
Outlet pressure: atmospheric pressure
Discharge rate from particle fractionating flow path end: 0.045 ml/minute
Subsequently, a particle tracking analysis was performed on a flow of microparticles using the above-described software.
Subsequently, a flow of microparticles when a piezoelectric element (PZT) provided in the particle fractionating flow path was simulated. In other words, a volume in the particle fractionating flow path (particularly, a volume of a pressure chamber) is increased by driving such a piezoelectric element. The pressure in the particle fractionating flow path is reduced due to a crease in the volume and thus a flow leading from the main flow path to the particle fractionating flow path via the orifice inlet is formed. A flow of microparticles when such a flow is formed was simulated.
A shape of a change in a voltage applied to the piezoelectric element due to such an increase in the volume was a pulse waveform. Driving conditions for the piezoelectric element were Tf/Tr: 15 μs, PW: 50 μs, pulse interval: 300 μs, and PZT displacement: 1 μm. Tf is a falling time of a voltage in a pulse waveform and Tr is a rising time of the voltage in the pulse waveform. PW is a time interval in which an amount of PZT displacement is 0.5 μm or more. At the falling time, the volume increases and thus a flow is formed to suction microparticles into the particle fractionating flow path. At the rising time, the volume decreases, the suctioning flow disappears, and thus a flow leading from an orifice section to the main flow path is formed.
Next,
A flow of a liquid in a microchip (hereinafter referred to as a “microchip of Example 1”) including a branch section with a flow path structure in which a cross-sectional area of a main flow path is constant up to a branch start position and a minimum cross-sectional area of a branch flow path is not more than half of the cross-sectional area of the main flow path at the branch start position was simulated using COMSOL Multiphysics v5.1. The branch section is as illustrated in
Analysis conditions in the above-described simulation are as follows.
Flow rate of sheath liquid: 5 ml/minute
Flow rate of sample liquid: 0.25 ml/minute
Flow rate of gate flow: 0.2 to 0.6 ml/minute
Outlet pressure: atmospheric pressure
Discharge rate from particle fractionating flow path end: 0.045 ml/minute
Also, a flow of microparticles was subject to a particle tracking analysis using the above-described software.
Simulation was performed under the same conditions as in Example 1 except that an angle of a branch flow path with respect to a main flow path was set to be 150 degrees. A branch section used in such simulation is as illustrated in
Next, a flow of microparticles was subject to a particle tracking analysis using the above-described software.
Simulation was performed under the same conditions as in Example 1 except that an angle of a branch flow path with respect to a main flow path was set to be 120 degrees. A branch section used in such simulation is as illustrated in
Next, a flow of microparticles was subject to a particle tracking analysis using the above-described software.
Simulation was performed under the same conditions as in Example 1 except that an angle of a branch flow path with respect to a main flow path was set to be 130 degrees immediately after branching and then was set to be 135 degrees. A branch section used in such a simulation is as illustrated in
Next, a flow of microparticles was subject to a particle tracking analysis using the above-described software.
A flow of microparticles was analyzed like in Comparative Example 1.
A flow rate in an x direction at a predetermined distance from an orifice inlet was measured like in Comparative Example 1.
The microchip of Example 4 and the microchip of Comparative Example 1 were actually subject to a microparticle fractionating experiment. In such an experiment, a capture rate of microparticles at a pulse drive interval, that is, various particle fractionating intervals, was measured. A PZT drive waveform was set to be Tf/Tr15 μs and PW35 μs.
An influence when a structure in which a cross-sectional area of a branch flow path continuously decreases in an advancing direction of a flow of a liquid from a position immediately after branching, the cross-sectional area of the branch flow path is minimized, and then the cross-sectional area of the branch flow path continuously increases in the advancing direction of the flow of the liquid again is provided was reviewed.
(a) of
Also,
Effects of bending a direction of a branch flow path toward a particle fractionating flow path were reviewed.
A flow rate in the branch flow path in a structure in which the direction of the branch flow path was bent toward the particle fractionating flow path was simulated using the software used in Example 1. Likewise, a flow rate in the branch flow path in a structure in which the direction of the branch flow path is not changed was simulated. Positions in which flow rates were measured are as illustrated in
It should be understood by those skilled in the art that various modifications, combinations, sub-combinations and alterations may occur depending on design requirements and other factors insofar as they are within the scope of the appended claims or the equivalents thereof.
Additionally, the present technology may also be configured as below.
(1)
A microchip comprising:
a main flow path through which a liquid containing microparticles flows; and
a branch flow path that branches from the main flow path;
wherein:
a cross-sectional area of a portion of the main flow path is substantially constant up to a branch start position or decreases toward the branch start position, and
a radius of curvature R of a side wall that connects a side wall of the main flow path and a side wall of the branch flow path is 0.5 mm or less and more than 0 mm.
(2)
The microchip according to (1), in which an orifice section which is coaxial with the main flow path is provided; and the side wall that connects the side wall of the main flow path and the side wall of the branch flow path is between 0 μm and 300 μm from an orifice inlet to the orifice section.
(3)
The microchip according to (1) or (2), in which the cross-sectional area of the portion of the main flow path leading up to the branch start position is substantially constant, or decreases, so that the radius of curvature R of the side wall begins the branching of the branch flow path from the main flow path.
(4)
The microchip according to any of (1) to (3), in which a maximum cross-sectional area of the branch flow path is not more than half of the cross-sectional area of the main flow path at the branch start position.
(5)
The microchip according to any of (1) to (4), in which the branch flow path branches from the main flow path in a Y shape.
(6)
The microchip according to any of (1) to (5), in which a cross-sectional area of the branch flow path continuously decreases in an advancing direction of a flow of a liquid from a start position of the branch flow path.
(7)
The microchip according to any of (1) to (5), in which a cross-sectional area of the branch flow path continuously decreases from a start position of the branch flow path to a predetermined position in an advancing direction of a flow of a liquid and continuously increases from the predetermined position in an advancing direction of a flow of a liquid.
(8)
The microchip according to any of (1) to (7), in which a side wall of the branch flow path connected to the orifice section is bent toward an axis of the main flow path.
(9)
The microchip according to any of (1) to (8), in which an orifice section which is coaxial with the main flow path is provided; and an angle of a side wall of the branch flow path connected to the orifice section with respect to an axis of the main flow path is 115 to 160 degrees.
(10)
The microchip according to any of (1) to (9), in which an orifice section which is coaxial with the main flow path is provided; and an angle of a side wall of the branch flow path connected to the orifice section with respect to an axis of the main flow path is 125 to 160 degrees.
(11)
The microchip according to any of (1) to (10), in which an orifice section which is coaxial with the main flow path is provided; and a pressure chamber communicates with the orifice section and a pressure in the pressure chamber is reduced or increased.
(12)
The microchip according to (11), in which microparticles are guided into the orifice section by reducing a pressure in the pressure chamber or microparticles are advanced to the branch flow path by increasing a pressure in the pressure chamber.
(13)
The microchip according to any of (1) to (12), in which an orifice section which is coaxial with the main flow path is provided; and a flow path configured to form a flow of a liquid leading from the orifice section to the main flow path is provided in the orifice section, such that microparticles are advanced to the branch flow path due to the formed flow.
(14)
The microchip according to any of (1) to (13), in which the cross-sectional area of the portion of the main flow path leading up to the branch start position is constant up to the branch start position; and the cross-sectional area of a second portion of the main flow path increases as it extends from the branch start position along the side wall.
(15)
A microparticle sorting device able to receive a microchip, the microchip including:
a main flow path through which a liquid containing microparticles flows; and
a branch flow path that branches from the main flow path;
wherein:
a cross-sectional area of a portion of the main flow path is substantially constant up to a branch start position or decreases toward the branch start position, and
a radius of curvature R of a side wall that connects a side wall of the main flow path and a side wall of the branch flow path is 0.5 mm or less.
(16)
The microparticle sorting device according to (15), including:
a light irradiation unit configured to irradiate microparticles flowing through the main flow path with light;
a detection unit configured to detect scattered light and/or fluorescence emitted from the microparticles; and
a control unit configured to control an advancing direction of the microparticles flowing through the main flow path on the basis of data detected by the detection unit.
(17)
The microparticle sorting device according to (16), in which the control unit controls a pressure in a pressure chamber communicating with the orifice section.
(18)
The microparticle sorting device according to (16) or (17), in which the control unit controls a flow of a liquid in a flow path provided to form a flow of a liquid leading from the orifice section to the main flow path.
| Number | Date | Country | Kind |
|---|---|---|---|
| JP2017-027583 | Feb 2017 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2018/005148 | 2/15/2018 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2018/151180 | 8/23/2018 | WO | A |
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| Japanese Office Action dated Dec. 25, 2020 in connection with Japanese Application No. 2017-027583 and English translation thereof. |
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| Number | Date | Country | |
|---|---|---|---|
| 20190366343 A1 | Dec 2019 | US |
