Droplet collection device and droplet collection method

Abstract

To provide a droplet dispensing apparatus and method that can appropriately and quickly dispense desired droplets. [Solution] A droplet sorting device comprising a microchannel, a first supply unit that supplies a first droplet containing a target substance of 1 or less, a second supply unit that supplies a reaction solution containing a target molecule, a fusion unit that forms a second droplet from the first droplet and the reaction solution, a first measurement unit that measures the intensity of first target light and reference light emitted from the target substance and reference particles in the first droplet, a second measurement unit that measures the intensity of second target light and reference light emitted from the target substance and reference particles in the second droplet, a sorting unit that sorts the second droplet, and a control unit that controls the sorting unit, wherein the control unit aligns the first target light and the second target light using a synchronization pattern that is commonly included in a first pattern, which is the correspondence between the measurement time of the first measurement unit and the intensity of the reference light, and a second pattern, which is the correspondence between the measurement time of the second measurement unit and the intensity of the reference light, as a reference, and based on the obtained ratio and the reference intensity ratio, supplies a control signal to the sorting unit to sort the second droplet.

Description

Technical Field

[0001] The present invention relates to a droplet separation device and a droplet separation method.

Background Art

[0002] Conventionally and currently, biosensors capable of highly sensitively detecting reactions occurring in a living body have been used for visualizing and analyzing phenomena occurring in a living body. Here, in the present specification, the “biosensor” refers to a substance that utilizes a molecular recognition mechanism by a substance of biological origin (biological origin substance) and changes in a form that can be optically detected, for example, when recognizing a molecule.

[0003] As a method for developing a biosensor, for example, a method using a microfluidic device as described in Non-Patent Document 1 is known. In the method described in Non-Patent Document 1, first, DNA is encapsulated in droplets so that each droplet contains one DNA, and then a protein is generated from the DNA. Thereafter, the target substance is injected into the droplets and reacted with the protein. The droplets into which the target substance has been injected are inserted into a microchannel device for droplet separation. In the microchannel device for droplet separation, the reaction state after mixing of the target substance and the protein is detected, and droplets containing a protein that is considered to have highly sensitively recognized the target substance are separated.

[0004] The protein separated together with the droplets becomes a candidate for a biosensor that detects the target substance. Further modification is made to the DNA that generated the protein thus separated and selected. A protein is generated from the obtained DNA, and the obtained protein is further selected by the above-described method as a candidate. In this way, by repeating the production and selection of candidate substances, a high-performance biosensor (protein translated from DNA) can be developed. The method of repeating such modification and selection is known as the “directed evolution method”, and the method of Non-Patent Document 1 can be said to be a method that enhances the directed evolution method using a microchannel device using droplets.

[0005] However, conventional methods, such as those described in Non-Patent Document 1 above, only detect the state of the target substance after it has been injected into a droplet and reacted with the protein, and do not evaluate the state of the protein before the reaction (background signal). Therefore, it has been difficult to accurately evaluate the sensitivity of the biosensor.

[0006] Therefore, methods are being researched to capture the state changes of substances within a droplet before and after a reaction, and to separate the droplet based on the results. For example, Non-Patent Document 2 describes a system that measures the fluorescence of a substance within a droplet flowing through a microchannel, then causes the droplet to flow through a delay channel, and measures it again after it has flowed through the delay channel. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] ACS Synthetic Biology, volume 10, pp. 252-257 (2021) [Non-Patent Document 2] Analytical Chemistry, volume 89, pp. 711-719 (2017) [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] In the method described in Non-Patent Document 2 above, it is necessary to ensure that the droplets being measured in both measurements are identical in order to capture the state change of the same substance. However, conventional methods, such as those described in Non-Patent Document 2 above, have the problem that if the throughput of droplets flowing in the microchannel is increased, it becomes difficult to ensure that the droplets measured before and after the reaction are identical. In other words, there is a trade-off relationship between droplet separation throughput and droplet identification accuracy. Therefore, improvements were needed to promote the development of biosensors using directed evolution methods.

[0009] The present invention has been made in view of these circumstances, and aims to provide a droplet separation device and a droplet separation method that enable the appropriate and rapid separation of desired droplets flowing through a microchannel. [Means for solving the problem]

[0010] To solve the above problems, one aspect of the present invention includes the following aspects.

[0011] [1] A channel member having a microchannel formed thereon; a first supply unit that supplies a first droplet containing a reference particle and one or fewer target substances into the microchannel; a second supply unit that supplies a reaction solution containing a target molecule to react with the target substance into the microchannel; a fusion unit that fuses the first droplet and the reaction solution to form a second droplet; a first measuring unit that irradiates the first droplet with a first measuring light to measure the intensity of the first target light emitted from the target substance and the intensity of the reference light emitted from the reference particle; a second measuring unit that irradiates the second droplet in the microchannel with a second measuring light to measure the intensity of the second target light emitted from the target substance and the intensity of the reference light emitted from the reference particle; a dispensing unit that dispenses the second droplet; and based on the measurement results in the first measuring unit and the second measuring unit... A droplet sorting device comprising: a control unit that controls the operation of the sorting unit, wherein the control unit determines a first pattern which is the correspondence between the measurement time in the first measurement unit and the intensity of the reference light measured in the first measurement unit, and a second pattern which is the correspondence between the measurement time in the second measurement unit and the intensity of the reference light measured in the second measurement unit, recognizes a synchronization pattern which is included in both the first and second patterns and corresponds to the location of the droplet containing the reference particle, determines the ratio of the intensity of the first target light emitted from the first droplet to the intensity of the second target light emitted from the second droplet corresponding to the first droplet, based on the measurement time of the synchronization pattern, and supplies a control signal to the sorting unit to sort the second droplet based on the ratio and a predetermined reference intensity ratio.

[0012] [2] The droplet dispensing apparatus according to [1], wherein the reference particle is a fluorescent particle, and the first measuring unit and the second measuring unit measure the fluorescence emitted by the target substance and the reference particle.

[0013] [3] The droplet dispensing device according to [1] or [2], wherein the microchannel has a meandering section between the position where the fusion section is provided and the position where the second measuring section is provided.

[0014] [4] A first supply step of supplying a first droplet containing a reference particle and one or fewer target substances into a microchannel; a first measurement step of irradiating the first droplet with a first measurement light and measuring the intensity of the first target light emitted from the target substance and the intensity of the reference light emitted from the reference particle; a second supply step of supplying a reaction solution containing a target molecule to be reacted with the target substance into the microchannel; a fusion step of fusing the first droplet and the reaction solution to form a second droplet; a reaction step of flowing the second droplet downstream of the microchannel and reacting the target substance and the target molecule; a second measurement step of irradiating the second droplet with a second measurement light and measuring the intensity of the second target light emitted from the target substance and the intensity of the reference light emitted from the reference particle; and the measurement results in the first and second measurement steps A droplet separation method comprising: a separation step of separating the second droplet containing the target substance that has undergone a desired reaction with the target molecule, wherein the separation step includes determining a first pattern which is the correspondence between the measurement time in the first measurement step and the intensity of the reference light measured in the first measurement step, and a second pattern which is the correspondence between the measurement time in the second measurement step and the intensity of the reference light measured in the second measurement step, recognizing a synchronization pattern which is included in both the first and second patterns and corresponds to the location of the droplet containing the reference particle, relating the first droplet and the second droplet with respect to the measurement time of the synchronization pattern, determining the ratio of the intensity of the first target light to the corresponding intensity of the second target light, and separating the second droplet based on the ratio and a predetermined reference intensity ratio.

[0015] [5] The reference particles are fluorescent particles, and in the first measurement step and the second measurement step, fluorescence emitted by the target substance and the reference particles is measured. The droplet sampling method described in [4].

[0016] [6] In the droplet sampling method according to [4] or [5], 20% or more and 80% or less of the first droplets of the entire first droplets contain the reference particles.

[0017] [7] In the droplet sampling method according to [6], 45% or more and 55% or less of the first droplets of the entire first droplets contain the reference particles.

Advantages of the Invention

[0018] According to the present invention, it is possible to provide a droplet sampling device and a droplet sampling method that enable appropriate and rapid sampling of desired droplets flowing through a microchannel.

Brief Description of the Drawings

[0019] [Figure 1] FIG. 1 is a schematic perspective view of a droplet sampling device 100. [Figure 2] FIG. 2 is a schematic plan view showing the droplet sampling device 100. [Figure 3] FIG. 3 is an explanatory diagram showing an example of a droplet forming device 90. [Figure 4] FIG. 4 is an explanatory diagram of light receiving parts 512 and 522. [Figure 5] FIG. 5 is an explanatory diagram for explaining a synchronous pattern recognition method in a control unit 70. [Figure 6] FIG. 6 is an explanatory diagram for explaining a synchronous pattern recognition method in a control unit 70. [Figure 7] FIG. 7 is a flowchart for explaining the droplet sampling method of the embodiment.

Embodiments for Carrying Out the Invention

[0020] The droplet collection apparatus and droplet collection method according to this embodiment will be described below with reference to Figures 1 to 7. Note that in all the following drawings, the dimensions and proportions of each component have been appropriately altered for clarity.

[0021] 《Droplet separation device》 Figures 1 and 2 are explanatory diagrams showing the droplet collection device 100 of this embodiment. Figure 1 is a schematic perspective view of the droplet collection device 100, and Figure 2 is a schematic plan view of the droplet collection device 100.

[0022] As shown in Figures 1 and 2, the droplet sorting device 100 includes a flow channel member 10, a first supply unit 20, a second supply unit 30, a fusion unit 40, a measuring unit 50, a sorting unit 60, and a control unit 70. The droplet sorting device 100 is used to measure the optical information of droplets flowing through a microchannel 110 formed in the flow channel member 10, and to sort and separate droplets based on the obtained results.

[0023] The operation of the droplet separator 100 is outlined as follows. First, in the droplet separator 100, a droplet (second droplet D2, described later) containing the target substance to be evaluated and the target molecule to react with the target substance is flowed through the microchannel 110 formed in the flow channel member 10, causing the target substance and the target molecule to react in the droplet. Subsequently, the droplet separator 100 identifies the droplet containing the target substance that has undergone the desired reaction by measurement and separates it downstream of the microchannel 110.

[0024] In this process, by controlling the droplet to contain only one type (or one molecule) of a target substance from among multiple target substances, it is possible to select and separate the target substance that will produce the desired reaction from among the multiple target substances. The details will be explained below in order.

[0025] (Target substance, target molecule) For example, when considering a sensor material for a biosensor that sensitively detects a specific compound in a droplet dispensing device 100, the specific compound to be detected by the biosensor is designated as the target molecule, and compounds or cells that can react with the target molecule are designated as the target substance. Proteins can be used as an example of the target substance.

[0026] Random mutations are introduced into DNA containing sequence information for candidate sensor materials (e.g., proteins) used in biosensors, and multiple candidate materials are created from this DNA. These multiple candidate materials are then used as target materials, and sensor materials are investigated by selecting those that react with the target molecule with high sensitivity from this group of target materials.

[0027] Other target substances that can be used include DNA, RNA, and peptides.

[0028] Both the target substance, DNA, and the DNA containing the sequence information of the target substance can be amplified through enzymatic reactions or by introducing them into E. coli bacteria.

[0029] (Flow channel member) The flow channel member 10 comprises a flow channel body 11 having grooves 110x corresponding to the microchannels 110, and a light-transmitting cover material 12 that covers the flow channel body 11. It is desirable that the flow channel body 11 be light-transmitting. The material used to form the flow channel body 11 can be a material known as a material for a member in which microchannels are formed. Such materials can include polymer materials such as polydimethylsiloxane (PDMS), polymethyl methacrylate, and polycarbonate, as well as inorganic materials such as glass and ceramics.

[0030] The grooves 110x in the channel body 11 can be formed using the above-mentioned material by known methods such as soft lithography, microimprinting, laser processing, or 3D printing. The space enclosed by the grooves 110x of the channel body 11 and the cover material 12 is the microchannel 110. The width of the microchannel 110 is, for example, 30 μm to 200 μm, and the depth is 30 μm to 200 μm.

[0031] The microchannel 110 includes a main channel 111, a side channel 112 connected to the main channel 111, and a branch channel 113 that branches off downstream of the microchannel 110.

[0032] (1st supply section) The first supply unit 20 supplies the first droplet D1 and the oil that disperses the first droplet D1 to the microchannel 110.

[0033] The first droplet D1 contains a reference particle Ref and a target substance of a value less than or equal to 1. If the target substance is a protein translated from dsDNA, the first droplet D1 may contain dsDNA instead of the target substance. In this case, the dsDNA is translated inside the first droplet D1 to produce the target substance, which is a protein.

[0034] For the reference particle Ref, for example, a fluorescent particle can be used. The fluorescent particle contains a fluorescent substance and emits fluorescence when irradiated with excitation light. As for the fluorescent particle, any known type can be used as long as it is of a size that does not obstruct the microchannel 110.

[0035] The first droplet D1 may contain a surfactant. As the surfactant, a known surfactant commonly used in the art of the present invention can be used.

[0036] As the oil used to disperse the first droplet D1, known oily solvents used in the technical field of the present invention, such as fluorine oil, hexadecane, and mineral oil, can be used.

[0037] The size of the first droplet D1 can be between 10 μm and 100 μm.

[0038] The first droplet D1 can be formed, for example, by a known droplet forming device that has a different configuration from the droplet dispensing device 100. Figure 3 is an explanatory diagram showing an example of a droplet forming device 90 that forms the first droplet D1.

[0039] The droplet forming apparatus 90 shown in Figure 3 causes an aqueous mother liquor M supplied from a storage unit (not shown) to flow into a pipe 91, and supplies oil O to the pipe 91 from side pipes 92 connected to both sides of the pipe 91. In the droplet forming apparatus 90, the supplied oil O divides the mother liquor M, and a first droplet D1 is formed.

[0040] The mother liquor M may be stored in a single storage section with the target substance and reference particles mixed together, or it may be stored separately in multiple storage sections and mixed and adjusted in the piping 91 upstream of the connection point of the side pipe 92.

[0041] The mother liquor contains more reference particles (Ref) than the target substance.

[0042] The first droplet D1 formed by the droplet formation apparatus 90 either does not contain the target substance or contains only one type (one molecule) of the target substance. Such first droplets D1 can be formed by using a mother liquor with a concentration such that, probabilistically, no more than one target substance is contained in a single droplet, based on the relationship between the concentration of the target substance in the mother liquor and the size of the first droplet D1 formed. It has been confirmed that the concentration of the target substance in the mother liquor roughly follows the concentration expected in the Poisson process (Non-patent literature: Nature Protocols, volume 8, pp.870-891 (2013)).

[0043] Furthermore, although the first droplet D1 formed by the droplet forming apparatus 90 contains a reference particle Ref, there may also be first droplets D1 that do not contain a reference particle Ref.

[0044] In other words, the first droplet D1 consists of four types: droplet D11 containing the target substance and the reference particle Ref; droplet D12 containing the target substance but not the reference particle Ref; droplet D13 containing the reference particle Ref but not the target substance; and droplet D14 containing neither the target substance nor the reference particle Ref.

[0045] The first supply unit 20 may also be configured to include the droplet forming device 90 described above and to supply the first droplet D1 to the microchannel 110 while forming the first droplet D1.

[0046] The first supply unit 20 is connected to the main channel 111 of the microchannel 110 and supplies the stored first droplet D1 to the microchannel 110. The first droplet D1 is arranged in a line in the direction in which the microchannel 110 extends and flows downstream.

[0047] (2nd supply section) The second supply unit 30 is connected to the side channel 112 of the microchannel 110 and supplies droplets of reaction solution (reaction droplets RD) containing target molecules to be reacted with the target substance to the microchannel 110. The second supply unit 30 may supply pre-prepared reaction droplets RD, or it may have a mechanism for generating reaction droplets RD. The mechanism for generating reaction droplets RD can be configured in the same way as the droplet forming device 90 described above. If the mother liquor M supplied in the droplet forming device 90 contains target molecules, the configuration of the mechanism for generating reaction droplets RD can be understood.

[0048] Furthermore, the second supply unit 30 may supply the reaction solution containing the target molecule in a liquid state without forming reaction droplets RD.

[0049] (fusion part) The fusion section 40 is located in the microchannel 110 at the point where the main channel 111 and the side channel 112 connect. The fusion section 40 applies a voltage to the first droplet D1 and the reaction droplet RD flowing within the microchannel 110.

[0050] When reaction droplets RD are supplied from the second supply unit 30, the fusion unit 40 applies a voltage to change the state of the surfactant on the surface of the first droplet D1 and the surface of the reaction droplet RD, fusing the first droplet D1 and the reaction droplet RD to form the second droplet D2.

[0051] When a reaction solution containing the target molecule is supplied in liquid form from the second supply unit 30, the fusion unit 40 applies a voltage to change the state of the surfactant on the surface of the first droplet D1, thereby introducing the reaction solution into the first droplet D1 and forming the second droplet D2.

[0052] The second droplet D2 that is formed, like the first droplet D1, will be of one of four types: droplet D21 containing the target substance and the reference particle Ref, droplet D22 containing the target substance but not the reference particle Ref, droplet D23 containing the reference particle Ref but not the target substance, and droplet D24 containing neither the target substance nor the reference particle Ref.

[0053] The second droplet D2 formed in the fusion section 40 flows through the microchannel 110. Similar to the first droplet D1, the second droplet D2 is arranged in a line in the direction in which the microchannel 110 extends and flows downstream. In other words, the arrangement of the second droplet D2 maintains the arrangement of the first droplet D1.

[0054] Furthermore, laminar flow is formed inside the microchannel 110. Therefore, the first droplet D1 and the second droplet D2 flow downstream of the microchannel 110 without diffusion.

[0055] The microchannel 110 has a meandering section 110L formed by a plurality of straight sections 110a and 110c and a plurality of curved sections 110b on the downstream side of the fusion section 40. The microchannel 110 in Figure 2 has channels A and B, in which five straight sections 110a are arranged in parallel and adjacent straight sections 110a are connected by curved sections 110b. Furthermore, channels A and B are connected by straight sections 110c that are longer than the straight sections 110a, forming a meandering section 110L.

[0056] In such a meandering section 110L, the flow of the second droplet D2 takes time. Therefore, the reaction between the target substance and the target molecule in the second droplet D2 can be accelerated. In addition, in a flow channel member 10 having a meandering section 110L, the microchannel 110 is folded in a meandering manner, so the planar area of ​​the flow channel member 10 can be reduced compared to a case where the microchannel 110 is not meandering. Note that, for example, if the time required for the reaction between the target substance and the target molecule is short, the droplet dispensing device 100 may be configured without a meandering section 110L.

[0057] The meandering section 110L may be provided with a configuration for adjusting the temperature of the second droplet D2. By heating the meandering section 110L using such a configuration, the reaction between the target substance and the target molecule in the second droplet D2 can be promoted.

[0058] Furthermore, the meandering section 110L may be provided with a configuration for adding liquid to the second droplet D2. By adding liquid reagents or reaction substrates to the second droplet D2 using such a configuration, multiple chemical reactions can be carried out within the second droplet D2. In addition, the pH of the liquid in the second droplet D2 can be changed.

[0059] Furthermore, the meandering section 110L may be provided with a laser light source for irradiating the second droplet D2 with laser light. By irradiating the second droplet D2 with laser light using such a configuration, for example, the reaction between the target substance and the target molecule in the second droplet D2 can be changed in a light-stimulation-dependent manner. The difference in the generated substances between the case where light stimulation is applied and the case where light stimulation is not applied can be used for sorting performed downstream.

[0060] (Measurement part) The measuring unit 50 has a first measuring unit 51 and a second measuring unit 52.

[0061] The first measuring unit 51 includes a light source 511 and a light receiving unit 512. The first measuring unit 51 irradiates a first measuring light ML1 from the light source 511 to a measuring point MP1 that overlaps with the microchannel 110, which is set upstream of the connection point between the main channel 111 and the side channel 112. A laser light source can be used as the light source 511.

[0062] When the first droplet D1 passes through the measurement point MP1, the first measurement light ML1 irradiates the target substance and reference particle Ref inside the first droplet D1. The target substance and reference particle Ref irradiated with the first measurement light ML1 emit a first signal light SL1. The first signal light SL1 includes a first target light TL1 emitted from the target substance and a reference light RL emitted from the reference particle, which has a different wavelength from the first target light TL1. The light receiving unit 512 receives the first signal light SL1 and measures its intensity.

[0063] Furthermore, if the reference particle Ref is a fluorescent particle, the reference light RL emitted from the reference particle Ref is fluorescent, and if the reference particle Ref is a light-scattering particle, the reference light RL is scattered light emitted from the reference particle Ref.

[0064] The second measuring unit 52 includes a light source 521 and a light receiving unit 522. The second measuring unit 52 irradiates a second measuring light ML2 from the light source 521 to a measurement point MP2 that overlaps with the microchannel 110, which is set downstream of the meandering section 110L. A laser light source can be used as the light source 521.

[0065] When the second droplet D2 passes through the measurement point MP2, the second measurement light ML2 irradiates the target substance and reference particle Ref inside the second droplet D2. The target substance and reference particle Ref irradiated with the second measurement light ML2 emit a second signal light SL2. The second signal light SL2 includes a second target light TL2 emitted from the target substance and a reference light RL emitted from the reference particle, which has a different wavelength from the second target light TL2. The light receiving unit 522 receives the second signal light SL2 and measures its intensity.

[0066] Figure 4 is an explanatory diagram of the light-receiving units 512 and 522. The light-receiving unit 512 includes an optical filter 512x that separates the first target light TL1 and the reference light RL, a light-receiving unit 512a that receives the separated first target light TL1, and a light-receiving unit 512b that receives the reference light RL.

[0067] The light-receiving unit 522 includes an optical filter 522x that separates the second target light TL2 and the reference light RL, a light-receiving unit 522a that receives the separated second target light TL2, and a light-receiving unit 522b that receives the reference light RL.

[0068] The light-receiving units 512 and 522 may have known optical systems such as a focusing optical system or a relay optical system. Furthermore, the light-receiving units 512 and 522 may have known optical systems that remove unwanted light not used for measurement, such as stray light originating from the measurement environment.

[0069] In Figure 1, the flow channel members 10 are positioned between the light source 511 and the light receiving unit 512, and between the light source 521 and the light receiving unit 522, but this is not limited to this arrangement. The light receiving units 512 and 522 should be positioned to appropriately receive the first signal light SL1 and the second signal light SL2.

[0070] The first measuring unit 51 and the second measuring unit 52 can use devices having known optical systems and detection systems, depending on the first measuring light ML1 and the second measuring light ML2 to be measured.

[0071] Furthermore, the measuring unit 50 may include optical systems not shown, such as a focusing optical system including a lens or concave mirror, a relay optical system, an optical filter, or a polarizing plate, depending on the intended measurement.

[0072] (Preparative separation section) The sorting unit 60 is located downstream of the measurement point MP2. The sorting unit 60 electrically attracts a desired second droplet D2 from among the second droplets D2 flowing in the microchannel 110 to the wall where the sorting unit 60 is located. In this way, the sorting unit 60 changes the flow direction of the second droplet D2 and supplies the second droplet D2 to one of the downstream branch channels 113 (branch channels 113a, 113b, 113c). Storage units 61, 62, and 63 are connected to the end of each branch channel 113, respectively, and the second droplet D2 is sorted via the branch channels 113.

[0073] The diagram shows the branch road 113 branching in three directions, but it could also branch in two directions.

[0074] Furthermore, the dispensing unit 60 may be configured to instantaneously supply oil from the side of the microchannel and change the flow direction of the second droplet D2 by the flow of the oil that is formed.

[0075] Furthermore, the separation unit 60 may be configured to generate surface acoustic waves on the wall surface of the microchannel, transmit longitudinal waves to the second droplet D2, and change the flow direction of the second droplet D2.

[0076] In the sorting unit 60, based on the measurement results from the first measuring unit 51 and the second measuring unit 52, the second droplet D2 containing the target substance that has undergone the desired reaction is selected and sorted. At this time, the sorting unit 60 may attract the second droplet D2 containing the target substance that has undergone the desired reaction and guide it to the storage unit 61, or it may attract the second droplet D2 that does not contain the target substance that has undergone the desired reaction and guide it to the storage unit 61.

[0077] (Control Unit) The control unit 70 controls the operation of the sorting unit 60 based on the measurement results from the first measuring unit 51 and the second measuring unit 52.

[0078] In droplets flowing through a microchannel, the target substance and target molecule are encapsulated, and the reactivity between the target substance and the target molecule is evaluated. To confirm that the target substance in the droplet has undergone the desired reaction with the target molecule, it is necessary to evaluate the changes in the same droplet between "the target substance before reaction with the target molecule" and "the target substance after reaction with the target molecule." However, it has been difficult to appropriately identify the same droplet while multiple droplets are flowing through a microchannel and to evaluate the reaction state with the target molecule.

[0079] Furthermore, as described above, when droplets are prepared from a mother liquor with a dilute concentration of the target substance and a reaction with the target molecule is induced in a microchannel, only a portion of the multiple droplets flowing in the microchannel actually contain the target substance, while the remaining droplets become "empty droplets" that do not contain the target substance. Therefore, it was extremely difficult to appropriately identify the droplets and evaluate the reaction state with the target molecule while multiple droplets were flowing in the microchannel.

[0080] In contrast, in the droplet dispensing apparatus of this embodiment, the first droplet D1 is formed from a mother liquor containing more reference particles Ref than the target substance, and used in the reaction. This enables the following control.

[0081] Figures 5 and 6 are explanatory diagrams illustrating the synchronization pattern recognition method in the control unit 70. First, the reference particle Ref contained in the first droplet D1 and the second droplet D2 is detected in the measurement unit 50 regardless of the presence or absence of the target substance, and regardless of the reaction state between the target substance and the target molecule. Therefore, the first droplet D1 (droplets D11, 13) and the second droplet D2 (droplets D21, 23) containing the reference particle Ref will have a signal originating from the reference particle Ref detected, while the first droplet D1 (droplets D12, 14) and the second droplet D2 (droplets D22, 24) not containing the reference particle Ref will not have a signal originating from the reference particle Ref detected.

[0082] In other words, as shown in Figure 5, the presence or absence of a reference particle Ref corresponds to the presence or absence of a signal in the light-receiving units 512b and 522b that receive the reference light RL. Therefore, if the droplet contains a reference particle Ref and the peak of the reference light RL is detected, it is represented as "1", and if the droplet does not contain a reference particle Ref and the peak of the reference light RL is not detected, it is represented as "0", and the signal corresponding to the reference particle Ref can be considered as a digital signal of 0 or 1.

[0083] In that case, for droplets flowing within the microchannel 110, the digital signals emitted from two adjacent droplets can be understood to be in four patterns: "0,0", "0,1", "1,0", and "1,1". Similarly, the reference light RL signals emitted from n consecutive droplets correspond to the positions of the droplets containing the reference particle Ref. n This results in a pattern of a certain type. Hereinafter, "a pattern formed from the signals of reference light RL emitted from multiple consecutive droplets" will be referred to as a synchronization pattern.

[0084] In addition to the presence or absence of a reference particle Ref (signal of reference light RL), information on the number of reference particles Ref (intensity of reference light RL) may also be used as information for the synchronization pattern.

[0085] In the measurement unit 50, the correspondence between the measurement time and the intensity of the resulting signal light is obtained as a measurement result. The obtained measurement result also includes the signal originating from the reference particle Ref (the signal of the reference light RL). At this time, it can be understood that as the number n of droplets defining the synchronization pattern increases, the probability of the same synchronization pattern appearing in the obtained measurement result decreases.

[0086] Furthermore, there is no difference in the signal related to the reference particle Ref between the measurement results from the first measurement unit 51 and the measurement results from the second measurement unit 52.

[0087] Therefore, the measurement results in the first measurement unit 51 (the first pattern, which is the correspondence between measurement time and the intensity of the reference light RL) and the measurement results in the second measurement unit 52 (the second pattern, which is the correspondence between measurement time and the intensity of the reference light RL) contain a common synchronization pattern. By using the synchronization pattern as a reference, the first droplet D1 measured in the first measurement unit 51 can be associated with the second droplet D2 corresponding to the first droplet D1. This makes it possible to identify the first target light TL1 emitted from the first droplet D1 and the second target light TL2 emitted from the second droplet D2 corresponding to the first droplet, based on the measurement time of the synchronization pattern.

[0088] As shown in Figure 6, the control unit 70 first determines the first pattern P1, which is the correspondence between the measurement time in the first measurement unit 51 and the intensity of the reference light RL, and the second pattern P2, which is the correspondence between the measurement time in the second measurement unit 52 and the intensity of the reference light RL. It also determines the correspondence between the measurement time in the first measurement unit 51 and the intensity of the first target light TL1, and the correspondence between the measurement time in the second measurement unit 52 and the intensity of the second target light TL2.

[0089] Next, we recognize the synchronization pattern SP that is common to both the first pattern P1 and the second pattern P2.

[0090] Next, the first droplet D1 measured in the first measurement unit 51 and the second droplet D2 measured in the second measurement unit 52 are correlated using the measurement time of the synchronization pattern SP as a reference. This makes it possible to identify the second droplet D2 corresponding to a specific first droplet D1 and to compare the intensities of the first target light TL1 and the second target light TL2 emitted from the target substance contained in the same droplet.

[0091] Next, the intensity ratio between the intensity of the first target light TL1 emitted from the first droplet D1 and the intensity of the second target light TL2 emitted from the second droplet D2 corresponding to the first droplet D1 is determined.

[0092] When the intensity of the target light measured by the reaction between the target substance and the target molecule increases, it is considered that the intensity of the second target light TL2 detected by the second measurement unit 52 increases as the reaction between the target substance and the target molecule proceeds favorably. Furthermore, if the intensity of the target light measured decreases due to the reaction between the target substance and the target molecule, it is considered that the intensity of the second target light TL2 detected by the second measurement unit 52 decreases as the reaction between the target substance and the target molecule proceeds favorably. Therefore, by pre-setting the signal intensity ratio to be separated (reference intensity ratio), the control unit 70 can determine that it should separate the second droplet D2 based on the obtained ratio and the predetermined reference intensity ratio.

[0093] Here, the distance between the measurement point MP2 and the dispensing unit 60 is known. Also, although the fluid velocity in the microchannel 110 is not perfectly constant (uniform motion), the change in fluid velocity is small enough to be negligible when determining the time it takes for the second droplet D2 to reach the dispensing unit 60 from the measurement point MP2 (arrival time). Therefore, the control unit 70 can pre-determine and store the time it takes for the second droplet D2 to reach the dispensing unit 60 after the measurement at the measurement point MP2 (arrival time).

[0094] Based on the above, the control unit 70 generates a control signal (timing signal) to drive the sorting unit 60 after the above arrival time has elapsed from the measurement at measurement point MP2, based on the measurement results from the first measurement unit 51 and the second measurement unit 52, and supplies it to the sorting unit 60. As a result, the sorting unit 60 can sort the second droplet D2. Furthermore, by using the above-mentioned synchronization pattern SP, it becomes possible to increase the processing speed of the sorting while maintaining high accuracy in identifying the same droplet D1 measured at measurement point MP1 at measurement point MP2.

[0095] 《Droplet separation method》 Figure 7 is a flowchart illustrating the droplet separation method of this embodiment. The droplet separation method may be carried out using the droplet separation device 100 described above, but is not limited to this, and can also be carried out using a droplet separation device with other device configurations. In the following description, the droplet separation device 100 described above will be used, and the reference numerals shown in Figures 1 to 6 will be used as appropriate.

[0096] In the droplet separation method of this embodiment, first, a first droplet D1 formed from a mother liquor containing the target substance or a substance that produces the target substance (DNA) and a larger number of reference particles Ref than these substances is supplied to the microchannel 110 (first supply step; step S1).

[0097] As mentioned above, fluorescent particles can be used as the reference particle Ref.

[0098] The first droplet D1 contains one or fewer units of the target substance. Preferably, 20% or less of the first droplets D1 contain the target substance, and more preferably 10% or less of the first droplets D1 contain the target substance. The proportion of first droplets D1 containing the target substance can be controlled by adjusting the concentration of the target substance in the mother liquor. When forming the first droplets D1 from a mother liquor diluted so that 20% or less of the first droplets D1 contain the target substance, the probability of each first droplet D1 containing two or more units (two molecules) of the target substance is extremely low. Therefore, each formed first droplet D1 will substantially contain one (one molecule) or less of the target substance.

[0099] Furthermore, in the plurality of first droplets D1 formed, it is preferable that 20% to 80% (number) of the first droplets D1 contain reference particles, and it is more preferable that 45% to 55% (number) of the first droplets D1 contain reference particles.

[0100] As described above, in the present invention, the presence or absence of a reference particle in the first droplet D1 and the second droplet D2 is used as a digital signal. Therefore, if there is an excess of the first droplet D1 and the second droplet D2 containing the reference particle Ref (the signal for the reference particle Ref is 1,1,1…) or an deficiency of the first droplet D1 and the second droplet D2 containing the reference particle Ref (the signal for the reference particle Ref is 0,0,0…), multiple identical synchronization patterns SP will appear in the first pattern P1.

[0101] In this case, the synchronization pattern SP in the second pattern P2 that should be used as a reference for measurement time becomes unknown to the synchronization pattern SP included in the first pattern P1. As a result, it becomes easy to mistakenly identify the synchronization pattern in the second pattern P2 that corresponds to the synchronization pattern SP in the first pattern P1, leading to misidentification.

[0102] Theoretical calculations and experimental results confirmed that the above-mentioned misidentification can be significantly reduced if 20% to 80% of the total number of first droplets D1 contain the reference particle Ref. Furthermore, a proportion of 45% to 55% containing the reference particle Ref is more preferable, and theoretically, 50% is the most preferable.

[0103] Next, the first droplet D1 is irradiated with the first measurement light ML1, and the intensity of the first signal light SL1 (first target light TL1, reference light RL) emitted from the target substance and the reference particle is measured (first measurement step; step S2). When a fluorescent particle is used as the reference particle Ref, the first measurement light ML1 is excitation light that excites the reference particle Ref (fluorescent particle) and the target substance, and the first signal light SL1 is fluorescence emitted from the target substance and the reference particle Ref.

[0104] Furthermore, reaction droplets containing the target molecule are supplied to the microchannel 110 (second supply step; step S3). There are no restrictions on the order in which the first and second supply steps are performed, and they may be performed simultaneously.

[0105] Next, the first droplet D1 and the reaction droplet RD are fused to form the second droplet D2 (fusion step, step S4). By synchronizing the supply of the first droplet D1 in the first supply step and the supply of the reaction droplet RD in the second supply step, the first droplet D1 and the reaction droplet RD fuse in a one-to-one ratio, forming the second droplet D2 containing an appropriate amount of target molecules.

[0106] Next, the second droplet D2 is flowed downstream of the microchannel 110 to react with the target substance and the target molecule (reaction step; step S5). The time the second droplet D2 flows through the microchannel 110 corresponds to the reaction time between the target substance and the target molecule. The reaction time can be controlled by selecting a microchannel 110 of an appropriate length and adjusting the fluid flow rate within the microchannel 110 as needed.

[0107] Next, the second droplet D2 is irradiated with the second measurement light ML2, and the intensity of the second signal light SL2 (second target light TL2, reference light RL) emitted from the target substance and the reference particle is measured (second measurement step; step S6). When a fluorescent particle is used as the reference particle Ref, the second measurement light ML2 is excitation light that excites the reference particle Ref (fluorescent particle) and the target substance, and the second signal light SL2 is fluorescence emitted from the target substance and the reference particle Ref.

[0108] Next, based on the measurement results from the first and second measurement steps, a second droplet D2 containing the target substance that has reacted with the target molecule as desired is separated (separation step; step S7).

[0109] In the preparative step, a first pattern P1 is determined, which is the correspondence between the measurement time in the first measurement step and the intensity of the reference light RL measured in the first measurement step, and a second pattern P2 is determined, which is the correspondence between the measurement time in the second measurement step and the intensity of the reference light RL measured in the second measurement step.

[0110] Next, the synchronization pattern SP, which is common to both the first pattern P1 and the second pattern P2, is recognized, and the first droplet D1 and the second droplet D2 are associated with each other based on the measurement time of the synchronization pattern SP, and the ratio of the intensity of the first target light TL1 to the intensity of the corresponding second target light TL2 is determined.

[0111] In this case, the number of droplets constituting the synchronization pattern SP (n in Figure 5) is preferably 6 or more, and more preferably 8 or more. If n is large, the computational burden on the control unit 70 increases, so for example, 14 or less is preferable, and 12 or less is more preferable.

[0112] Next, the second droplet D2 is dispensed based on the calculated intensity ratio and a predetermined standard intensity ratio. In this way, the desired second droplet D2 can be dispensed appropriately and quickly.

[0113] According to the droplet separation device and droplet separation method described above, it is possible to appropriately and quickly separate desired droplets flowing through a microchannel.

[0114] Preferred embodiments of the present invention have been described above with reference to the attached drawings, but the present invention is not limited to these examples. The shapes and combinations of the constituent members shown in the above examples are merely examples, and can be modified in various ways based on design, specifications, etc., without departing from the spirit of the present invention. [Explanation of Symbols]

[0115] 10...Flow channel member, 20...First supply unit, 30...Second supply unit, 40...Fusion unit, 50...Measurement unit, 51...First measurement unit, 52...Second measurement unit, 60...Dispensing unit, 70...Control unit, 100...Droplet dispensing device, 110...Microchannel, 110L...Meandering unit, D1, D11, D12, D13, D14...First droplet, D2, D21, D22, D23, D24...Second droplet, ML1...First measurement light, ML2...Second measurement light, P1...First pattern, P2...Second pattern, RD...Reaction droplet, Ref...Reference particle, SL1...First signal light, SL2...Second signal light, SP...Synchronization pattern

Claims

1. A channel member in which microchannels are formed, A first supply unit that supplies a first droplet containing a reference particle and a target substance of one or less to the microchannel, A second supply unit supplies a reaction solution containing a target molecule to be reacted with the aforementioned target substance to the microchannel, A fusion unit that fuses the first droplet and the reaction solution to form a second droplet, A first measuring unit irradiates the first droplet with a first measuring light to measure the intensity of the first target light emitted from the target substance and the intensity of the reference light emitted from the reference particle. A second measuring unit irradiates the second droplet in the microchannel with a second measuring light to measure the intensity of the second target light emitted from the target substance and the intensity of the reference light emitted from the reference particle. A dispensing unit for dispensing the second droplet, The system includes a control unit that controls the operation of the sorting unit based on the measurement results from the first and second measurement units, The control unit determines a first pattern which is the correspondence between the measurement time in the first measurement unit and the intensity of the reference light measured in the first measurement unit, and a second pattern which is the correspondence between the measurement time in the second measurement unit and the intensity of the reference light measured in the second measurement unit. The first and second patterns share a common synchronization pattern that corresponds to the location of the droplet containing the reference particle, Based on the measurement time of the synchronization pattern, the ratio of the intensity of the first target light emitted from the first droplet to the intensity of the second target light emitted from the second droplet corresponding to the first droplet is determined. A droplet dispensing device that supplies a control signal to the dispensing unit for dispensing the second droplet based on the aforementioned ratio and a predetermined standard intensity ratio.

2. The aforementioned reference particle is a fluorescent particle, The droplet dispensing apparatus according to claim 1, wherein the first measuring unit and the second measuring unit measure the fluorescence emitted by the target substance and the reference particles.

3. The droplet dispensing device according to claim 1 or 2, wherein the microchannel has a meandering section between the position where the fusion section is provided and the position where the second measuring section is provided.

4. A first supply step involves supplying a first droplet containing a reference particle and a target substance of one or less into a microchannel, A first measurement step involves irradiating the first droplet with a first measurement light and measuring the intensity of the first target light emitted from the target substance and the intensity of the reference light emitted from the reference particle. A second supply step involves supplying a reaction solution containing a target molecule to be reacted with the aforementioned target substance into the microchannel. A fusion step in which the first droplet and the reaction solution are fused to form a second droplet, A reaction step in which the second droplet is flowed downstream of the microchannel to react the target substance with the target molecule, A second measurement step involves irradiating the second droplet with a second measurement light and measuring the intensity of the second target light emitted from the target substance and the intensity of the reference light emitted from the reference particle. The process includes a sorting step of sorting the second droplet containing the target substance that has reacted with the target molecule as desired, based on the measurement results in the first and second measurement steps, The sorting step determines a first pattern which is the correspondence between the measurement time in the first measurement step and the intensity of the reference light measured in the first measurement step, and a second pattern which is the correspondence between the measurement time in the second measurement step and the intensity of the reference light measured in the second measurement step. The first and second patterns share a common synchronization pattern that corresponds to the location of the droplet containing the reference particle, Based on the measurement time of the synchronization pattern, the first droplet and the second droplet are associated, and the ratio of the intensity of the first target light to the corresponding intensity of the second target light is determined. A droplet separation method comprising the step of separating the second droplet based on the aforementioned ratio and a predetermined standard intensity ratio.

5. The aforementioned reference particle is a fluorescent particle, The droplet separation method according to claim 4, wherein the fluorescence emitted by the target substance and the reference particle is measured in the first and second measurement steps.

6. The droplet separation method according to claim 4 or 5, wherein 20% to 80% of the entire first droplet contains the reference particle.

7. The droplet separation method according to claim 6, wherein 45% to 55% of the entire first droplet contains the reference particle.

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