Droplet separation device, separation signal generation device, separation signal generation method, and program

Abstract

This droplet separation device comprises: an upstream part; a chamber part provided downstream from the upstream part and having a larger cross-sectional area than the upstream part; a branch part provided downstream from the chamber part; two branch flow paths branched by the branch part; a micro flow path in which droplets flow together with a first fluid; a droplet interval adjustment flow path that is connected to the upstream part so as to flow thereinto and causes a second fluid having the same composition as the first fluid to flow into the upstream part; an electric field generating electrode that is provided adjacent to the chamber part and that generates an electric field by applying a voltage controlled by an external signal; and a plurality of reference electrodes provided to generate an electric field gradient in the chamber part in response to the generation of the electric field by the electric field generating electrode, wherein the branch part has an asymmetric shape so that the droplet flows into only one of the two branch flow paths without being broken when the electric field generating electrode does not generate an electric field.

Description

Droplet sorting device, sorting signal generating device, sorting signal generating method, and program

[0001] This application claims priority to U.S. Provisional Application No. 63 / 422,611, filed November 4, 2022, the contents of which are incorporated herein by reference.

[0002] Microfluidic systems utilizing droplets with diameters of 100 micrometers or less allow the compartmentalization of molecules or particles, chemical reactions, or bioassays in sub-nanoliter volumes. These systems have been used to achieve rapid screening of drug molecules and single-cell analysis. These analyses may require measuring the contents or state of individual droplets and selectively aliquoting them for further manipulation or analysis.

[0003] A method utilizing dielectrophoresis is widely used as a method for sorting droplets in a microfluidic device (see, for example, Patent Document 1). In this method, electrodes are formed near a branched microchannel, and a voltage is applied to the electrodes as the droplets to be sorted pass near the electrodes, thereby generating an electric field. The dielectrophoresis caused by the generated electric field causes the droplets to change their course, allowing them to be sorted. In this method, the droplets can be selectively sorted without splitting or fusing with other droplets by adjusting the spacing between droplets, the droplet speed, the applied voltage, the channel structure, and the electrode structure.

[0004] High-speed droplet sorting is important because it enables shortening of the total analysis time, discovery and analysis of rare events from a large population, and preparation of a large number of diverse subsets for subsequent analysis. For example, in order to perform gene expression analysis or gene function analysis on extracellular particles, which are much smaller than cells, the speed of 10 ６ From 10 ７ To achieve this, it is necessary to prepare a subset of particles of about 10 ... ７ From 10 ８It is necessary to measure approximately 100 droplets and selectively separate droplets containing the particles to be analyzed from the measured droplets.

[0005] JP 2014-178119 A

[0006] However, achieving both high-speed droplet sorting and accurate sorting has been difficult. This is because increasing droplet processing speed inevitably involves increasing droplet velocity within the channel or decreasing the spacing between droplets. The former, increasing droplet velocity within the channel, makes sorting itself difficult because it does not provide sufficient dielectrophoretic time for sorting in branched microchannels. The latter, decreasing the spacing between droplets, makes it difficult to selectively sort only the droplets to be sorted from among droplets adjacent in the flow direction. In response to this, because sorting performance is highly dependent on fluid conditions and the distribution of the electric field generated by the electrodes, droplet sorting devices with various channel and electrode structures have been proposed. However, these conventional droplet sorting devices achieve a sorting speed of approximately several thousand droplets per second. There is also a known droplet sorting device that can sort 30,000 droplets per second by lowering the channel height of the branching part of the microchannel compared to the surrounding area. However, this method requires a complex channel structure and deformation of the droplets, which poses problems in terms of versatility and stability.

[0007] As described above, conventional droplet sorting devices using microchannel devices have the problem of being insufficient in versatility and stability for high-speed droplet sorting. There is a demand for a droplet sorting device that can achieve both high-speed droplet sorting and accurate sorting with a simple configuration.

[0008] The present invention has been made in consideration of the above points, and provides a sorting signal generating device, a sorting signal generating method, and a program that can achieve both high-speed droplet sorting and accurate sorting with a simple configuration.

[0009] The present invention has been made to solve the above-mentioned problems, and one aspect of the present invention is a droplet sorting device comprising: an upstream section; a chamber section located downstream of the upstream section and having a larger cross-sectional area than the upstream section; a branch section located downstream of the chamber section; and two branch flow paths branched by the branch section, wherein droplets flow together with a first fluid; a droplet spacing adjustment flow path connected to join the upstream section and allowing a second fluid having the same composition as the first fluid to flow into the upstream section; an electric field generating electrode located adjacent to the chamber section and controlled by an external signal to generate an electric field when a voltage is applied; and a plurality of reference electrodes located so as to generate an electric field gradient in the chamber section in response to the generation of the electric field by the electric field generating electrode, wherein the branch section has an asymmetric shape so that when the electric field generating electrode is not generating an electric field, the droplets flow into only one of the two branch flow paths without breaking up.

[0010] Furthermore, one aspect of the present invention is that, in the above-mentioned droplet sorting device, the distance between the connection portion where the droplet spacing adjustment flow path is connected to the upstream portion and the chamber portion is the distance obtained by multiplying the spacing between the droplets upstream of the connection portion in the upstream portion by the ratio of the flow rate upstream of the connection portion in the upstream portion to the flow rate downstream of the connection portion.

[0011] Furthermore, one aspect of the present invention is that in the above-mentioned droplet sorting device, the width of the inlet section, which is the most upstream part of the chamber section, is identical to the diameter of the droplet, and the width of the outlet section, which is the most downstream part of the chamber section, is identical to the sum of twice the diameter of the droplet and the width of the branch section.

[0012] Furthermore, one aspect of the present invention is that, in the above-mentioned droplet sorting device, the cross-sectional area of ​​the chamber portion is a cross-sectional area that corresponds to the cross-sectional area of ​​the upstream portion and the flow rate of the second fluid that the droplet spacing adjustment flow path causes to flow into the upstream portion.

[0013] Furthermore, one aspect of the present invention is that, in the above-mentioned droplet sorting device, the length of the chamber section corresponds to the flow rate downstream of the connection section where the droplet spacing adjustment flow path in the upstream section is connected to the upstream section, and is a length such that the number of droplets flowing through the chamber section at the same time is one on average over time.

[0014] In one aspect of the present invention, in the droplet sorting device, the width of the branching portion is approximately the same as or equal to the diameter of the droplets.

[0015] Furthermore, one aspect of the present invention is that in the droplet sorting device described above, the shape of the most upstream part of the branching section is a curved surface on one side of the two branching flow paths through which the droplets flow when the electric field generating electrode is not generating an electric field.

[0016] Furthermore, one aspect of the present invention is that, in the above-mentioned droplet sorting device, the tip portion of the electric field generating electrode faces a first side surface of the chamber portion, the surface orientation of the tip portion is approximately parallel to the surface orientation of the first side surface, and the surface area of ​​the tip portion is approximately the same as the area of ​​the first side surface.

[0017] In one aspect of the present invention, in the droplet sorting device described above, some of the plurality of reference electrodes are provided adjacent to the electric field generating electrode on the side of the microchannel where the electric field generating electrode is provided, and some of the remaining plurality of reference electrodes are provided on the side of the microchannel where the electric field generating electrode is not provided.

[0018] In one aspect of the present invention, in the droplet sorting device, the width of the microchannel is approximately the same as the diameter of the droplets in a portion other than the chamber portion.

[0019] Another aspect of the present invention is a sorting signal generating device comprising: a first trigger signal generating unit that generates a first trigger signal when a sorting target, enclosed in a droplet and flowing through a microchannel together with a first fluid, is detected; a second trigger signal generating unit that generates a second trigger signal when the droplet flowing through the microchannel passes a predetermined position in the direction of flow velocity through the microchannel; a determination unit that determines whether a determination signal, which is the sum of the magnitude of the first trigger signal and the magnitude of the second trigger signal, is equal to or greater than a predetermined threshold; and a sorting signal output unit that outputs a sorting signal for sorting the sorting target when the determination signal is equal to or greater than the threshold.

[0020] In addition, in one aspect of the present invention, in the above-mentioned sorting signal generating device, the fact that the droplets flowing through the microchannel have passed the position in the flow velocity direction of the microchannel is detected based on scattered light from the droplets flowing through the microchannel.

[0021] Another aspect of the present invention is a sorting signal generating method including: a first trigger signal generating step of generating a first trigger signal when a sorting target, enclosed in a droplet and flowing through a microchannel together with a first fluid, is detected; a second trigger signal generating step of generating a second trigger signal when the droplet flowing through the microchannel passes a predetermined position in the direction of flow velocity of the microchannel; a determination step of determining whether a determination signal, which is the sum of the magnitude of the first trigger signal and the magnitude of the second trigger signal, is equal to or greater than a predetermined threshold; and a sorting signal output step of outputting a sorting signal for sorting the sorting target when the determination signal is equal to or greater than the threshold.

[0022] Another aspect of the present invention is a program for causing a computer to execute the following steps: a first trigger signal generating step of generating a first trigger signal when a separation target, enclosed in a droplet and flowing through a microchannel together with a first fluid, is detected; a second trigger signal generating step of generating a second trigger signal when the droplet flowing through the microchannel passes a predetermined position in the direction of flow velocity of the microchannel; a determination step of determining whether a determination signal, which is the sum of the magnitude of the first trigger signal and the magnitude of the second trigger signal, is equal to or greater than a predetermined threshold; and a separation signal output step of outputting a separation signal for separating the separation target when the determination signal is equal to or greater than the threshold.

[0023] According to the present invention, both high speed droplet dispensing and accurate dispensing can be achieved with a simple configuration.

[0024] 1 is a plan view showing the configuration of a micro-channel chip according to an embodiment of the present invention. FIG. 1 is a plan view showing an outline of the configuration of a micro-channel chip according to an embodiment of the present invention. FIG. 2 is a diagram showing an example of an outline of detection of a measurement sample by a measurement sample detection device according to an embodiment of the present invention. FIG. 3 is a diagram showing an example of an outline of alignment of measurement samples in an alignment unit according to an embodiment of the present invention. FIG. 4 is a diagram showing the focusing performance by an alignment unit according to an embodiment of the present invention. FIG. 5 is a diagram showing an example of time change in signal intensity of fluorescence from a measurement sample detected by a detector according to an embodiment of the present invention. FIG. 6 is a diagram showing an example of the count number of a measurement sample versus signal intensity according to an embodiment of the present invention. FIG. 1 is a plan view showing an outline of the configuration of a droplet sorting unit according to an embodiment of the present invention. FIG. 2 is a plan view showing an enlarged view of the periphery of a chamber unit according to an embodiment of the present invention. FIG. 3 is a diagram showing a three-dimensional model of a micro-channel used in a simulation of an electric field gradient according to an embodiment of the present invention. FIG. 4 is a diagram showing the calculation results of an electric field gradient calculated by a simulation of an electric field gradient according to an embodiment of the present invention. FIG. 5 is a diagram showing an outline of determination of conditions for sorting based on a measurement signal according to an embodiment of the present invention. FIG. 6 is a diagram showing an outline of detection of a measurement signal according to an embodiment of the present invention. FIG. 7 is a diagram showing an outline of signal processing related to output of a sorting signal according to an embodiment of the present invention. FIG. 8 is a diagram showing an example of the functional configuration of a sorting signal generating device according to an embodiment of the present invention. FIG. 9 is a diagram showing an example of the flow of a sorting signal generation process according to an embodiment of the present invention. FIG. 10 is a diagram showing an example of an optical system according to an embodiment of the present invention. FIG. FIG. 1 is a diagram showing an example of the results of imaging droplet generation according to an embodiment of the present invention; FIG. 2 is a diagram showing the results of imaging droplets flowing through a microchannel when no voltage is applied to an electric field generating electrode according to an embodiment of the present invention; FIG. 3 is a diagram showing the results of imaging droplets flowing through a microchannel when a voltage of 1000 volts is applied to an electric field generating electrode according to an embodiment of the present invention; FIG. 4 is a diagram showing the results of fractionation at 50 microsecond time intervals according to an embodiment of the present invention; and FIG. 5 is a diagram showing a time series of captured images when fractionation is performed at 50 microsecond time intervals according to an embodiment of the present invention.1 is a diagram showing the results of fractionation at a time interval of 100 microseconds according to an embodiment of the present invention; FIG. 2 is a diagram showing a time series of captured images when fractionation is performed at a time interval of 100 microseconds according to an embodiment of the present invention; FIG. 3 is a diagram showing fractionation results when the time from detection of a signal detection signal to generation of a first trigger signal according to an embodiment of the present invention is shifted; FIG. 4 is a histogram showing, for each measurement sample, how many frames before the frame in which the measurement sample was detected according to an embodiment of the present invention was the frame in which fractionation was performed; FIG. 4 is a diagram showing fractionation accuracy versus fractionation delay time according to an embodiment of the present invention; FIG. 5 is a diagram showing the results of fractionation of nanoparticles using a droplet detection device according to an embodiment of the present invention; FIG. 6 is a diagram showing the results of evaluation of fractionation performance according to an embodiment of the present invention; FIG. 7 is a diagram showing the ratio of the number of counted fractionation targets to the number of counted droplets according to an embodiment of the present invention; FIG. 8 is a diagram showing detection results when two types of nanoparticles are used before fractionation according to an embodiment of the present invention; FIG. 9 is a diagram showing detection results when two types of nanoparticles are used after fractionation according to an embodiment of the present invention; FIG. 10 is a diagram showing the results of fractionation of extracellular microparticles according to an embodiment of the present invention;

[0025] Hereinafter, an embodiment of the present invention will be described in detail with reference to the drawings. In this embodiment, unless otherwise specified, a certain amount being approximately the same as another amount means that the difference between the certain amount and the other amount is within 20% of the other amount.

[0026] Fig. 1 is a plan view showing the configuration of a micro-channel chip 1 according to this embodiment. Fig. 2 is a plan view showing the outline of the configuration of the micro-channel chip 1 according to this embodiment.

[0027] The microchannel chip 1 includes a sample flow supply unit 2, a sample channel 21, a first sheath liquid supply unit 3, a first sheath liquid inlet channel 31, a first oil supply unit 4, an oil inlet channel 41, a second oil supply unit 5, and a droplet sorting unit 6.

[0028] In the drawings, an xyz coordinate system is shown as a three-dimensional Cartesian coordinate system where appropriate. In this embodiment, the y-axis direction is the width direction of the sample channel 21. The x-axis direction is the length direction of the sample channel 21. The length direction of the sample channel 21 is also referred to as the flow direction of the sample channel 21 or the flow direction of the sample flow. The z-axis direction is perpendicular to the sample channel 21 and is the height direction of the sample channel 21. In this embodiment, the height of the bottom surface of the sample channel 21 does not change in the flow direction of the sample channel 21. The flow of liquid in the sample channel 21 moves the measurement sample A1 in the +x direction of the x-axis. In other words, the width direction of the sample channel 21 is the direction perpendicular to the flow line of the fluid flowing with the measurement sample A1.

[0029] One end of the sample flow channel 21 communicates with the sample flow supply unit 2. The other end of the sample flow channel 21 communicates with the droplet sorting unit 6. The sample flow F1 is supplied from the sample flow supply unit 2 and flows from one end of the sample flow channel 21 to the other end.

[0030] The downstream end of the sample flow supply unit 2 in the flow direction of the sample flow F1 is connected to the upstream end of the sample flow channel 21 in the flow direction of the sample flow F1. The sample flow supply unit 2 supplies the sample flow F1 to the sample flow channel 21. The sample flow F1 is a fluid containing a plurality of measurement samples A1 (also referred to as measurement targets). The amount of sample flow F1 supplied by the sample flow supply unit 2 per minute is 0.2 microliters per minute.

[0031] The measurement sample A1 is a microparticle. In this embodiment, the diameter of the microparticle is 1 nanometer or less. In other words, the measurement sample A1 is a nanoparticle. As an example, the measurement sample A1 is an extracellular microparticle or a microbead (e.g., a polystyrene bead).

[0032] The first sheath liquid inlet channel 31 is formed alongside the sample flow channel 21. Two first sheath liquid inlet channels 31 are formed. The two first sheath liquid inlet channels 31 are formed symmetrically with the sample flow channel 21 in between. A first sheath liquid B1 flows through the first sheath liquid inlet channel 31. The first sheath liquid B1 aligns the measurement samples A1 in a row in the alignment section 211 and flows continuously through the alignment section 211.

[0033] The first sheath liquid B1 flows through the first sheath liquid inlet channel 31 in the same direction as the sample flow F1, from upstream to downstream in the flow direction of the sample flow F1. The two first sheath liquid inlet channels 31 are connected at their upstream ends and downstream ends in the flow direction of the first sheath liquid B1. The downstream ends of the two first sheath liquid inlet channels 31 are connected to the alignment section 211 of the sample flow channel 21.

[0034] The first sheath fluid supply unit 3 is provided at the upstream ends of the two first sheath fluid inlet channels 31. The first sheath fluid supply unit 3 is in communication with the upstream ends of the two first sheath fluid supply units 3. The first sheath fluid supply unit 3 supplies the first sheath fluid B1 to the two first sheath fluid supply units 3. The amount of first sheath fluid B1 supplied by the first sheath fluid supply unit 3 per hour is 2 microliters per minute.

[0035] A detection unit 212 is provided downstream of the alignment unit 211. The detection unit 212 is the portion of the sample flow channel 21 where detection of the measurement sample A1 is performed. A measurement sample detection device 9 (not shown) is provided near the detection unit 212. The measurement sample detection device 9 is a device separate from the micro-channel chip 1. The measurement sample detection device 9 includes, for example, a laser light source, a detector, and a control unit.

[0036] 3 to 7, the alignment of measurement samples A1 in alignment unit 211 and detection of measurement samples A1 by measurement sample detection device 9 will be described. Fig. 3 is a diagram showing an example of an outline of detection of measurement samples A1 by measurement sample detection device 9 according to this embodiment. Fig. 4 is a diagram showing an example of an outline of alignment of measurement samples A1 in alignment unit 211 according to this embodiment.

[0037] The alignment unit 211 rectifies the sample flow F1 using hydrofocusing technology. The alignment unit 211 focuses the sample flow F1 to the center of the sample flow channel 21 using hydrofocusing technology. Here, the center of the sample flow channel 21 refers to the center of the sample flow channel 21 in both the height and width directions. Therefore, the alignment unit 211 rectifies the sample flow F1 in both the height and width directions of the sample flow channel 21. The rectification by the alignment unit 211 is also referred to as three-dimensional focusing. The sample flow channel 21 communicates with the first sheath fluid inlet channel 31 at the alignment unit 211. The height (depth) of the first sheath fluid inlet channel 31 is greater than the height of the sample flow channel 21. The sample flow channel 21 communicates with the first sheath fluid inlet channel 31 at the center position of the first sheath fluid inlet channel 31 in the height direction.

[0038] In the alignment section 211, the first sheath fluid B1 flows in from both side surfaces of the sample flow channel 21. The inflowing first sheath fluid B1 focuses the sample flow F1 toward the center of the sample flow channel 21. As shown in Figure 4, upstream of the alignment section 211 in the sample flow channel 21, the measurement sample A1 varies in position in both the height and width directions.

[0039] Downstream of the alignment section 211, the measurement sample A1 is prevented from varying in position in both the height and width directions of the sample flow channel 21. In other words, by focusing the sample flow F1 at the center of the sample flow channel 21, the position of the measurement sample A1 is aligned in both the height and width directions of the sample flow channel 21. In the alignment section 211, the sample flow F1 is hardly diluted by the inflow of the first sheath fluid B1, and the sample flow F1 can be focused at the center of the sample flow channel 21. Note that the sample flow F1 flowing downstream of the alignment section 211 flows through the sample flow channel 21 together with the first sheath fluid B1, but the sample flow F1 flowing downstream of the alignment section 211 together with the first sheath fluid B1 will also be referred to as the sample flow F1.

[0040] Next, we will explain the detection of measurement sample A1 by measurement sample detection device 9. Measurement sample A1 aligned by alignment unit 211 flows through sample flow path 21 together with sample flow F1 and reaches detection unit 212 downstream of alignment unit 211. In detection unit 212, measurement sample detection device 9 detects measurement sample A1.

[0041] The measurement sample detection device 9 detects the measurement sample A1 flowing through the sample flow path 21 together with the sample flow F1. The measurement sample detection device 9 uses a detector to detect fluorescence or scattered light generated from the measurement sample A1 by irradiation with laser light, and acquires, for example, information about the measurement sample A1. In this embodiment, the measurement sample detection device 9 detects the fluorescence generated from the measurement sample A1. Based on the acquired information, the cell information acquisition device 300 uses a control unit to identify a separation target A2 from among the multiple measurement samples A1 contained in the sample flow F1. Upon detecting the separation target A2, the control unit outputs a signal (referred to as a signal detection signal SS1) indicating that the separation target A2 has been detected to the droplet sorting unit 6.

[0042] The measurement sample detection device 9 includes an irradiation optical system that irradiates the measurement sample A1 with laser light and a detection optical system that detects fluorescence or scattered light generated from the measurement sample A1 using a detector. The irradiation optical system includes optical elements such as a dichroic mirror and an objective lens, as well as a laser light source. The detection optical system includes optical elements such as an objective lens, a dichroic mirror, and a filter, as well as a detector. Some optical elements may be shared between the irradiation optical system and the detection optical system. The wavelength of the laser emitted by the laser light source is, for example, 488 nm. The detector included in the measurement sample detection device 9 is, for example, a photomultiplier tube (PMT). The measurement sample detection device 9 may also have the function of identifying the characteristics of the sample A2 through machine learning.

[0043] Figure 5 shows the focusing performance of the alignment unit 211. As described above, the flow of first sheath liquid B1 into the sample flow channel 21 focuses the sample flow F1 at the center of the sample flow channel 21. The graph shown in Figure 5 is a histogram showing the distribution of the widthwise positions of the measurement sample A1 in the sample flow channel 21 after it has passed a certain position downstream of the alignment unit 211. This graph shows that the variation in the widthwise positions of the measurement sample A1 from the center is kept to 2 micrometers or less.

[0044] FIG. 6 shows an example of the change over time in the signal intensity of fluorescence from measurement sample A1 detected by the detector. The results shown in FIG. 6 are detection results obtained using fluorescent beads with a diameter of 40 nanometers for measurement sample A1. There were 20 measurement samples A1. FIG. 7 shows an example of the count number for measurement sample A1 versus signal intensity. The results shown in FIG. 7 are detection results obtained using fluorescent beads with a diameter of 40 nanometers for measurement sample A1, just like FIG. 6. There were 15,000 measurement samples A1. The histogram shown in FIG. 7 shows that the measurement sample detection device 9 was able to distinguish between fractionation targets A2 and non-fractionation targets A3 for 15,000 measurement samples A1.

[0045] 1 and 2, the description of the configuration of the micro-channel chip 1 will continue. The oil inlet channel 41 is formed alongside the first sheath liquid inlet channel 31. Two oil inlet channels 41 are formed. The two oil inlet channels 41 are formed symmetrically with the first sheath liquid inlet channel 31 in between. The first oil C1 flows through the oil inlet channels 41.

[0046] The first oil C1 flows through the oil inlet channel 41 in the same direction as the sample flow F1, from upstream to downstream in the flow direction of the sample flow F1. The two oil inlet channels 41 are connected at their upstream ends and downstream ends in the flow direction of the first oil C1. The downstream ends of the two oil inlet channels 41 are connected to the droplet generation unit 213 of the sample flow channel 21.

[0047] The first oil C1 forms droplets D1 that envelop the sample flow F1 in the droplet generating unit 213. Depending on the time when the measurement sample A1 reaches the position of the droplet generating unit 213 and the time when the droplets D1 are formed, the droplets D1 may contain the measurement sample A1 or not. The first oil C1 and the droplets D1 flowing downstream from the droplet generating unit 213 are collectively referred to as a phase fluid G1. The first oil C1 corresponds to a continuous phase fluid. The droplets D1 correspond to a dispersed phase fluid.

[0048] The first oil supply unit 4 is provided at the upstream ends of the two oil inlet passages 41. The first oil supply unit 4 is connected to the upstream ends of the two oil inlet passages 41. The first oil supply unit 4 supplies the first oil C1 to the two oil inlet passages 41. The amount of the first oil C1 supplied by the first oil supply unit 4 per hour is 12 microliters per minute.

[0049] The second oil supply unit 5 is provided at the upstream end of the droplet interval adjustment flow path 62. The second oil supply unit 5 supplies the second oil E1 to the droplet interval adjustment flow path 62. The amount of the second oil E1 supplied by the second oil supply unit 5 per hour is 20 microliters per minute.

[0050] The droplet sorting unit 6 sorts out droplets D1 that surround a sorting target A2 from among the droplets D1. The droplet sorting unit 6 includes a microchannel 61, a droplet spacing adjustment channel 62, an electric field generating electrode 63, and a plurality of reference electrodes 64.

[0051] The waste-side discharge port 7 is provided at the downstream end of the waste liquid-side flow path 613. A phase fluid G1 containing droplets D1 that were not sorted by the droplet sorting unit 6 flows through the waste-side discharge port 7. The phase fluid G1 that passes through the waste-side discharge port 7 is discharged into a test tube (not shown) or the like that is arranged downstream of the downstream end of the waste-side discharge port 7.

[0052] The sorting outlet 8 is provided at the downstream end of the sorting flow path 614. A phase fluid G1 containing droplets D1 sorted by the droplet sorting unit 6 flows through the sorting outlet 8. The phase fluid G1 that passes through the sorting outlet 8 is discharged into a test tube (not shown) or the like that is located downstream of the downstream end of the sorting outlet 8.

[0053] 8 to 10, the configuration of the droplet sorting unit 6 will be described in detail. Fig. 8 is a plan view showing the outline of the configuration of the droplet sorting unit 6 according to this embodiment. Fig. 9 is a plan view of the droplet sorting unit 6 according to this embodiment.

[0054] A droplet D1 flows through the microchannel 61 together with a first oil C1. A measurement sample A1 is enveloped in the droplet D1 and flows through the microchannel 61 together with the first oil C1. The first oil C1 flows through the sample channel 21 as a continuous phase fluid. The droplet D1 flows through the sample channel 21 as a dispersed phase fluid. The microchannel 61 is in communication with the sample channel 21 on the upstream side.

[0055] The microchannel 61 has an upstream section 610, a chamber section 611, a branch section 612, a waste liquid side channel 613, and a separation side channel 614. The upstream section 610, the chamber section 611, and the branch section 612 are provided in this order from upstream to downstream. Therefore, the chamber section 611 is provided downstream of the upstream section 610. The branch section 612 is provided downstream of the chamber section 611. The waste liquid side channel 613 and the separation side channel 614 are provided downstream of the branch section 612. The waste liquid side channel 613 and the separation side channel 614 are two branch channels branched by the branch section 612.

[0056] The droplet spacing adjustment flow path 62 is connected to merge with the upstream section 610. The droplet spacing adjustment flow path 62 causes the second oil E1 to flow into the upstream section 610. The second oil E1 is a fluid with the same composition as the first oil C1. Note that the first oil C1 is an example of a first fluid, and the second oil E1 is an example of a second fluid.

[0057] The droplet spacing adjustment channel 62 allows the second oil E1 to flow into the upstream section 610, thereby finely adjusting the speed of the droplets D1 flowing through the microchannel 61 and the spacing between the droplets D1. The spacing between the droplets D1 refers to the spacing between adjacent droplets D1 in the flow direction of the sample channel 21 among the multiple droplets D1 flowing through the sample channel 21. Finely adjusting the speed of the droplets D1 means increasing the speed of the droplets D1 flowing through the microchannel 61 together with the first oil C1 as the phase fluid G1 by a predetermined percentage relative to the flow rate of the phase fluid G1 when the droplet spacing adjustment channel 62 is not provided, by a percentage corresponding to the flow rate of the second oil E1. Similarly, finely adjusting the spacing between the droplets D1 means changing the spacing between the droplets D1 by a percentage corresponding to the flow rate of the second oil E1 relative to the spacing between the droplets D1 when the droplet spacing adjustment channel 62 is not provided.

[0058] The chamber portion 611 is wider than the upstream portion 610. The chamber portion 611 is deeper than the upstream portion 610. In other words, the chamber portion 611 has a larger cross-sectional area than the upstream portion 610. By making the cross-sectional area of ​​the microchannel 61 larger than that of the upstream portion 610, the microchannel 61 decelerates the droplets D1 that flow through the microchannel 61 together with the first oil C1 as the phase fluid G1. Here, the cross-sectional area of ​​each portion of the microchannel 61, such as the chamber portion 611 or the upstream portion 610, refers to the area of ​​a cross section perpendicular to the flow direction of the microchannel 61.

[0059] The electric field generating electrode 63 is provided adjacent to the chamber portion 611 and generates an electric field by applying a voltage controlled by an external signal. The plurality of reference electrodes 64 are provided so as to generate an electric field gradient in the chamber portion 611 in response to the electric field generated by the electric field generating electrode 63. The electric field gradient is locally high enough to allow the droplet D1 to be separated by dielectrophoretic force. The plurality of reference electrodes 64, for example, includes a first reference electrode 641, a second reference electrode 642, a third reference electrode 643, and a fourth reference electrode 644.

[0060] The branching section 612 has an asymmetric shape so that when the electric field generating electrode 63 is not generating an electric field, the droplets D1 flow only through the waste side flow path 613 out of the waste side flow path 613 and the separation side flow path 614 without breaking up.

[0061] The widths of the upstream section 610, the separation-side channel 614, and the waste-side channel 613 are approximately the same as the diameter of the droplet D1. In other words, the width of the microchannel 61 is approximately the same as the diameter of the droplet D1 in the portions other than the chamber section 611. In this embodiment, the widths of the upstream section 610, the separation-side channel 614, and the waste-side channel 613 are each 12 micrometers. With this configuration, the droplet D1 flowing through the microchannel 61 is less likely to break at the branch section 612, making it easier to separate.

[0062] The width of the microchannel 61 does not have to be approximately the same as the diameter of the droplet D1 in the portion other than the chamber portion 611. However, in order to make the droplet D1 flowing through the microchannel 61 less likely to be broken at the branching portion 612 and to facilitate sorting, it is preferable that the width of the microchannel 61 be approximately the same as the diameter of the droplet D1 in the portion other than the chamber portion 611.

[0063] Here, the configuration of the droplet spacing adjustment flow path 62 will be described in more detail. The droplet spacing adjustment flow path 62 is connected to the upstream section 610 at a connection section 621. The connection section 621 is the section to which the droplet spacing adjustment flow path 62 of the upstream section 610 is connected. Here, the distance between the connection section 621 and the chamber section 611 is a distance (referred to as a first distance) obtained by multiplying the distance between droplets D1 upstream of the connection section 621 in the upstream section 610 by the ratio of the flow rate upstream of the connection section 621 to the flow rate downstream of the connection section 621 in the upstream section 610.

[0064] By setting the distance between the connection part 621 and the chamber part 611 to the first distance, the distance from the droplet detection part 214 to the droplet sorting part 6 can be made as short as possible, and the time from when the droplet D1 is detected to when the droplet D1 is sorted can be made as short as possible. Furthermore, by setting the distance between the connection part 621 and the chamber part 611 to the first distance, when sorting the droplet D1 to be sorted, the influence of the electric field generated by the electric field generating electrode 63 on the droplet D1 adjacent to the droplet D1 to be sorted in the flow direction of the sample flow channel 21 can be made as small as possible. The droplet D1 adjacent to the droplet D1 to be sorted includes both the droplet D1 flowing upstream of the droplet D1 to be sorted and the droplet D1 flowing downstream of the droplet D1 to be sorted.

[0065] The distance between the connection part 621 and the chamber part 611 may be longer or shorter than the first distance. However, as described above, in order to simultaneously shorten the time from when the droplet D1 is detected until the droplet D1 is dispensed and to reduce the effect of the electric field on the droplet D1 to be dispensed and on the droplets D1 adjacent to the droplet D1, it is preferable that the distance between the connection part 621 and the chamber part 611 be set to the first distance.

[0066] Next, the configuration of the chamber section 611 will be described in more detail. The shape of the chamber section 611 is trapezoidal when the micro-channel chip 1 is viewed from above. FIG. 10 shows an enlarged plan view of the periphery of the chamber section 611. The inlet section 6111 is the most upstream part of the chamber section 611. The outlet section 6112 is the most downstream part of the chamber section 611. The inlet section 6111 corresponds to the upper side of the trapezoid in the plan view. The outlet section 6112 corresponds to the lower side of the trapezoid in the plan view. A first side surface 6113 and a second side surface 6114, which are side surfaces that make up the chamber section 611, respectively correspond to the legs of the trapezoid. The first side surface 6113 faces an electrode tip section 631, which is the tip section of the electric field generating electrode 63.

[0067] The width of the inlet portion 6111 is the same as the diameter of the droplet D1. In this embodiment, the diameter of the droplet D1 is about 10 micrometers (9 micrometers to 12 micrometers). The width of the outlet portion 6112 is the same as the sum of twice the diameter of the droplet D1 and the width of the branch portion 612. The width of the branch portion 612 is 10 micrometers. Therefore, the sum of twice the diameter of the droplet D1 and the width of the branch portion 612 is 34 micrometers.

[0068] The bottom surface of the chamber portion 611 is deeper than the bottom surface of the upstream portion 610. The ceiling of the chamber portion 611 is the same height as the ceiling of the upstream portion 610. The depth of the chamber portion 611 is about two to three times the depth of the upstream portion 610. In this embodiment, the depth of the upstream portion 610 is 18 micrometers. Therefore, the depth of the chamber portion 611 is 30 to 40 micrometers. By making the depth of the chamber portion 611 about two to three times the depth of the upstream portion 610, it is possible to temporarily decelerate the droplet D1 in the chamber portion 611, where a strong dielectrophoretic force acts.

[0069] The length of the chamber 611 corresponds to the flow rate downstream of the connection part 621, and is a length that allows the number of droplets D1 flowing through the chamber 611 at the same time to be one on average over time. This configuration makes it possible to prevent the droplet D1 to be collected from being mistakenly collected with another droplet D1 adjacent to it in the flow direction of the microchannel 61. In this embodiment, the length of the chamber 611 is 55 micrometers.

[0070] In the droplet sorting section 6 of this embodiment, the configuration of the chamber section 611 described above makes it possible to increase the speed of the droplet D1 flowing through the microchannel 61 as a whole, while applying a dielectrophoretic force on the droplet D1 that is large enough for sorting.

[0071] The configuration (shape and size) of chamber 611 is not limited to the configuration described above, as long as the cross-sectional area of ​​chamber 611 is larger than the cross-sectional area of ​​upstream section 610. For example, the ceiling of chamber 611 may be higher than the ceiling of upstream section 610, and the bottom surface of chamber 611 may be at the same depth as the bottom surface of upstream section 610. The ceiling of chamber 611 may be higher than the ceiling of upstream section 610, and the bottom surface of chamber 611 may be deeper than the bottom surface of upstream section 610. Furthermore, the ceiling of chamber 611 may be at the same height as the ceiling of upstream section 610, the bottom surface of chamber 611 may be at the same depth as the bottom surface of upstream section 610, and the width of chamber 611 may be wider than the width of upstream section 610.

[0072] The depth of the chamber portion 611 may be other than approximately two to three times the depth of the upstream portion 610. When the depth of the chamber portion 611 is greater than approximately two to three times the depth of the upstream portion 610, the droplets D1 are decelerated more than in this embodiment. In this case, by increasing the flow rate of the second oil E1 introduced by the droplet spacing adjustment flow path 62, the degree of deceleration of the droplets D1 in the chamber portion 611 can be made to be the same as in this embodiment. On the other hand, when the depth of the chamber portion 611 is shallower than approximately two to three times the depth of the upstream portion 610, the droplets D1 are not decelerated as much as in this embodiment. In this case, by reducing the flow rate of the second oil E1 introduced by the droplet spacing adjustment flow path 62, the degree of deceleration of the droplets D1 in the chamber portion 611 can be made to be the same as in this embodiment.

[0073] Therefore, it is preferable that the cross-sectional area of ​​the chamber 611 corresponds to the cross-sectional area of ​​the upstream section 610 and the flow rate of the second oil E1 that the droplet spacing adjustment channel 62 causes to flow into the upstream section 610. By adjusting the cross-sectional area of ​​the chamber 611 according to the cross-sectional area of ​​the upstream section 610 and the flow rate of the second oil E1, the velocity of the droplets D1 in the chamber 611 can be set to a desired velocity that allows a dielectrophoretic force large enough for separation to act on the droplets D1. Note that the cross-sectional area of ​​the chamber 611 does not have to correspond to the cross-sectional area of ​​the upstream section 610 and the flow rate of the second oil E1 that the droplet spacing adjustment channel 62 causes to flow into the upstream section 610.

[0074] Furthermore, the shape of the chamber 611 may be a shape other than the trapezoid shown in Fig. 8. The shape of the chamber 611 may be a shape that is line-symmetric with respect to the flow path direction. For example, the shape of the chamber 611 may be a so-called isosceles trapezoid, in which the lengths of the non-parallel sides are equal and the interior angles at both ends of the base are equal.

[0075] Next, the configuration of the branching portion 612 will be described in more detail. The width of the branching portion 612 is approximately the same as the diameter of the droplet D1 or less than the diameter of the droplet D1. The width of the branching portion 612 is the distance between the two branching flow paths branched by the branching portion 612. In this embodiment, the width of the branching portion 612 is 10 micrometers. The branching tip 6121 of the branching portion 612 is the most upstream portion of the branching portion 612. Depending on whether the width of the branching portion 612 is approximately the same as the diameter of the droplet D1 or less than the diameter of the droplet D1, the branching tip 6121 has a shape that is less sharp with respect to the flowing droplet D1.

[0076] The branch tip 6121 is composed of a waste liquid side surface 6122 and a fractionation side surface 6123. The waste liquid side surface 6122 is the side surface of the branch tip 6121 that faces the waste liquid flow path 613. The fractionation side surface 6123 is the side surface of the branch tip 6121 that faces the fractionation flow path 614. The waste liquid side surface 6122 is a gently curved surface. On the other hand, the fractionation side surface 6123 is a flat surface. In the plan view shown in FIG. 8 , the waste liquid side surface 6122 has a gently curved shape, and the fractionation side surface 6123 has a straight shape. Therefore, the branch tip 6121 of the branch section 612 has an asymmetric shape.

[0077] As described above, the waste liquid side surface 6122 has a gently curved shape in the plan view. A gently curved shape is, for example, a shape that is convex toward the waste liquid flow path 613 (convex in the +y direction of the y-axis direction) as shown in FIG. 8 . The shape of the waste liquid side surface 6122 is not limited to the shape shown in FIG. 8 as long as it satisfies the condition that, when the electric field generating electrode 63 is not generating an electric field, droplets D1 flow only through the waste liquid flow path 613 among the waste liquid flow path 613 and the separation flow path 614 without being broken up. This condition is achieved by ensuring that the degree of sharpness of the branch tip 6121 is equal to or less than a predetermined value. For example, the shape of the waste liquid side surface 6122 may be a curved surface other than the curved surface (curved in the plan view) shown in FIG. 8 , a flat surface (straight in the plan view), or a shape consisting of multiple flat surfaces (broken lines in the plan view). When the shape of the waste liquid side surface 6122 is a shape consisting of multiple flat surfaces, it is preferable that the corners where the multiple flat surfaces connect to each other be rounded.

[0078] Therefore, in the shape of the most upstream part of the branch section 612 (branch tip 6121), the part (waste side side 6122) on the side of one of the two branch flow paths (waste side flow path 613 and separation side flow path 614) through which droplet D1 flows when the electric field generating electrode 63 is not generating an electric field, is curved.

[0079] The branch tip 6121 of the branch section 612 is shaped so that the resistance ratio between the waste flow path 613 and the separation flow path 614 is approximately 1:1.1 to 1:1.2. The branch tip 6121 of the branch section 612 may be shaped so that the resistance ratio between the waste flow path 613 and the separation flow path 614 is a ratio other than approximately 1:1.1 to 1:1.2.

[0080] Due to the configuration of the branching section 612 described above, when the electric field generating electrode 63 is not generating an electric field, the droplet D1 can flow only into the waste side flow path 613 of the two branching flow paths (in this embodiment, the waste side flow path 613 and the separation side flow path 614) without being broken up or merging with other droplets D1 flowing adjacent to the droplet D1 in the flow path direction.

[0081] Next, the configuration of the electric field generating electrode 63 and the multiple reference electrodes 64 will be described in more detail. As described above, the electrode tip 631, which is the tip portion of the electric field generating electrode 63, faces the first side surface 6113 of the chamber section 611. The surface of the electrode tip 631 is approximately parallel to the first side surface 6113. In the plan view shown in FIG. 8 , the surface of the electrode tip 631 and the first side surface 6113 are oblique to each other. Furthermore, the surface area of ​​the electrode tip 631 is approximately the same as the area of ​​the first side surface 6113.

[0082] Some of the multiple reference electrodes 64 (first reference electrode 641 and second reference electrode 642) are provided adjacent to the electric field generating electrode 63 on the side of the microchannel 61 where the electric field generating electrode 63 is provided. Meanwhile, the remaining some of the multiple reference electrodes 64 (third reference electrode 643 and fourth reference electrode 644) are provided on the side of the microchannel 61 where the electric field generating electrode 63 is not provided. The first reference electrode 641 is provided to face the third reference electrode 643 across the microchannel 61. The second reference electrode 642 is provided to face the fourth reference electrode 644 across the microchannel 61.

[0083] The above-described configuration makes it possible to generate a locally high electric field gradient throughout the chamber 611, to the extent that the droplets D1 can be separated by dielectrophoretic force. As described above, in this embodiment, the length of the chamber 611 is such that the number of droplets D1 flowing simultaneously through the chamber 611 is one on average over time. Therefore, being able to generate a locally high electric field gradient throughout the chamber 611 means that a locally high electric field gradient can be generated over an area approximately equal to the spacing between the droplets D1.

[0084] 11 and 12 show the results of simulation of the electric field gradient generated by the electric field generating electrode 63 and the plurality of reference electrodes 64. FIG. 11 is a diagram showing a three-dimensional model of the microchannel 61 used in the electric field gradient simulation. FIG. 12 shows the calculation results of the electric field gradient calculated by the electric field gradient simulation. COMSOL (registered trademark) Multiphysics was used for the electric field gradient simulation. The results shown in FIG. 12 show that a high electric field gradient is generated locally in the chamber portion 611.

[0085] The above-described arrangement of the electric field generating electrode 63 and the plurality of reference electrodes 64, and the number of the plurality of reference electrodes 64 are merely examples and are not limited to these. As long as a high electric field gradient can be generated locally in the chamber portion 611, other configurations may be used for the arrangement of the electric field generating electrode 63 and the plurality of reference electrodes 64, and the number of the plurality of reference electrodes 64.

[0086] Next, with reference to Figures 13 to 17, a process for dispensing droplets D1 based on the timing at which the dispensing target A2 and droplets D1 are detected will be described. Figure 13 is a diagram showing an overview of determining conditions for dispensing based on a measurement signal. Figure 14 is a diagram showing an overview of detection of a measurement signal according to this embodiment. Figure 15 is a diagram showing an overview of signal processing related to output of a dispensing signal according to this embodiment.

[0087] The measurement sample detection device 9 detects the fractionation target A2 flowing through the microflow path 61 in the detection unit 212. As described above, the detection unit 212 is located upstream of the droplet generation unit 213 in the sample flow path 21.

[0088] When the measurement sample detection device 9 detects the separation target A2, it generates a signal detection signal SS1 as a measurement signal. The signal detection signal SS1 is a signal that indicates the change in signal intensity of the fluorescence from the measurement sample A1 over time, as shown in FIG. 6. The fluorescence from the separation target A2 is detected by the PMT 120. When the signal detection signal SS1 exceeds a predetermined threshold, the separation signal generation device 11 generates a first trigger signal T1. The first trigger signal T1 is a pulse signal that rises in response to the signal detection signal SS1 exceeding the predetermined threshold. The first trigger signal T1 is generated by the FPGA 121.

[0089] The time when the pulse of the first trigger signal T1 rises is synchronized with the time when the fractionation target A2 is detected, although there is a time delay between the time when the fractionation target A2 is detected and the time when the pulse of the first trigger signal T1 rises due to processing by the FPGA 121.

[0090] The droplet detection device 10 detects the droplet D1 flowing through the microchannel 61 in the droplet detection unit 214. As described above, the droplet detection unit 214 is located downstream of the droplet generation unit 213 in the sample channel 21. Therefore, the droplet D1 is detected after the separation target A2 has been detected.

[0091] When the droplet detection device 10 detects a droplet D1, it generates a droplet detection signal DS1 as a measurement signal. The droplet detection signal DS1 is a signal that indicates the change over time in the signal intensity of the scattered light from the droplet D1. The scattered light from the droplet D1 is detected by the PMT 122. When the droplet detection signal DS1 exceeds a predetermined threshold, the sorting signal generation device 11 generates a second trigger signal T2. The second trigger signal T2 is a pulse signal that rises in response to the droplet detection signal DS1 exceeding the predetermined threshold. The second trigger signal T2 is generated by a function generator 123.

[0092] The time at which the pulse of the second trigger signal T2 rises is synchronized with the time at which the droplet D1 is detected, although there is a time delay between the time at which the droplet D1 is detected and the time at which the pulse of the second trigger signal T2 rises, due to processing by the function generator 123.

[0093] When a determination signal T3, which is the sum of the first trigger signal T1 and the second trigger signal T2, is equal to or greater than a predetermined threshold (hereinafter referred to as the "sorting threshold"), the sorting signal generating device 11 outputs a sorting signal T4 for sorting the sorting target A2. The sorting signal T4 is an external signal that controls the application of a voltage to the electric field generating electrode 63. Here, the first trigger signal T1 output from the FPGA 121 and the second trigger signal T2 output from the function generator 123 are combined by the split connector 124 and output to the function generator 125 as a determination signal T3. The function generator 125 determines whether the determination signal T3 is equal to or greater than the sorting threshold. When the function generator 125 determines that the determination signal T3 is equal to or greater than the sorting threshold, it outputs the sorting signal T4 to the amplifier circuit 126. The sorting signal T4 amplified by the amplifier circuit 126 is output to the electric field generating electrode 63 as an external signal that controls the application of a voltage to the electric field generating electrode 63.

[0094] Here, we will explain the relationship between the pulse width of the first trigger signal T1 and the time interval TD1 during which the droplet D1 passes through the droplet detection unit 214 of the sample flow channel 21. The time interval TD1 is the time interval between droplets D1 adjacent in the flow velocity direction during which the droplets D1 flowing through the microflow channel 61 pass a predetermined position (the position of the droplet detection unit 214).

[0095] In this embodiment, as an example, the pulse width of the first trigger signal T1 is shorter than the time interval TD1. This configuration allows only the droplets D1 containing the dispensing target A2 to be separated from the droplets D1. If the pulse width of the first trigger signal T1 is longer than the time interval TD1, the pulse of the second trigger signal T2, which is generated in response to the detection of the next droplet D1 adjacent to the droplet D1 containing the dispensing target A2 in the flow path direction, may overlap with the pulse of the first trigger signal T1. In this case, even if the dispensing target A2 is not detected in the next droplet D1, the determination signal T3 exceeds the dispensing threshold, causing dispensing to occur.

[0096] On the other hand, if the pulse width of the first trigger signal T1 is too narrow, the pulse of the first trigger signal T1 and the pulse of the second trigger signal T2 will be unlikely to overlap, and even if a droplet D1 containing the target A2 to be separated is detected, it may not be possible to separate the droplet D1.

[0097] In the present embodiment, an example of detecting the collection target A2 and then collecting droplets D1 containing the collection target A2 has been described, but this is not limiting. The droplets D1 may be collected regardless of the detection result of the collection target A2. In this case, the collected droplets D1 include droplets D1 containing the collection target A2 and droplets D1 not containing the collection target A2. After the droplets D1 are discharged from the collection-side outlet 8, the droplets D1 are destroyed, and the collection targets A2 contained in the droplets D1 are recovered. Collecting droplets D1 regardless of the detection result of the collection target A2 is suitable when it is desired to collect as many droplets D1 as possible, even if the collection target A2 cannot be identified.

[0098] When the droplet D1 is collected regardless of the detection result of the collection target A2, the pulse width of the first trigger signal T1 may be longer than the time interval TD1. With this configuration, the pulse of the first trigger signal T1 and the pulse of the second trigger signal T2 tend to overlap in time, making it easier to collect the detected droplet D1.

[0099] In this embodiment, an example in which the second trigger signal T2 is generated based on the droplet detection signal DS1 has been described. That is, an example in which the second trigger signal T2 is generated in response to the detection of scattered light from the droplet D1 has been described, but this is not limiting. The second trigger signal T2 may also be generated based on the cycle in which the droplets D1 are generated. In the droplet generating unit 213, the cycle in which the droplets D1 are generated is determined to some extent depending on the flow rate of the first oil C1 from the oil inlet channel 41 and the flow rate of the sample flow F1 in the sample flow channel 21. Therefore, the cycle in which the droplets D1 are generated may be acquired in advance, and the second trigger signal T2 may be generated based on this cycle. In this case, the configuration of the droplet detection device 10 may be omitted.

[0100] 16 is a diagram showing an example of the functional configuration of the sorting signal generating device 11 according to this embodiment. The sorting signal generating device 11 includes a sorting target detection signal acquiring unit 110, a first trigger signal generating unit 111, a droplet detection signal acquiring unit 112, a second trigger signal generating unit 113, a determining unit 114, and a sorting signal output unit 115.

[0101] The separation target detection signal acquisition unit 110 acquires a signal detection signal SS1 from the measurement sample detection device 9. The first trigger signal generation unit 111 generates a first trigger signal T1 when a separation target A2 surrounded by a droplet and flowing through the microchannel 61 together with a first fluid (in this embodiment, first sheath fluid B1) is detected. The first trigger signal generation unit 111 includes an FPGA 121.

[0102] The droplet detection signal acquisition unit 112 acquires a droplet detection signal DS1 from the droplet detection device 10. The second trigger signal generation unit 113 generates a second trigger signal T2 when a droplet D1 flowing through the microchannel 61 passes a predetermined position in the flow velocity direction of the microchannel 61. The second trigger signal generation unit 113 includes a function generator 123.

[0103] The determination unit 114 determines whether a determination signal T3, which is the sum of the first trigger signal T1 and the second trigger signal T2, is equal to or greater than a predetermined threshold (sorting threshold). The determination unit 114 includes a function generator 125. The sorting signal output unit 115 outputs a sorting signal T4 for sorting the sorting target A2 when the determination signal T3 is equal to or greater than the threshold. The sorting signal output unit 115 includes the function generator 125.

[0104] As described above, when the second trigger signal T2 is generated based on the period at which the droplets D1 are generated, the sorting signal generating device 11 pre-stores the period at which the droplets D1 are generated, and the second trigger signal generating unit 113 generates the second trigger signal T2 based on the pre-stored period.

[0105] 17 is a diagram showing an example of the flow of the sorting signal generation process by the sorting signal generation device 11 according to this embodiment. The sorting signal generation device 11 repeatedly executes the sorting signal generation process while the microfluidic device including the micro-channel chip 1 is operating.

[0106] Step S10: The first trigger signal generation unit 111 determines whether the sorting target detection signal acquisition unit 110 has acquired a signal detection signal SS1 from the measurement sample detection device 9. If the first trigger signal generation unit 111 determines that the signal detection signal SS1 has been acquired (Step S10; YES), it generates a first trigger signal T1 (Step S20). Thereafter, the determination unit 114 executes the process of Step S50. On the other hand, if the first trigger signal generation unit 111 determines that the signal detection signal SS1 has not been acquired (Step S10; NO), the determination unit 114 executes the process of Step S50.

[0107] Step S30: The second trigger signal generation unit 113 determines whether the droplet detection signal acquisition unit 112 has acquired the droplet detection signal DS1 from the droplet detection device 10. If the second trigger signal generation unit 113 determines that the droplet detection signal DS1 has been acquired (Step S30; YES), it generates a second trigger signal T2. Thereafter, the determination unit 114 executes the process of Step S50. On the other hand, if the second trigger signal generation unit 113 determines that the droplet detection signal DS1 has not been acquired (Step S30; NO), the determination unit 114 executes the process of Step S50.

[0108] Step S50: The determination unit 114 generates a determination signal T3, which is the sum of the first trigger signal T1 and the second trigger signal T2. Step S60: The determination unit 114 determines whether the determination signal T3 is equal to or greater than a predetermined threshold (sorting threshold). If the determination unit 114 determines that the determination signal T3 is equal to or greater than the sorting threshold (Step S60; YES), the sorting signal output unit 115 outputs a sorting signal T4. The sorting signal output unit 115 outputs the sorting signal T4 to the electric field generating electrode 63 via the amplifier circuit 126. On the other hand, if the determination unit 114 determines that the determination signal T3 is not equal to or greater than the sorting threshold (Step S60; NO), the sorting signal generating device 11 ends the sorting signal generation process.

[0109] FIG. 18 is a diagram showing an example of an optical system according to this embodiment. The optical system includes an irradiation optical system and a detection optical system. The optical elements included in the irradiation optical system and the optical elements included in the detection optical system may be interchangeable. Note that the optical system shown in FIG. 18 is merely an example, and an optical system other than the optical system shown in FIG. 18 may be used. In the example shown in FIG. 18, the irradiation optical system includes two light sources, a laser light source 127a and a laser light source 127b. The measurement sample A1 is irradiated with laser light of two wavelengths, a 488 nanometer laser light and a 640 nanometer laser light, from the laser light source 127a and the laser light source 127b, respectively.

[0110] The detection optical system includes two PMTs 120, PMT 120a and PMT 120b, as detectors. PMT 120a detects fluorescence from measurement sample A1 irradiated with laser light emitted from laser light source 127a as excitation light. PMT 120b detects fluorescence from measurement sample A1 irradiated with laser light emitted from laser light source 127b as excitation light. The detection optical system also includes a laser light source 128 and a PMT 122. Laser light of 561 nanometers is irradiated onto droplet D1 from laser light source 128. PMT 122 detects scattered light resulting from scattering of the laser light from laser light source 128 by droplet D1.

[0111] 19 shows the actual measured values ​​of the signal detection signal SS1, the determination signal T3, and the sorting signal T4. As described above, the determination signal T3 is the sum of the first trigger signal T1 synchronized with the signal detection signal SS1 and the second trigger signal T2 synchronized with the droplet detection signal DS1. As shown in FIG. 19, the sorting signal generating device 11 according to this embodiment generates the sorting signal T4 in synchronization with both the detection of the sorting target A2 and the detection of the droplet D1.

[0112] FIG. 20 is a diagram showing an example of the results of imaging the state of droplets D1 being generated. The size of the microchannel is 12 micrometers wide and 18 micrometers high. The flow rate of water is 2 microliters per second. The flow rate of oil is 10 microliters per second. The diameter of the generated droplets D1 is 9 micrometers to 10 micrometers. The volume of the generated droplets D1 is 400 femtoliters to 500 femtoliters. The generation rate of droplets D1 is 40,000 droplets per second.

[0113] 21 to 26 are diagrams showing examples of the results of imaging the separation process. FIG. 21 shows the result of imaging the droplet D1 flowing through the microchannel 61 when no voltage is applied to the electric field generating electrode 63. The droplet D1 flows toward the waste flow channel 613 without breaking up. On the other hand, FIG. 22 shows the result of imaging the droplet D1 flowing through the microchannel 61 when a voltage of 1000 volts is applied to the electric field generating electrode 63. The droplet D1 flows toward the separation flow channel 614 without breaking up. It can be seen that the droplet D1 flowing at high speed through the microchannel 61 is separated by dielectrophoretic force.

[0114] The results shown in Figure 23 are the result of dispensing at time intervals of 50 microseconds, which is the time interval corresponding to dispensing every third droplet D1. Figure 24 shows a time series of captured images when dispensing at time intervals of 50 microseconds. The results shown in Figure 25 are the result of dispensing at time intervals of 100 microseconds, which is the time interval corresponding to dispensing every fifth droplet D1. Figure 26 shows a time series of captured images when dispensing at time intervals of 100 microseconds.

[0115] Next, the relationship between fractionation timing and fractionation performance will be described with reference to Figures 27 to 29. Figure 27 shows fractionation results when the time from detection of the signal detection signal SS1 to generation of the first trigger signal T1 (referred to as fractionation delay time) is shifted. Figure 27 shows images of the measurement sample A1 flowing through the microchannel chip 1 when the fractionation delay time is appropriately adjusted, when it is shortened from the appropriately adjusted fractionation delay time, and when it is lengthened from the appropriately adjusted fractionation delay time. When the fractionation delay time is appropriately adjusted, fractionation is successful, whereas when it is lengthened from the appropriately adjusted fractionation delay time and when it is lengthened from the appropriately adjusted fractionation delay time, fractionation fails.

[0116] Figure 28 is a histogram showing, for each measurement sample A1, how many frames before the frame in which the measurement sample A1 was detected was the frame in which the fraction was collected when the fractionation delay time was changed in 5-microsecond increments from 85 microseconds to 110 microseconds. Figure 29 shows the fractionation accuracy as a function of the fractionation delay time. The fractionation accuracy is the percentage of successful fractionation. It can be seen that when the fractionation delay time is approximately 95 microseconds, a fractionation accuracy of 90 percent or more is achieved.

[0117] FIG. 30 shows the results of nanoparticle separation using the droplet detection device 10. The results shown in FIG. 30 were obtained by using two types of nanoparticles as measurement sample A1: beads with a diameter of 110 nanometers and PbS nanoparticles. FIG. 30 also shows the time change in the detected fluorescent signal, the distribution of signal intensity, and the time change in brightness for each of the two types of nanoparticles. FIG. 30 also shows a graph evaluating the time change in the number of droplets generated per second in frequency units, and a graph showing the time change in throughput (the number of nanoparticles separated per second). The graph showing the time change in throughput reveals that approximately 5,000 nanoparticles were separated per second.

[0118] Figure 31 shows the results of evaluating the separation performance. Droplets D1 discharged into a test tube from the separation outlet 8 were collected, and after the collected droplets D1 were broken, nanoparticles (beads with a diameter of 110 nanometers), which were the separation target A2, were extracted. The results shown in Figure 31 are the results of counting the collected droplets D1 and the separation target A2. The respective counting results are for the cases where separation was not performed, where separation was performed and the sample was separated, and where separation was performed and the sample was not separated.

[0119] 32 shows the ratio of the number of counted droplets D1 containing the target A2 to the number of counted droplets D1 for the cases where no fractionation was performed, where fractionation was performed and the droplets were collected, and where fractionation was not performed and the droplets D1 containing the target A2 were collected with an accuracy of 80 percent or more.

[0120] Figures 33 and 34 show detection results when two types of nanoparticles were used as measurement sample A1. The two types of nanoparticles used as measurement sample A1 were yellow beads with a diameter of 110 nanometers and sky blue beads with a diameter of 260 nanometers. Figure 33 shows the results before fractionation. The wavelength of fluorescence emitted from the yellow beads is 640 nanometers. The wavelength of fluorescence emitted from the sky blue beads is 488 nanometers. The yellow beads are the fractionation target A2, and the sky blue beads are the non-fractionation target A3. Before fractionation, measurement sample A1 contains yellow beads and sky blue beads in a 1:1 ratio.

[0121] 34 shows the results after sorting. The results before sorting are based on the results of flowing measurement sample A1 through the flow path of micro-channel chip 1 and detecting fluorescence with detection unit 212. The results after sorting are the results of recovering and destroying droplets D1 sorted by droplet sorting unit 6, and then flowing measurement sample A1 contained in droplets D1 again through the flow path of micro-channel chip 1 and detecting fluorescence with detection unit 212.

[0122] As shown in Figure 33, before sorting, the proportions of yellow beads and sky blue beads detected by fluorescence were 57.1 percent and 42.9 percent, respectively. On the other hand, as shown in Figure 34, after sorting, the proportions of yellow beads and sky blue beads detected by fluorescence were 92.7 percent and 7.3 percent, respectively. Therefore, it can be seen that the proportion of yellow beads, which are sorting target A2, has increased as a result of sorting compared to before sorting.

[0123] Figures 35 and 36 show the results of sorting extracellular vesicles using the droplet sorting unit 6. In the results shown in Figure 35, CD9 and CD147 contained in exosomes from human colon cancer cells (HCT116) were fluorescently stained to emit fluorescence at 640 nanometers and 488 nanometers, respectively. The diameter of HCT116 exosomes is approximately 100 to 200 nanometers. Figure 35 is a scatter plot plotting the signal intensities of fluorescence at two wavelengths emitted by CD9 and CD147 contained in HCT116 exosomes. Figure 35 also shows the results for HCT116, along with the results of a similar measurement of PbS nanoparticles for HCT116 exosomes for comparison.

[0124] Figure 36 shows an image of CD9 contained in HCT116 exosomes being fluorescently stained to emit fluorescence at 640 nanometers, and the exosomes being sorted based on the results of detecting the fluorescence as a signal.

[0125] As described above, the droplet sorting device according to this embodiment (in this embodiment, the droplet sorting unit 6) includes a microchannel 61, a droplet interval adjustment channel 62, an electric field generating electrode 63, and a plurality of reference electrodes 64 (in this embodiment, a first reference electrode 641, a second reference electrode 642, a third reference electrode 643, and a fourth reference electrode 644). The microchannel 61 includes an upstream section 610, a chamber section 611 that is provided downstream of the upstream section 610 and has a larger cross-sectional area than the upstream section 610, a branch section 612 that is provided downstream of the chamber section 611, and two branch channels branched by the branch section 612 (in this embodiment, a waste liquid-side channel 613 and a sorting-side channel 614). The droplets D1 flow through the microchannel 61 together with a first fluid (in this embodiment, a first oil C1). The droplet spacing adjustment channel 62 is connected to the upstream section 610 so as to merge therewith, and a second fluid (in this embodiment, a second oil E1) having the same composition as a first fluid (in this embodiment, a first oil C1) flows into the upstream section 610. The electric field generating electrode 63 is provided adjacent to the chamber section 611 and generates an electric field by applying a voltage controlled by an external signal. Multiple reference electrodes 64 (in this embodiment, a first reference electrode 641, a second reference electrode 642, a third reference electrode 643, and a fourth reference electrode 644) are provided to generate an electric field gradient in the chamber section 611 in response to the electric field generated by the electric field generating electrode 63. The branch section 612 has an asymmetric shape so that the droplets D1 flow into only one of the two branch channels (in this embodiment, a waste liquid-side channel 613 and a fraction-side channel 614) without breaking up when the electric field generating electrode 63 is not generating an electric field.

[0126] With this configuration, the droplet sorting device according to this embodiment (in this embodiment, the droplet sorting unit 6) can sort the droplets D1 in the chamber unit 611 by dielectrophoretic force while decelerating the droplets D1, and the droplets D1 can flow downstream of the chamber unit 611 without breaking up, so it is possible to achieve both high-speed droplet sorting and accurate sorting. With a simple configuration, high-speed droplet sorting means, for example, sorting 10,000 or more droplets per second.

[0127] The droplet sorting device of the present invention can sort 30,000 or more droplets per second because of the fluid conditions, fluid structure, and electrode arrangement that enable sorting without crushing droplets by applying voltage in a short period of time. Note that the droplet sorting device, droplet sorting unit 6, may be provided in a microchannel other than the microchannel chip 1 according to this embodiment.

[0128] The sorting signal generating device 11 according to this embodiment also includes a first trigger signal generating unit 111, a second trigger signal generating unit 113, a determination unit 114, and a sorting signal output unit 115. The first trigger signal generating unit 111 generates a first trigger signal T1 when a sorting target A2, encased in a droplet and flowing through the microchannel 61 together with a first fluid (in this embodiment, first sheath fluid B1), is detected. The second trigger signal generating unit 113 generates a second trigger signal T2 when a droplet D1 flowing through the microchannel 61 passes a predetermined position in the flow velocity direction of the microchannel 61. The determination unit 114 determines whether a determination signal T3, which is the sum of the first trigger signal T1 and the second trigger signal T2, is equal to or greater than a predetermined threshold. The sorting signal output unit 115 outputs a sorting signal T4 for sorting the sorting target A2 when the determination signal T3 is equal to or greater than the threshold.

[0129] With this configuration, the sorting signal generating device 11 according to this embodiment can determine the time of sorting based on the time when the sorting target A2 is detected and the time when the droplet D1 is generated, thereby improving the accuracy of sorting. As described above, in the micro-channel chip 1 according to this embodiment, after the sorting target A2 is detected, the droplet D1 surrounding the sorting target A2 is formed. Here, the time when the desired sorting target A2 is detected from the measurement sample A1 flowing at high speed through the sample channel 21 is not predetermined. Meanwhile, although the cycle at which the droplets D1 are generated is approximately fixed, the droplets D1 are generated at an extremely high frequency of 30,000 or more droplets per second. Therefore, in the past, it was difficult to determine the time to sort the droplet D1 containing the sorting target A2 after the sorting target A2 was detected.

[0130] In addition, each part of each device in the above embodiment (measurement sample detection device 9, droplet detection device 10, sorting signal generation device 11) may be realized by dedicated hardware, or may be realized by memory and a microprocessor.

[0131] In addition, each part of each device may be composed of a memory and a CPU (central processing unit), and the functions of each part of each device may be realized by loading a program into memory and executing it.

[0132] In addition, a program for realizing the functions of each unit of each device may be recorded on a computer-readable recording medium, and the program recorded on the recording medium may be read into a computer system and executed to perform processing by each unit of the control unit. Note that the term "computer system" here includes hardware such as the OS and peripheral devices.

[0133] Furthermore, if a WWW system is used, the term "computer system" also includes the homepage provision environment (or display environment). Furthermore, "computer-readable recording medium" refers to portable media such as flexible disks, optical magnetic disks, ROMs, and CD-ROMs, as well as storage devices such as hard disks built into computer systems. Furthermore, "computer-readable recording medium" also includes devices that dynamically store programs for a short period of time, such as communication lines used when transmitting programs over networks like the Internet or over communication lines like telephone lines, and devices that store programs for a fixed period of time, such as volatile memory within the computer systems that serve as servers or clients in such cases. Furthermore, the above-mentioned programs may be those that implement some of the aforementioned functions, or may be those that can realize the aforementioned functions in combination with programs already stored in the computer system.

[0134] One embodiment of the present invention has been described in detail above with reference to the drawings, but the specific configuration is not limited to that described above, and various design changes and the like are possible within the scope that does not deviate from the gist of the present invention.

[0135] 1...microchannel chip, 6...droplet sorting section, 61...microchannel, 62...droplet spacing adjustment channel, 63...electric field generating electrode, 64...reference electrode, 610...upstream section, 611...chamber section, 612...branch section, 613...waste liquid side channel, 614...separation side channel, C1...first oil, E1...second oil, D1...droplet, A2...separation target, 11...separation signal generating device, 111...first trigger signal generating section, 113...second trigger signal generating section, 114...determination section, 115...separation signal output section, T1...first trigger signal, T2...second trigger signal, T3...determination signal, T4...separation signal

Claims

1. a microflow channel including an upstream section, a chamber section provided downstream of the upstream section and having a larger cross-sectional area than the upstream section, a branch section provided downstream of the chamber section, and two branch flow channels branched by the branch section, and through which droplets flow together with a first fluid; a droplet interval adjustment flow path connected to the upstream portion so as to join the upstream portion and allowing a second fluid having the same composition as the first fluid to flow into the upstream portion; an electric field generating electrode provided adjacent to the chamber portion and configured to generate an electric field by applying a voltage thereto under control of an external signal; a plurality of reference electrodes disposed in the chamber portion to generate an electric field gradient in response to the electric field generated by the electric field generating electrodes; Equipped with The branching portion has an asymmetric shape so that the droplets flow in only one of the two branching channels without being broken up when the electric field generating electrodes are not generating an electric field. Droplet separation device.

2. The distance between the connection part where the droplet interval adjustment flow path is connected to the upstream part and the chamber part is a distance obtained by multiplying the interval between the droplets on the upstream side of the connection part in the upstream part by the ratio of the flow rate on the upstream side of the connection part to the flow rate on the downstream side of the connection part in the upstream part. The droplet sorting device according to claim 1 .

3. The width of the inlet portion, which is the most upstream portion of the chamber, is the same as the diameter of the droplet, and the width of the outlet portion, which is the most downstream portion of the chamber, is the same as the sum of twice the diameter of the droplet and the width of the branch portion. The droplet sorting device according to claim 1 or 2.

4. The cross-sectional area of ​​the chamber portion corresponds to the cross-sectional area of ​​the upstream portion and the flow rate of the second fluid that the droplet interval adjustment channel causes to flow into the upstream portion. The droplet sorting device according to claim 1 .

5. The length of the chamber section corresponds to the flow rate downstream of a connection section where the droplet interval adjustment flow path in the upstream section is connected to the upstream section, and is a length such that the number of droplets flowing through the chamber section at the same time is one on average over time. The droplet sorting device according to claim 1 .

6. The width of the branch is equal to or less than the diameter of the droplet. The droplet sorting device according to claim 1 .

7. In the shape of the most upstream portion of the branching section, the portion on one side through which the droplets flow when the electric field generating electrode does not generate an electric field in one of the two branching channels is a curved surface. The droplet sorting device according to claim 1 .

8. A tip portion of the electric field generating electrode faces a first side surface of the chamber portion, the surface of the tip portion is oriented substantially parallel to the first side surface, and the area of ​​the surface of the tip portion is approximately the same as the area of ​​the first side surface. The droplet sorting device according to claim 1 .

9. Some of the plurality of reference electrodes are provided adjacent to the electric field generating electrode on a side of the microchannel where the electric field generating electrode is provided, and the remaining some of the plurality of reference electrodes are provided on a side of the microchannel where the electric field generating electrode is not provided. The droplet sorting device according to claim 1 .

10. The width of the microchannel is approximately the same as the diameter of the droplet in the portion other than the chamber portion. The droplet sorting device according to claim 1 .

11. a first trigger signal generating unit that generates a first trigger signal when a sample to be sorted is detected, the sample being surrounded by a droplet and flowing through the microchannel together with the first fluid; a second trigger signal generating unit that generates a second trigger signal when the droplet flowing through the microchannel passes a predetermined position in a flow velocity direction of the microchannel; a determination unit that determines whether a determination signal, which is the sum of a magnitude of the first trigger signal and a magnitude of the second trigger signal, is equal to or greater than a predetermined threshold; a fractionation signal output unit that outputs a fractionation signal for fractionating the fractionation target when the determination signal is equal to or greater than the threshold value; A fractionation signal generating device comprising:

12. The fact that the droplet flowing through the microchannel has passed through the position in the flow velocity direction of the microchannel is detected based on scattered light from the droplet flowing through the microchannel. The sorting signal generating device according to claim 11.

13. a first trigger signal generating step of generating a first trigger signal when a sorting target that is surrounded by a droplet and flows through the microchannel together with the first fluid is detected; a second trigger signal generating step of generating a second trigger signal at a timing when the droplet flowing through the microchannel passes a predetermined position in a flow velocity direction of the microchannel; a determination step of determining whether a determination signal, which is the sum of the magnitude of the first trigger signal and the magnitude of the second trigger signal, is equal to or greater than a predetermined threshold; a fractionation signal output step of outputting a fractionation signal for fractionating the fractionation target when the determination signal is equal to or greater than the threshold value; A method for generating a preparative signal comprising:

14. On the computer, a first trigger signal generating step of generating a first trigger signal when a sorting target that is surrounded by a droplet and flows through the microchannel together with the first fluid is detected; a second trigger signal generating step of generating a second trigger signal at a timing when the droplet flowing through the microchannel passes a predetermined position in a flow velocity direction of the microchannel; a determination step of determining whether a determination signal, which is the sum of the magnitude of the first trigger signal and the magnitude of the second trigger signal, is equal to or greater than a predetermined threshold; a fractionation signal output step of outputting a fractionation signal for fractionating the fractionation target when the determination signal is equal to or greater than the threshold value; A program for executing the above.