Patent Application
20250186907
The present technology relates to a microparticle sorting device and a microparticle sorting method.
There is known a microparticle sorting device that forms a sheath flow containing microparticles in a channel, irradiates the microparticles in the sheath flow with light to detect fluorescence and scattered light emitted from the microparticles, and separately recovers a microparticle group (population) exhibiting a predetermined optical characteristic. For example, in a flow cytometer, a plurality of types of cells contained in a sample is labeled with fluorescent dyes, and the fluorescent dye labeled on each cell is optically identified, so that only a specific type of cell is separately recovered.
Patent Documents 1 and 2 each disclose a microchip-type microparticle sorting device that forms a sheath flow in a channel formed in a microchip such as a plastic microchip or a glass microchip and performs analysis.
The microparticle sorting device disclosed in Patent Document 1 is configured to regulate a voltage applied to an actuator to generate a pressure change containing a step waveform component and an undershoot waveform component in a branch channel. This microparticle sorting device can not only introduce, into the branch channel, a fluid having a volume necessary for drawing microparticles but also perform an operation of capturing microparticles into the branch channel with higher throughput.
The microparticle sorting device disclosed in Patent Document 2 is configured to apply, to an actuator, a drive waveform that is any one a pulse waveform, a step waveform, or a step waveform with undershoot and control the application of the pulse waveform separately for a falling waveform section and a rising waveform section. This microparticle sorting device can avoid a return operation before it happens that hinders the capture of the next microparticle to be sorted and thus increase the yield of microparticles to be sorted.
For even faster and more accurate analysis, a technique for extracting, with higher throughput and stability, a microparticle of interest from a sheath flow flowing through a channel is required for a microparticle sorting device. The conventional techniques, however, have a problem that when sorting a microparticle, a pulsating flow (also referred to as unwanted oscillation) in which a flow in a suction direction and a flow in a discharge direction opposite to the suction direction are alternately repeated is generated in a channel due to a pressure change caused by a suction operation and a discharge operation of an actuator, which prevents the microparticle of interest from being selectively sorted with high efficiency and high purity. It is therefore a main object of the present technology to provide a microparticle sorting device capable of selectively sorting a microparticle of interest with high efficiency and high purity.
Therefore, the inventors have found that it is possible to suppress unwanted flow oscillation generated in a channel after a suction operation and a discharge operation performed by an actuator during a microparticle sorting operation by performing the suction operation and the discharge operation at time intervals that cause their respective oscillation characteristics to cancel each other out and sort a microparticle of interest with high efficiency and high purity.
That is, the present technology provides a microparticle sorting device including:
The suction operation may be performed by generating a negative pressure in the recovery channel.
The actuator may increase a volume of an inner space of the recovery channel.
The control unit may control the drive interval and/or the drive time so as to reduce an amplitude of changes in flow velocity of a pulsating flow that is generated by the suction operation and in which a flow in a suction direction and a flow in a direction opposite to the suction direction are alternately repeated, and/or an amplitude of changes in flow velocity of a pulsating flow that is generated by the discharge operation and in which a flow in a discharge direction and a flow in a direction opposite to the discharge direction are alternately repeated.
The control unit may control the drive interval and/or the drive time so as to cause a peak of changes in flow velocity of a pulsating flow that is generated by the suction operation and in which a flow in a suction direction and a flow in a direction opposite to the suction direction are alternately repeated, and a peak of changes in flow velocity of a pulsating flow that is generated by the discharge operation and in which a flow in a discharge direction and a flow in a direction opposite to the discharge direction are alternately repeated to cancel each other out. The control unit may apply, to the actuator, a drive voltage with a drive waveform including a falling waveform and a rising waveform to perform the suction operation and the discharge operation, so as to make a drive voltage application time of the falling waveform and a drive voltage application time of the rising waveform different from each other.
The control unit may apply, to the actuator, a drive voltage with a drive waveform including a falling waveform and a rising waveform to perform the suction operation and the discharge operation, so as to make a drive voltage application time of the falling waveform and a drive voltage application time of the rising waveform identical to each other.
In the microparticle sorting device according to the present technology, in the recovery channel, a microparticle to be sorted among the microparticles and/or an emulsion in which the first fluid is contained in a second fluid immiscible with the first fluid may be recovered.
Furthermore, the microparticle sorting device according to the present technology may further include an emulsion detection unit that detects the microparticles and/or the emulsion in the recovery channel.
The control unit may automatically control the drive voltage, the drive interval, and the drive time of the actuator on the basis of optical information from the microparticles and/or the emulsion detected by the emulsion detection unit.
In the microparticle sorting device according to the present technology, the main channel may further include a particle detection unit that detects the microparticle to be sorted in the first fluid flowing through the main channel, and
The present technology provides a microparticle sorting method including:
Hereinafter, preferred modes for carrying out the present technology will be described. Note that embodiments described below are representative embodiments of the present technology, and the scope of the present technology is not limited only to these embodiments. Note that the present technology will be described in the following order.
A microparticle sorting device according to the present technology includes: a main channel through which a first fluid containing microparticles flows; a recovery channel communicating with the main channel; an actuator that performs a suction operation and a discharge operation by generating a pressure change in the recovery channel; and a control unit that applies a drive voltage to the actuator to control a drive interval between the suction operation and the discharge operation performed by the actuator and/or a drive time from the suction operation to the end of the discharge operation performed by the actuator.
Hereinafter, first, a configuration example of the microparticle sorting device according to the first embodiment of the present technology will be described.
The microparticle sorting device according to the present technology may be configured as a device that sorts a microparticle to be sorted in a closed space, and may be configured as a device that sorts the microparticle to be sorted by controlling a channel through which the microparticles travel, for example.
As illustrated in
Hereinafter, first, the microparticle sorting microchip 150 will be described, and next, sorting processing performed by the microparticle sorting device 100 will be described together with a description of other components of the device. Note that, in the following drawings, the same reference numerals denote the same components, so that the description of such components will be omitted after the first description.
As illustrated in
The microparticle sorting microchip 150 has a channel structure in which the sample fluid channel 152 through which the sample fluid flows and the sheath fluid channel 154 through which the sheath fluid flows are joined at a junction 162 to become a main channel 155. As illustrated in
The laminar flow flows through the main channel 155 toward a particle sorting section 157. Preferably, the microparticles flow in a line in the main channel 155. Therefore, when a particle detection region 156 to be described below is irradiated with light, light generated by irradiating one microparticle with light and light generated by irradiating another microparticle with light can be easily distinguished from each other.
The microparticle sorting microchip 150 includes the particle detection region 156. In the particle detection region 156, the first light irradiation unit 101 irradiates a microparticle flowing through the main channel 155 with light, and the particle detection unit 102 detects light generated by the light irradiation. On the basis of characteristics of the light detected by the particle detection unit 102, the determination unit 105 included in the control unit 103 determines whether or not the microparticle is the microparticle to be sorted. For example, the determination unit 105 may make a determination on the basis of scattered light such as forward-scattered light, side-scattered light, or back-scattered light, a determination on the basis of fluorescence having the same or a plurality of wavelengths, or a determination on the basis of an image (for example, a dark field image and/or a bright field image, or the like).
The microparticle sorting microchip 150 illustrated in
In the particle sorting section 157, the laminar flow flowing through the main channel 155 flows into the waste channel 158. Furthermore, in the particle sorting section 157, only in a case where the microparticle Po to be sorted flows, a flow into the recovery channel 159 is formed, and the microparticle Po is sorted. When the microparticle Po is sucked into the recovery channel 159, the sample fluid forming the laminar flow or the sample fluid and the sheath fluid forming the laminar flow may also flow into the recovery channel 159.
In order to prevent the microparticle Pw not to be sorted from entering the recovery channel 159, the fluid supply channel 161 may be connected to the connection channel 170 as illustrated in
The microparticle sorting device 100 includes the actuator 107 that performs a suction operation and a discharge operation by generating a pressure change in the recovery channel 159. In order to suck the microparticle to be sorted into the recovery channel 159, the actuator 107 generates a negative pressure in the recovery channel 159 to suck the sample fluid containing the microparticle and the sheath fluid into the recovery channel 159. That is, the actuator 107 performs the suction operation of sucking the microparticle by generating a negative pressure in the recovery channel 159. The actuator 107 is a piezoelectric element such as a piezo element. The actuator 107 is arranged in contact with a surface of the microchip 150 and is arranged at a position corresponding to the recovery channel 159. More specifically, the actuator 107 is arranged at a position corresponding to a pressure chamber 165 provided as an inner space obtained by expanding a part of the recovery channel 159 (see
The inner space of the pressure chamber 165 is expanded in a planar direction (width direction of the recovery channel 159) as illustrated in
The actuator 107 generates an expansion/contraction force along with a change in an applied drive voltage to cause a pressure change in the recovery channel 159 via a surface (contact surface) of the microchip 150. When a flow occurs in the recovery channel 159 along with the pressure change in the recovery channel 159, a volume in the recovery channel 159 changes at the same time. The volume in the recovery channel 159 changes until reaching a volume defined by a displacement amount of the actuator 107 corresponding to the applied drive voltage. More specifically, the actuator 107 presses a displacement plate 167 (see
In order to efficiently transmit the expansion/contraction force of the actuator 107 into the pressure chamber 165, it is preferable that the surface of the microchip 150 is recessed at a position corresponding to the pressure chamber 165, and the actuator 107 is arranged in the recess as illustrated in
In order to return, to the original state, the pressure chamber 165 deformed for the sucking of the microparticle to be sorted into the recovery channel 159, the actuator 107 reduces the volume of the pressure chamber 165 by a certain amount to generate a positive pressure. The suction force may be adjusted by means of a drive waveform of the drive voltage applied to the piezo element, or may be adjusted by means of the drive voltage applied to the piezo element. Note that the recovery channel 159 itself may function as a pressure chamber. The pressure in the pressure chamber 165 may be reduced. A suction force is generated by the reduction in the pressure in the pressure chamber 165 to introduce the microparticle Po to be sorted into the recovery channel 159. Furthermore, the pressure in the pressure chamber 165 may be increased. A suction force is generated by the increase of the pressure in the pressure chamber 165 to prevent the microparticle Pw not to be sorted from entering the recovery channel 159. As described above, it is possible to sort, by adjusting the pressure in the pressure chamber 165, only the microparticle Po to be sorted.
In
In order to capture the microparticle Po to be sorted from the main channel 155 into the recovery channel 159 with ease, the communication port 169 is desirably opened at a position corresponding to a sample fluid laminar flow S in the sheath flow formed in the main channel 155 as illustrated in
The microparticle sorting microchip used in the microparticle sorting device according to the present technology may be manufactured by a method known in the technical field. For example, the microparticle sorting microchip can be manufactured by bonding two substrates in which the channels as described in the above 1. are formed. The channels may be formed in both of the two substrates, or may be formed in only one of the substrates. In order to make alignment when bonding the substrates easier, the channels may be formed in only one of the substrates.
Examples of the material for forming the microparticle sorting microchip used in the microparticle sorting device according to the present technology may include materials known in the technical field. The examples include, but are not limited to, for example, polycarbonate, cycloolefin polymer, polypropylene, polydimethylsiloxane (PDMS), polymethylmethacrylate (PMMA), polyethylene, polystyrene, glass, and silicon. Especially, polymer materials such as polycarbonate, cycloolefin polymer, and polypropylene are particularly preferable because they are excellent in processability and allow a microchip to be manufactured using a molding device with less cost.
The microparticle sorting device according to the present technology includes the control unit 103 that controls a sorting condition applied to the particle sorting section 157 on the basis of information regarding light detected by the emulsion detection unit 108. As illustrated in
The control unit 103 can change the piezo drive height, detect any one of fluorescence, side-scattered light, or back-scattered light, acquire a signal intensity of the emulsion detected by the emulsion detection unit 108, and determine a piezo drive height at which a signal intensity higher than a predetermined signal intensity threshold at which it is determined that the emulsion is generated is obtained as a piezo drive height D1 at which the emulsion can be generated. The signal intensity may be an area signal, a peak signal, or a width signal.
The control unit 103 can change the drive time interval, detect any one of fluorescence, side-scattered light, or back-scattered light, count the number of emulsions detected by the emulsion detection unit 108, and determine a time at which the counted number is minimized as the drive time interval Th at which unwanted flow oscillation is suppressed.
The control unit 103 can change the piezo drive height, detect any one of fluorescence, side-scattered light, or back-scattered light, acquire the signal intensity of the emulsion detected by the emulsion detection unit 108, and determine, on the basis of a correlation between the signal intensity and the emulsion size, a piezo drive height at which a signal intensity close to a signal intensity that is detected on the basis of a desired emulsion size is obtained as the piezo drive height D at which the desired emulsion size can be acquired.
The control unit 103 can change the recovery start time from the microparticle detection to the suction operation, detect any one of fluorescence, side-scattered light, or back-scattered light, count the number of microparticles in the emulsion detected by the emulsion detection unit 108, and determine a recovery start time at which the counted number is maximized as an optimal recovery start time Td.
The microparticle sorting device 100 according to the present technology may further include, in order to confirm whether or not the microparticle to be sorted has been sorted, an information processing unit (not illustrated) that integrates information regarding the microparticles detected by the particle detection unit 102 and information regarding the microparticles in the emulsion detected by the emulsion detection unit 108.
In the microparticle sorting microchip 150 having such a channel structure, in a case where the microparticle Po to be sorted is sorted, a flow from the main channel 155 into the recovery channel 159 through the connection channel 170 (hereinafter, also referred to as “flow at the time of microparticle sorting”) is formed. The flow at the time of microparticle sorting is formed only in a case where the microparticle Po to be sorted is sorted. The pressure in the pressure chamber 165 may be reduced to form the flow at the time of microparticle sorting. The reduction in the pressure forms a flow from the main channel 155 toward the recovery channel 159, the flow being stronger than the flow from the connection channel 170 toward the main channel 155 generated by the flow of the second fluid from the fluid supply channel 161, and as a result, the microparticle Po to be sorted is sorted into the recovery channel 159.
Making the pressure in the recovery channel 159 negative may form the flow at the time of microparticle sorting. That is, making the pressure in the recovery channel 159 negative causes the microparticle Po to be sorted to be sucked into the recovery channel 159. The sucking of the microparticle Po is performed at a time point when a predetermined time has elapsed since the microparticle passed through the particle detection region 156 in a case where the determination unit 105 included in the control unit 103 determines that the microparticle should be sorted on the basis of the light detected by the particle detection unit 102 in the particle detection region 156. In order to perform microparticle sorting with higher accuracy, it is necessary to optimize a time elapsed before the time point when the suction should be performed. That is, it is necessary to optimize the recovery start time, which is the time from the detection of the microparticle in the particle detection unit 102 to the sucking of the microparticle in the particle sorting section 157. In the connection channel 170 included in the particle sorting section 157, the first fluid is contained in the second fluid, and an emulsion containing the microparticle and an emulsion not containing the microparticle are generated.
In a case where the microparticle Po to be sorted is sucked into the recovery channel 159, together with the microparticle Po, the sample fluid including the first fluid containing the microparticle Po and/or the sheath fluid including only the first fluid not containing the microparticle Po are sucked into the recovery channel 159. A suction force above a certain level is required for generating the emulsion. The size of the emulsion varies in a manner that depends on the suction force, and in a case where the applied suction force is too large, an amount of the sample fluid and/or the sheath fluid sucked into the recovery channel 159 together with the microparticle Po increases, and a density of the microparticle Po to be sorted decreases, which is not preferable. Furthermore, in a case where the suction force is too large, the emulsion is split, and a plurality of emulsions is generated by one suction, which is not preferable. On the other hand, in a case where the applied suction force is too small, a possibility that the microparticle Po to be sorted is not sorted increases. Therefore, it is desirable to optimize the applied suction force, too.
The microparticle Po to be sorted sucked into the connection channel 170 is captured into the pressure chamber 165 as illustrated in
In order to draw the microparticle Po to be sorted from the main channel 155 into the pressure chamber 165, the amount of increase in the volume of the inner space of the pressure chamber 165 is set larger than the volume (see
As described above, the microparticle Po to be sorted is captured into the back part of the pressure chamber 165 whose inner space has been expanded in the recovery channel 159, so that it is possible to prevent the microparticle Po from flowing out from the pressure chamber 165 toward the main channel 155 again even in a case where the pressure in the recovery channel 159 is reversed to a positive pressure. That is, as illustrated in
A base drive waveform of the piezo drive voltage applied to the actuator 107 will be described with reference to
As illustrated in
It is possible to suppress, by adjusting the drive interval between the suction operation and the discharge operation to an appropriate time interval, the generation of unwanted flow oscillation in the connection channel 170 and shorten a time interval between a sorting operation of sorting the microparticle Po to be sorted and a sorting operation of sorting the next microparticle Po to be sorted, which allows highly efficient sorting. Furthermore, it is possible to prevent, by suppressing unwanted oscillation, the microparticle Pw not to be sorted from being sucked due to unwanted oscillation and sort the microparticle Po to be sorted with high purity.
In order to suppress generation of unwanted flow oscillation in the connection channel 170, the drive voltage application time (falling slope) of the falling waveform and the drive voltage application time (rising slope) of the rising waveform in the drive waveform may be different from each other.
As illustrated in
The microparticle sorting microchip 150 has an emulsion detection region 164 located downstream of the particle sorting section 157. In the emulsion detection region 164, the second light irradiation unit 109 irradiates the emulsion flowing through the recovery channel 159 with light, and the emulsion detection unit 108 detects light generated by the light irradiation. Note that in a case of detection only with forward-scattered light, the presence or absence of the emulsion and the size of the emulsion can be detected, but the microparticle contained in the emulsion causes scattered light to occur on the surface of the emulsion, which prevents the emulsion containing the microparticle and the emulsion not containing the microparticle from being distinguished from each other. In order to detect the microparticle Po to be sorted in the emulsion, to detect a type (for example, a cell type) of the microparticle, and to detect the state (for example, life or death of a cell) of the microparticle, the emulsion detection unit 108 may preferably use a combination of forward-scattered light detection and fluorescence detection, a combination of forward-scattered light detection and side-scattered light detection, a combination of forward-scattered light detection and back-scattered light detection, a combination of forward-scattered light detection, side-scattered light detection, and the fluorescence detection, or a combination of forward-scattered light detection, back-scattered light detection, and the fluorescence detection. Note that the fluorescence may have the same wavelength or a plurality of wavelengths. On the basis of characteristics of the light detected by the emulsion detection unit 108, the determination unit 105 included in the control unit 103 determines whether or not the microparticle Po to be sorted is contained in the emulsion. For example, the determination unit 105 may make the determination on the basis of the detection combinations.
Hereinafter, a light irradiation unit, a particle detection unit, and an emulsion detection unit in the microparticle sorting device according to the present technology will be described.
The microparticle sorting device according to the present technology includes the first light irradiation unit 101 and the second light irradiation unit 109 as the light irradiation unit. The first light irradiation unit 101 irradiates microparticles flowing through the main channel 155 with fluorescent excitation light for detection of the microparticle in the sample fluid channel 152. The second light irradiation unit 109 irradiates the emulsion E flowing through the recovery channel 159 with fluorescent excitation light for detection of the microparticle Po to be sorted in the recovery channel 159.
The first light irradiation unit 101 and the second light irradiation unit 109 irradiate, with light (for example, excitation light), the microparticles flowing in the channels in the microparticle sorting microchip 150. The light irradiation unit may include a light source that emits light, and an objective lens that concentrates the excitation light onto the microparticle flowing through the detection area. The light source may be appropriately selected by one skilled in the art according to the purpose of an analysis, and may be, for example, a laser diode, an SHG laser, a solid-state laser, a gas laser, a high brightness LED, or a halogen lamp, or may be a combination of two or more of them. The light irradiation unit may include another optical element, as needed, in addition to the light source and the objective lens. A numerical aperture (NA) of the objective lens may be preferably 0.1 to 1.5, more preferably 0.5 to 1.0. Furthermore, the light irradiation position may be within a field of view of these objective lenses, or preferably, both the light irradiation position and a branch portion may be within the field of view.
The particle detection unit 102 may detect scattered light and fluorescence generated as a result of causing the first light irradiation unit 101 to irradiate the microparticle with light. The determination unit 105 included in the control unit 103 may determine whether or not the microparticle should be sucked (sorted) on the basis of the detected scattered light and fluorescence. Furthermore, the control unit 103 may detect the passage of the microparticle through the predetermined position on the basis of the detected scattered light and fluorescence. Furthermore, the control unit 103 may calculate a passing velocity of the microparticle on the basis of the detected scattered light and fluorescence.
In the emulsion detection unit 108, at least one of the different optical detection systems can detect scattered light. The scattered light may be any one of forward-scattered light, back-scattered light, or side-scattered light. Furthermore, in the emulsion detection unit 108, at least one of the different optical detection systems may detect fluorescence. The fluorescence detected may have the same wavelength or a plurality of wavelengths.
The emulsion detection unit 108 may detect information regarding the presence or absence of the emulsion, the shape of the emulsion, the number of emulsions, and the like on the basis of the forward-scattered light, and the emulsion detection unit 108 may detect information regarding the presence or absence of the microparticle in the emulsion, the shape of the emulsion, the number of emulsions, and the like on the basis of the fluorescence, the back-scattered light, or the side-scattered light. Hereinafter, the detection in the emulsion detection unit 108 will be described in more detail.
The emulsion detection unit 108 may detect scattered light and fluorescence generated as a result of causing the second light irradiation unit 109 to irradiate the microparticle with light. The emulsion detection unit 108 may detect a combination of forward-scattered light detection and fluorescence detection, a combination of forward-scattered light detection and side-scattered light detection, a combination of forward-scattered light detection and back-scattered light detection, a combination of forward-scattered light detection, side-scattered light detection, and fluorescence detection, and a combination of forward-scattered light detection, back-scattered light detection, and fluorescence detection. The emulsion detection unit 108 detects an emulsion using forward-scattered light and a sorted microparticle using fluorescence, for example. Note that, in a case where only the sorted microparticles are counted (only optimization of a sorting time (suction start time) is performed), the scattered light detection system need not be provided. In the forward-scattered light detection, the emulsion size can be detected, and in the side-scattered light detection or the back-scattered light detection, the microparticle in the emulsion can be detected by detection of the scattered light generated in the emulsion.
The particle detection unit 102 and the emulsion detection unit 108 detect scattered light and/or fluorescence generated from the microparticle as a result of causing the first light irradiation unit 101 and the second light irradiation unit 109 to irradiate the microparticle with light. The particle detection unit 102 and the emulsion detection unit 108 may include a condenser lens that condenses the fluorescence and/or the scattered light generated from the microparticle and a detector. Examples of the detector include, but not limited to, a PMT, a photodiode, a CCD, a CMOS, and the like. The particle detection unit 102 and the emulsion detection unit 108 may include another optical element, as needed, in addition to the condenser lens and the detector. The particle detection unit 102 and the emulsion detection unit 108 may further include, for example, a spectroscopic unit. Examples of optical components that form the spectroscopic unit include a grating, a prism, and an optical filter, for example. The spectroscopic unit can detect, for example, light having a wavelength to be detected separately from light having other wavelengths. The particle detection unit 102 and the emulsion detection unit 108 can convert the detected light into an analog electric signal by photoelectric conversion. The particle detection unit 102 and the emulsion detection unit 108 can further convert the analog electric signal into a digital electric signal by AD conversion.
The sorting operation of the microparticle sorting device according to the present technology forms an emulsion containing the second fluid as a dispersion medium and the first fluid as a dispersoid in the connection channel 170.
The kinematic viscosity of the first fluid and the kinematic viscosity the second fluid at 25° C. may be preferably 0.3 cSt to 5 cSt, more preferably 0.4 cSt to 4 cSt, and still more preferably 0.5 cSt to 3 cSt, for example. The kinematic viscosity of the second fluid is preferably 1/1000 to 1000 times, more preferably 1/100 to 100 times, still more preferably 1/10 to 10 times, still more preferably ⅕ to 5 times, and particularly preferably ½ to 2 times the kinematic viscosity of the first fluid. In the present technology, it is preferable that the kinematic viscosity of the first fluid and the kinematic viscosity of the second fluid are substantially the same. This makes it easy to form the emulsion.
The density of the first fluid and the density of the second fluid at 25° C. both may be preferably 0.5 g/cm3 to 5 g/cm3, more preferably 0.6 g/cm3 to 4 g/cm3, and still more preferably 0.7 g/cm3 to 3 g/cm3, for example.
Furthermore, the density of the second fluid is preferably 1/100 to 100 times, more preferably 1/10 to 10 times, still more preferably ⅕ to 5 times, and particularly preferably ½ to 2 times the density of the first fluid. In the present technology, it is preferable that the density of the first fluid and the density of the second fluid are substantially the same. This makes it easy to form the emulsion.
The first fluid and the second fluid have the above physical properties, so that the emulsion is easily formed in the particle sorting section 157. Furthermore, such physical properties allow these fluids to easily flow in a microchannel.
In one implementation of the present technology, the first fluid may be a hydrophilic fluid, and the second fluid may be a hydrophobic fluid. In this implementation, an emulsion containing the hydrophobic fluid as a dispersion medium and the hydrophilic fluid as a dispersoid may be formed in the particle sorting section 157. For example, it is desirable that a biological particle such as a cell be present in a state of being contained in the hydrophilic fluid such as a buffer or a culture solution. This implementation is therefore suitable for recovering microparticles, particularly biological particles, more particularly cells, which are desired to be present in the hydrophilic fluid.
The hydrophilic fluid contains, for example, water and a fluid miscible with water. For example, the hydrophilic fluid may be a fluid containing, as a main component, one or a mixture of two or more selected from the group consisting of water, a hydrophilic alcohol, a hydrophilic ether, ketone, a nitrile-based solvent, dimethyl sulfoxide, and N, N-dimethylformamide. In the present specification, the main component refers to a component that accounts for, for example, 50 mass % or more, particularly 60 mass % or more, more particularly 70 mass % or more, and still more particularly 80 mass % or more, 85 mass % or more, or 90 mass % or more of the fluid. Examples of the hydrophilic alcohol include ethanol, methanol, propanol, and glycerin. Examples of the hydrophilic ether include tetrahydrofuran, polyethylene oxide, and 1,4-dioxane. Examples of the ketone include acetone and methyl ethyl ketone. Examples of the nitrile-based solvent include acetonitrile.
The hydrophilic fluid may be preferably a fluid containing water as a main component, and may be, for example, water, an aqueous solution, or an aqueous dispersion. The hydrophilic fluid may be, for example, the sheath fluid and/or the sample fluid. The hydrophilic fluid is preferably a hydrophilic fluid that does not adversely affect microparticles (for example, biological particles, particularly, cells).
The hydrophilic fluid may be, for example, a fluid containing a biomolecule. The biomolecule may be, for example, one or a combination of two or more selected from amino acids, peptides, and proteins.
Furthermore, the hydrophilic fluid may contain, for example, a surfactant, particularly a nonionic surfactant. Examples of the nonionic surfactant include a triblock copolymer of polyethylene oxide and polypropylene oxide, and the triblock copolymer is also referred to as poloxamer or Pluronic (registered trademark)-based surfactant. A more specific example of the Pluronic (registered trademark)-based surfactant is Pluronic (trademark) F68.
Examples of the hydrophilic fluid include, but are not limited to, a culture solution and a buffer. The buffer is preferably Good's buffer.
When the culture solution is used as the hydrophilic fluid, a cell sorted as the microparticle to be sorted can be cultured while being held in emulsion particles.
Furthermore, when the hydrophilic fluid (particularly, the sheath fluid) contains a cell stimulating component, the cell sorted as the microparticle to be sorted can be stimulated while being held in the emulsion particles. Moreover, characteristics (for example, morphology, and the like) of the stimulated cell can also be observed under a microscope or the like.
Furthermore, the hydrophilic fluid (for example, the sheath fluid or the sample fluid) may include an assay system that enables observation of a response of cell stimulation. The use of the assay system allows the response from the cell sorted as the microparticle to be sorted to be detected, for example, optically while being held in the emulsion particles. The assay system is preferably a wash-free assay system, and for example, a system using fluorescence resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET), or the like is preferable.
As described above, in the present technology, in a case where the microparticle is a biological particle (particularly, a cell), it is possible to perform various analyses of a single biological particle (particularly, single cell analysis, for example, single cell imaging or the like).
The density of the hydrophilic fluid at 25° C. may be preferably 0.5 g/cm3 to 5 g/cm3, more preferably 0.6 g/cm3 to 4 g/cm3, and still more preferably 0.7 g/cm3 to 3 g/cm3, for example.
The kinematic viscosity of the hydrophilic fluid at 25° C. may be preferably 0.3 cSt to 5 cSt, more preferably 0.4 cst to 4 cSt, and still more preferably 0.5 cSt to 3 cSt, for example. The hydrophilic fluid has the above physical properties, so that the hydrophilic fluid easily flows in a microchannel, and an emulsion is easily formed in the particle sorting section 157.
The hydrophobic fluid may be any fluid selected from fluids that are immiscible with the hydrophilic fluid. The hydrophobic fluid may be, for example, a fluid containing, as a main component, one or a mixture of two or more selected from the group consisting of an aliphatic hydrocarbon, a fluorine-based oil, a low molecule or polymer containing a fluorine atom, a silicone oil, an aromatic hydrocarbon, an aliphatic monohydric alcohol (for example, n-octanol or the like), and a fluorinated polysaccharide.
The aliphatic hydrocarbon is preferably an aliphatic hydrocarbon having carbon atoms ranging from 7 to 30 inclusive. The number of carbon atoms is in a range of 7 to 30 inclusive, so that the kinematic viscosity of the hydrophobic fluid is suitable for flowing in the microchannel. Examples of the aliphatic hydrocarbon include mineral oils; for example, oil derived from animals and plants such as squalane oil and olive oil; for example, a paraffinic hydrocarbon having 10 to 20 carbon atoms such as decane and hexadecane; and an olefin-based hydrocarbon having 10 to 20 carbon atoms.
In the present technology, from the viewpoint of good immiscibility with the hydrophilic fluid, the hydrophobic fluid is preferably a fluorine-based oil. Examples of the fluorine-based oil include perfluorocarbon (PFC), perfluoropolyether (PFPE), and hydrofluoroether (HFE). Examples of the perfluorocarbon include Fluorinert (trademark) FC40 and Fluorinert FC-770 (manufactured by 3M Company). Examples of the perfluoropolyether include Krytox (manufactured by DuPont). Examples of the hydrofluoroether include HFE7500 (manufactured by 3M Company).
The density of the hydrophobic fluid at 25° C. may be preferably 0.5 g/cm3 to 5 g/cm3, more preferably 0.6 g/cm3 to 4 g/cm3, and still more preferably 0.7 g/cm3 to 3 g/cm3, for example.
The kinematic viscosity of the hydrophobic fluid at 25° C. may be preferably 0.3 cSt to 5 cSt, more preferably 0.4 cSt to 4 cSt, and still more preferably 0.5 cst to 3 cSt, for example.
The hydrophobic fluid has the above physical properties, so that an emulsion is easily formed in the particle sorting section 157. For example, in a case where the density or the kinematic viscosity is too high, a possibility that the fluid does not flow smoothly in the connection channel 170 increases.
In a preferred implementation of the present technology, either or both of the first fluid and the second fluid may contain a surfactant. In particular, either or both of the hydrophobic fluid and the hydrophilic fluid contain a surfactant, more particularly the hydrophobic fluid contains a surfactant. The surfactant allows emulsion particles to be not only formed easily but also maintained stably. Examples of the surfactant include a nonionic surfactant and a fluorine-based surfactant. Examples of the nonionic surfactant include, but are not limited to, Span80 and Abil EM. A type of the surfactant may be selected as appropriate by one skilled in the art. Examples of the fluorine-based surfactant include a perfluoropolyether-based surfactant and a pseudosurfactant. Examples of the former include Krytox (manufactured by DuPont), and examples of the latter include perfluorooctanol.
The surfactant may be present, for example, in the hydrophobic fluid at or above a critical micelle concentration of the surfactant. The critical micelle concentration may be preferably 1 μM to 1000 μM, particularly 10 μM to 100 mM, for example. Furthermore, the interfacial tension of the surfactant is preferably 40 mN/m or less, and may be particularly 20 mN/m or less, for example.
In another implementation of the present technology, the first fluid may be a hydrophobic fluid, and the second fluid may be a hydrophilic fluid.
In this implementation, an emulsion containing the hydrophilic fluid as a dispersion medium and the hydrophobic fluid as a dispersoid may be formed in the particle sorting section 157. Examples of the hydrophobic fluid and the hydrophilic fluid are as described above.
Furthermore, this implementation may be applied to, for example, a case where only the microparticle of interest is further sorted from an emulsion in which the dispersion medium and the dispersoid are the hydrophobic fluid and the hydrophilic fluid, respectively and that contains the microparticle.
Furthermore, as an assay system that may be used in the present technology, it is possible to use not only a system in which a microparticle emits fluorescence but also a system in which an emulsion particle emits fluorescence. Therefore, in order to recover the emulsion containing the microparticle, the determination unit 105 may make a determination on the microparticle, may make a determination on the emulsion, or may make a determination on both the microparticle and the emulsion. As described above, in the present technology, whether the microparticle and/or the emulsion is to be sorted or not may be determined on the basis of information obtained from the microparticle and/or the emulsion.
In the recovery channel 159, the microparticle to be sorted among microparticles and/or the emulsion in which the first fluid is contained in the second fluid immiscible with the first fluid is recovered.
A microparticle sorting method according to a second embodiment of the present technology includes a recovery step and a control step. The microparticle sorting method according to the present embodiment will be described below with reference to
In a recovery step S1901 in
The suction operation of sucking the microparticle by generating a negative pressure in the recovery channel, and the suction operation and the discharge operation by generating a pressure change in the recovery channel are performed in the recovery step, and in a control step S1902 in
A microparticle sorting method according to a third embodiment of the present technology allows an automatic adjustment to the drive condition of the actuator 107 under which unwanted flow oscillation generated in the connection channel 170 during the recovery operation can be suppressed, and the emulsion E containing the microparticle Po to be sorted can be formed with a desired emulsion size. In the present embodiment, it is possible to reduce, by automatically adjusting the drive condition of the actuator 107, variations in unwanted oscillation suppressing effect due to variations between individuals or variations between lots of microchips. The microparticle sorting method according to the third embodiment of the present technology includes a determination step of determining a first piezo drive height D1, a determination step of determining a drive time interval Th, a determination step of determining a second piezo drive height D, and a determination step of determining a recovery start time Td. The microparticle sorting method in the present embodiment will be described below with reference to
In a determination step S2001 of determining the first piezo drive height D1 in
In the acquiring step S2101 of acquiring the signal intensity of the emulsion in
In a determination step S2002 of determining the drive time interval Th in
In the emulsion counting step S2104 of counting the number of emulsions in
In a determination step S2003 of determining the second piezo drive height D in
In the acquiring step S2107 of acquiring the signal intensity of the emulsion in
In a determination step S2004 of determining the recovery start time Td in
In the particle counting step S2110 of counting the number of particles, the number of particles passing through the emulsion detection region 164 located downstream of the recovery channel 159 is counted in a case where the sorting operation is performed under conditions of the recovery start time from the detection of microparticles to the suction operation set to a predetermined recovery start time Td0, and the drive time interval Th and the second piezo drive height D thus determined. Here, in the particle counting step S2110 of counting the number of particles, the sorting operation may be performed for a known number of particles (for example, 10 to 1000) to count the number of recovered particles. In the repeating step S2111 of repeating the particle counting step of counting the number of particles, the particle counting step of counting the number of particles is repeatedly performed under the same conditions except that the recovery start time is changed to a time longer or shorter than Td0. As a result, the counted numbers of particles recovered in the recovery channel 159 at various recovery start times Td are obtained as illustrated in
Note that the present technology may also have following configurations.
[1]
A microparticle sorting device including:
The microparticle sorting device according to [1], in which
The microparticle sorting device according to [1] or [2], in which
The microparticle sorting device according to any one of [1] to [3], in which
The microparticle sorting device according to any one of [1] to [4], in which
The microparticle sorting device according to any one of [1] to [5], in which
The microparticle sorting device according to any one of [1] to [5], in which
The microparticle sorting device according to any one of [1] to [7], in which
The microparticle sorting device according to any one of [1] to [8], further including an emulsion detection unit that detects the microparticles and/or the emulsion in the recovery channel.
[10]
The microparticle sorting device according to [9], in which
The microparticle sorting device according to [9] or [10], in which
A microparticle sorting method including:
| Number | Date | Country | Kind |
|---|---|---|---|
| 2022-038878 | Mar 2022 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2023/006138 | 2/21/2023 | WO |
