Apparatus and method for generating and collecting droplets

Abstract

The invention relates to a device (100) for generating droplets (41), comprising an emulsification junction (50) in which a first input (51) is configured to be supplied with at least one first immiscible fluid (21-25) and a second input (52) is configured to be connected to at least one first flow means (32) arranged to supply a flow of a second immiscible fluid (31) to the second input (52). The output (53) is connected to at least one second flow device (33). The device (100) is configured to operate a droplet generation mode in which the first input (51) is supplied by the second flow means (33) and the second input (52) is simultaneously supplied by the first flow means (32) such that droplet generation occurs and generated droplets (41) are removed from the emulsification junction (50) by the second flow means (33).

Description

Technical Field

[0001] This invention relates to the field of microfluidics. More specifically, it relates to an apparatus for generating droplets, the apparatus comprising an emulsification junction. Such droplets can be advantageously used to generate droplet pools from single or multiple reagents. For example, emulsions of identical droplets or highly diverse droplet libraries can be obtained. Background Technology

[0002] A "droplet library" is a group of microfluidic droplets comprising at least two identical groups of droplets. Each group of identical droplets can encapsulate at least one component having predetermined characteristics. For example, a first group of identical droplets can encapsulate a first drug at a first dose, and a second group of identical droplets can encapsulate a second drug at a second dose.

[0003] Droplet libraries can be advantageously used in a variety of applications, such as high-throughput screening. More specifically, droplet libraries can be used to perform thousands of parallel tests with different drugs and dosages to determine which drugs and dosages are most effective for a patient's disease.

[0004] Droplets can be generated through emulsification, a phenomenon that occurs when two immiscible fluids are mixed and droplets of one immiscible fluid are dispersed in the second immiscible fluid. The resulting droplets, ranging in size from nanoliters to microliters, are then collected in vials, thus creating a droplet library.

[0005] One approach to generating droplets involves using a microarray spotter and configuring it to automatically deliver a predetermined amount of at least one first fluid into a vial containing a second fluid immiscible with the first fluid. However, microarray spotters lack speed because they require frequent cleaning and tip changes, as well as movement in three-dimensional space. Furthermore, it requires the droplets to pass through air, which can be a source of contamination and leads to challenges in terms of aggregation.

[0006] Another approach is to use an emulsification junction, which is a system comprising two microfluidic inlet channels configured to deliver two immiscible fluids to the emulsification zone. The geometry at the intersection of the two immiscible fluids produces reproducible droplet breakup.

[0007] Several emulsion junctions of this type can be placed in parallel to produce different groups of droplets with varying contents.

[0008] However, such systems typically require frequent cleaning to avoid contamination, which is time-consuming. Furthermore, these systems necessitate the use of valves to collect fluid or direct fluid into and out of the emulsion junction. This can lead to droplet deformation and / or contamination, particularly through wetting within the valve's internal volume, potentially affecting droplet transport in the microfluidic system and the integrity of related experiments.

[0009] Therefore, the technical problem solved by the present invention is to design an integrated device that is not prone to contamination between droplets. Summary of the Invention

[0010] To address this problem, the applicant has developed an apparatus for generating droplets, comprising an emulsification junction configured to generate droplets by contacting a flow of at least one first immiscible fluid with a flow of at least one second immiscible fluid. The emulsification junction includes a first input terminal, a second input terminal, an output terminal, and an emulsification zone in which droplet generation occurs, wherein: - The first input terminal is configured to be supplied with the at least one first immiscible fluid. - The second input terminal is configured to be connected to at least one first flow device, the first flow device being configured to supply a flow of the second immiscible fluid to the second input terminal.

[0011] The invention is characterized in that the output terminal is connected to at least one second flow device. The device is configured to operate in a droplet generation mode, wherein the first input terminal is supplied with a flow of the first immiscible fluid by the second flow device, while the second input terminal is supplied with a flow of the second immiscible fluid by the first flow device, such that droplet generation occurs in the emulsification zone, and the generated droplets are removed from the emulsification junction by the second flow device.

[0012] In other words, the second flow device is located downstream of the output end of the emulsification junction. In the droplet generation mode, the second flow device is configured to draw in the flow of the first immiscible fluid through the first input end of the emulsification junction, while the first flow device pushes the second immiscible fluid through the second input end of the emulsification junction. In this way, droplets are generated in the emulsification zone and extracted through the output end by means of the suction of the second flow device. Therefore, in the droplet generation mode, the droplets never need to pass through any valve. In fact, inside the valve assembly, droplets may wet surfaces present in the valve assembly. Once the surface is wetted, it may more easily merge with other droplets passing through the valve, thus forming droplets of different sizes. It is impossible to control this wetting phenomenon within a closed valve. Therefore, removing the valve allows for the production of uncontaminated droplets of the same shape and size throughout the droplet generation process.

[0013] Advantageously, the device also includes: - A shut-off device, disposed upstream of the first input terminal, the shut-off device having an open state and a closed state, the shut-off device being configured to block flow through the first input terminal, and - An outlet, disposed downstream of the output end, having an open state and a closed state, configured to allow extraction of the generated droplets from the device.

[0014] The device is further configured to operate in a droplet collection mode, wherein the shut-off device is in the shut-off state and the outlet is in the open state, and a flow of the second immiscible fluid is generated to extract the generated droplets from the device through the open outlet.

[0015] In other words, the device has two modes: a generation mode, in which droplets are generated in the emulsification junction and extracted from the emulsification junction by a suction flow from downstream of the junction; and a collection mode, in which droplet generation is complete and the generated droplets are collected from the device into a vial, for example, to create a droplet library. Therefore, these two modes cannot be activated simultaneously.

[0016] To collect the droplets, a flow of a second immiscible fluid can be generated. According to one embodiment, the flow of the second immiscible fluid is generated by a second flow device. According to another embodiment, the flow of the second immiscible fluid is generated by a first flow device. The generated flow can push or draw the produced droplets through the device outlet.

[0017] According to an embodiment, the emulsified junction includes a shrinkage portion having a shrinkage output end configured to guide the at least one first immiscible fluid to the emulsified zone.

[0018] According to another embodiment, the generated droplets can be accumulated in a tube connected to the output end of the emulsification junction. Optionally, the device may further include a reservoir comprising a storage region configured to capture the generated droplets, the reservoir being positioned downstream of the output end.

[0019] Advantageously, the accumulator is positioned between the output end and the second flow device, so that the second flow device can propel the generated droplets toward the accumulator area.

[0020] In practice, accumulators are specifically designed to allow the accumulation of generated droplets. Advantageously, the accumulator region is a dead volume in which droplets are guided by natural phenomena such as buoyancy or gravity or by external forces such as flow, magnetism, acoustic forces or dielectric forces.

[0021] According to a preferred embodiment, the accumulator is a gravity-based accumulator configured to capture gravity-driven droplets generated within the accumulator.

[0022] The size of the liquid storage area is determined by the maximum number of droplets that need to be generated.

[0023] Advantageously, the reservoir has a conical shape, including a base configured to allow passage of the generated droplet and a apex positioning the reservoir area; or the reservoir has a pointed dome shape, including a base configured to allow passage of the generated droplet and a apex positioning the reservoir area. In some embodiments, the tip includes the outlet. In this way, the accumulated droplet is close to the outlet and can be rapidly extracted from the device. For example, extraction can be performed via a collection conduit connected to the outlet and configured to guide the generated droplet into a vial. Alternatively, the generated droplet can be directly injected into another device, such as a microfluidic chip or fluid conduit.

[0024] According to another embodiment, the device further includes a third flow device connected to the outlet of the accumulator. The third flow device is configured to generate a flow that is configured to mix the generated droplets contained within the accumulator zone. In practice, the flow generated by the third flow device is configured to push the generated droplets against gravity towards the base of the accumulator, thereby enabling the mixing of droplets with different properties. This mixing occurs in a manner similar to that of a fluidized bed.

[0025] In a preferred embodiment, the second immiscible fluid is an oil phase selected from fluorinated oil, silicone oil, vegetable oil, mineral oil, and hydrocarbon oil, and the first immiscible fluid is an aqueous phase comprising at least one of cell culture medium, drug solution, antibody solution, cell suspension, organoid or cell tissue suspension, hydrogel solution, microsphere or particle suspension. The droplets may be stabilized in the oil phase with a surfactant (such as PEG-di-Krydox fluorinated surfactant, perfluorooctyl alcohol, perfluorododecyl alcohol, perfluoropolyether (PFPE) derivatives including PFPE-PEG triblock copolymer, span 80, Abil EM90, monolein, oleic acid, n-butanol) or in the aqueous phase with a surfactant (phospholipid, Triton-X-100, SDS, Pluronic, Tween 20 / 80).

[0026] Alternatively, the second immiscible fluid can be an aqueous phase, and the first immiscible fluid can be an oil phase.

[0027] In practice, the first input terminal is configured to be supplied with multiple sections of at least one first immiscible fluid, including at least a first section and a second section, the two consecutive sections being separated by a third section of at least one second immiscible fluid.

[0028] According to the invention, the segment is a predetermined amount of a first immiscible fluid. Since the first immiscible fluids are miscible with each other, spacers made of a material immiscible with the first immiscible fluids can be added to maintain subsequent separation. These spacers can be made of at least one second immiscible fluid used for emulsifying the joint. Alternatively, they can be made of a third immiscible fluid to further prevent undesirable coalescence between different segments of the first immiscible fluids. These spacers can be added in liquid (mineral oil, vegetable oil) or gas (bubbles or nitrogen, etc.).

[0029] Such multiple segments can advantageously be obtained using a robotic arm configured to first extract a first segment of a first immiscible fluid (e.g., a chemotherapy or cell therapy fluid) at a predetermined concentration, then extract a segment of a second immiscible fluid, such as oil, then extract a second segment of another first immiscible fluid (e.g., another type of chemotherapy or cell therapy) at a different concentration, and so on. Alternatively, the second segment of the first immiscible fluid can be made from the same first immiscible fluid as the first segment of the first immiscible fluid. The obtained multiple segments can be converted into droplets and collected in vials, thereby creating a droplet library that can be tested in a microfluidic device.

[0030] This apparatus can be used to generate suspensions of identical droplets. Alternatively, the present invention relates to the use of the above-described apparatus for generating a droplet library comprising at least two sets of droplets, wherein a first set of droplets is generated using a first immiscible fluid of a first type, and a second set of droplets is generated using a first immiscible fluid of a second type. Such a droplet library can advantageously be obtained using a single emulsification junction.

[0031] According to another aspect, the present invention relates to a method for generating droplets using an apparatus such as those described above, wherein the method comprises the following steps: - Supplying a flow of at least one second immiscible fluid to the second input terminal, the second input terminal being configured to deliver the second immiscible fluid to the emulsification zone, and - Simultaneously, at least one flow of a first immiscible fluid is supplied to the first input end by drawing the first immiscible fluid downstream of the output end. The first input end is configured to deliver the first immiscible fluid to the emulsification zone, such that droplets are generated in the emulsification zone, and the generated droplets are drawn out from the emulsification junction by drawing from the downstream of the output end.

[0032] Advantageously, the method further includes the step of accumulating the generated droplets in, for example, the accumulator described above.

[0033] Advantageously, the suction downstream of the output is sequential suction, with the first input configured to be supplied with multiple segments of the at least one first immiscible fluid, including at least one first segment and at least one second segment. The sequential suction is configured to insert at least one third segment of the at least one second immiscible fluid between the at least one first segment and the at least one second segment, thereby generating a segment string including the at least one first segment, the at least one second segment, and the at least one third segment. The at least one first segment, the at least one second segment, and the at least one third segment of the segment string can be randomly distributed. The at least one first segment has a first volume, the at least one second segment has a second volume, and the third segment has a third volume. The first volume, the second volume, and the third volume can be different. This allows for droplet “mixing” or random accumulation without requiring any modification to the hardware setup or any post-processing during library collection.

[0034] According to another embodiment, the present invention also relates to a method for collecting droplets from the above-described device, wherein the method includes the following steps: - Use the shut-off device to shut off the first input terminal. - Open the exit, and - A flow of a second immiscible fluid is supplied, such that the generated droplets are extracted from the device through an open outlet.

[0035] Advantageously, the flow of the second immiscible fluid is supplied from downstream of the output end. Attached Figure Description

[0036] Figure 1 This is a schematic front view of the apparatus according to an embodiment of the present invention in droplet generation mode. Figure 2 This is a schematic front view of the apparatus according to an embodiment of the present invention in droplet collection mode. Figure 3 This is a schematic cross-sectional view of the emulsified joint according to an embodiment of the present invention. Figure 4 yes Figure 3 A schematic cross-sectional view of the emulsion joint, magnified to show the contraction section. Figure 5 This is a schematic top view of the emulsified joint according to an embodiment of the present invention. Figure 6 This is a schematic diagram illustrating the steps of a method for generating droplets using the apparatus according to an embodiment of the present invention. Figure 7 This is a schematic diagram illustrating the steps of a method for collecting droplets using the apparatus according to an embodiment of the present invention. Figure 8It comes from Figure 5 A schematic front view of the first step of the method, wherein a first section of a first immiscible fluid is aspirated in an apparatus according to an embodiment of the invention. Figure 9 It comes from Figure 5 A schematic front view of the first step of the method, wherein a first section of the second immiscible fluid is aspirated in an apparatus according to an embodiment of the invention. Figure 10 It comes from Figure 5 A schematic front view of the first step of the method, wherein several sections of first and second immiscible fluids are aspirated in an apparatus according to an embodiment of the invention. Figure 11 It comes from Figure 5 A schematic front view of the first step of the method, in which the segment is driven toward the emulsified joint. Figure 12 It comes from Figure 5 Schematic front views of the second and third steps of the method. Figure 13 For from Figure 5 A schematic front view of the fourth step of the method. Figure 14 For from Figure 5 A schematic front view of the first step of the method. Figure 15 For from Figure 5 A schematic front view of the first step of the method. Figure 16 for Figure 6 A schematic front view of the steps of the method. Figure 17 This is a diagram with experimental images showing the effect of the shrinkage of the droplet emulsion junction on the droplet volume generated by the segment. Figure 18 This is a schematic diagram of several methods for generating the random accumulation of droplets. Figure 19 These are cross-sectional views of different embodiments of the reservoir shape, and Figure 20 A schematic diagram of a method for controlling droplet accumulation in multiple liquid storage zones. Detailed Implementation

[0037] like Figure 1 and Figure 2 As shown, the device 100 according to the present invention includes an emulsification junction 50 having a first input terminal 51, a second input terminal 52 and an output terminal 53.

[0038] Emulsion joint The emulsification junction 50 is a system comprising two input ends 51, 52 configured to deliver at least one first immiscible fluid 21-25 and at least one second immiscible fluid 31 to the emulsification zone 55. The geometry of the emulsification zone 55 is such that at least two immiscible fluids 21-25, 31 meet and droplet breakage occurs therein.

[0039] The emulsification joint 50 can be of any type, such as a T-joint, Y-joint, flow-focusing joint, co-flow-focusing joint, inclined emulsification joint, stepped emulsification joint, buoyancy emulsification joint, centrifugal stepped emulsification joint, electrohydrodynamic emulsification joint, or acoustic emulsification. Specifically, such as... Figure 3 and Figure 4 As shown, the emulsion junction 50 can advantageously be a 3D flow-focusing junction, wherein the first and second immiscible phases are simultaneously forced through the narrow opening of the collection tube, resulting in the first phase being uniformly broken up as it is surrounded by the second phase. Such an emulsion junction 50 may optionally include a contraction portion 54.

[0040] The 3D flow focusing junction is made of at least two delivery tubes 56, 57, wherein at least a first delivery tube 56 is located inside at least a second delivery tube 57. Advantageously, the delivery tubes 56, 57 are concentric. The delivery tubes 56, 57 may be capillaries made of glass, plastic, resin or fluoropolymer. The 3D flow focusing junction includes a first delivery tube 56, which includes a first input end 51, a second input end 52 and an output end 53. The first input end 51 and the output end 53 are preferably aligned along a straight line d2, while the second input end 52 is angled relative to the straight line d2 and aligned along a straight line d3.

[0041] The inner diameter of the first input terminal 51 and the second input terminal 52 may be between 0.1 mm and 2 mm, preferably 1 mm, and the outer diameter is preferably between 0.2 mm and 3 mm, preferably 1.6 mm. The output terminal 53 has a smaller inner diameter and outer diameter, with its inner diameter preferably between 0.1 mm and 2 mm, preferably 0.75 mm, and its outer diameter preferably between 0.2 mm and 3 mm, preferably 1.6 mm.

[0042] The second delivery tube 57 is positioned inside the first delivery tube 56 along a straight line d2. It includes a first end 59 and a second end 58. It preferably has an inner diameter between 0.1 mm and 1 mm, more preferably 0.25 mm, and an outer diameter between 0.5 mm and 1.6 mm, more preferably 0.75 mm. The length of the second tube 57 is advantageously between 25 mm and 75 mm.

[0043] Preferably, the inner diameter of the output end 53 is equal to the outer diameter of the second delivery pipe 57. In this way, a fluid protection cavity is created between the inner diameter of the first delivery pipe 56 and the outer diameter of the second delivery pipe 57, in which fluid can flow.

[0044] The emulsification zone 55 configured for generating droplets can be defined in front of the first end 59 of the second delivery tube.

[0045] Advantageously, the emulsifying joint 50 of the present invention includes a contraction portion 54, which includes a first end 541 and a second end 542, the second end 542 being located in front of the first end 59 of the second delivery tube. In this case, the emulsifying zone 55 can be defined between the contraction portion 54 and the first end 59 of the second delivery tube.

[0046] The geometry and position of the contraction portion 54 relative to the emulsification zone 55 play a role in achieving the desired droplet size and droplet uniformity.

[0047] For example, such as Figure 4 As shown, a cylindrical constriction section can be used. In Figure 4 In the diagram, d1 defines the inner diameter of the second end 542 of the contraction section, L defines the length of the contraction section 54, and w defines the distance between the second end 542 of the contraction section and the first end 59 of the second delivery pipe.

[0048] The geometry of the contraction section 54 is constrained by the desired droplet size. The inner diameter d1 of the contraction section is preferably equal to or less than the desired droplet diameter D. In other words, the following formula can be defined: d1 ≲ D. The length L of the contraction section is preferably equal to or greater than the desired droplet diameter D. In other words, the following formula can be defined: L ≳ D. The gap between the end of the contraction section and the end of the emulsion capillary is preferably less than 10 times the desired droplet diameter D. In other words, the following formula can be defined: w ≲ 10D. The inner diameter of the contraction section 54 can be constant along the length of the contraction section, or alternatively, it can have a tapered shape with a larger diameter at the first end 541 and a smaller diameter at the second end 542 of the contraction section.

[0049] The contraction section 54 allows for minimization of polydispersity between segments of the first immiscible fluid 23 in the first input end 51. The presence of the contraction section 54 reduces the difference between the cross-sectional diameter of the first input end 51 (typically 1 mm) and the cross-sectional diameter of the first end 59 of the second delivery tube (typically 250 µm). The contraction section 54 prevents the formation of large droplets 47 in the last set of droplets at the end of the first input end 51. This improvement can... Figure 17 As can be seen, a contraction section with an inner diameter d1 of 500µm is used. Figure 17The effect of the contraction on droplet uniformity is shown. The graph above represents the volume V of each number of droplets (#) in nanoliters. In this graph, dots represent droplets without contractions (A), while crosses represent droplets with contractions (B). Images of the first group (I), the middle group (II), and the last group (III) of droplets without contractions (A) and with contractions (B). In the last group (III) of droplets without contractions (A), large droplets 47 are formed.

[0050] Figure 5 An example of an emulsion joint 50 that can be obtained through 3D printing is shown. More precisely, the emulsion joint 50 can take the shape of a microfluidic boat comprising a body 73 made of a rigid material configured to hold delivery tubes 56, 57 together. Delivery tubes 56, 57 can also be printed using plastics such as polyetheretherketone (PEEK) or tetrafluoroethylene (TEFLON) or combinations thereof. Silicone tubing 71 and adhesive 72 can be used to hold / assemble delivery tubes 56, 57 together. The use of silicone tubing 71 and adhesive 72 allows for better concentric alignment of delivery tubes 56, 57 and stable control of distance w to allow for reproducibility of droplet generation.

[0051] The body 73 may include a cavity 74 configured to allow visual access to the emulsified area 55 and to allow direct microscopic measurements, such as distance w. The cavity 74 may be an opening or filled with a transparent material, such as glass, plastic, or polydimethylsiloxane (PDMS).

[0052] First input terminal 51 like Figure 1 As shown, for the purpose of droplet generation mode, the first input terminal 51 is configured to be supplied with at least one first immiscible fluid 21-25. For this purpose, the first input terminal 51 can be connected to at least one first tank 61, which contains at least one first immiscible fluid 21-25. In one embodiment, the first input terminal 51 can be permanently connected to several first tanks 61 containing different first immiscible fluids 21-25. Therefore, a device such as a multi-directional control valve can be used to select the desired first tank 61.

[0053] Alternatively, when needed, a device can be used to operate the connection between the desired first tank 61 and the first input terminal 51, such as a robotic arm movable in at least two spatial directions.

[0054] In the first embodiment, the first input terminal 51 can be supplied with a continuous flow of a first immiscible fluid 21-25, such as Figure 18As shown in Figure A. Alternatively, the first input terminal 51 can be supplied with different sections of the first immiscible fluid 21-25, separated from the section of the second immiscible fluid 31 supplied by the second tank 62, thereby producing as... Figure 18 BD shows a series of segments of immiscible fluids. Therefore, the segments are typically generated sequentially. The order of the segments can be periodic, where the segments of the first immiscible fluid 21-25 are constantly arranged, and the volume of the segments of the first immiscible fluid 21-25 is constant, as shown... Figure 18 As shown in B, this sequential aspiration of the sections of the first immiscible fluid 21-25 separated by the spacer of the second immiscible fluid 31 will result in the accumulation of droplets in a stratified distribution within the reservoir, as shown in Figure B. Figure 18 As shown on the right side of B. Droplet accumulation can be random, causing the droplets to mix. During the collection of segments, one way to randomize droplet accumulation without modifying hardware settings or performing any post-processing is to randomly distribute segments of the first immiscible fluid 21-25, each segment being separated by a segment of the second immiscible fluid 31, as shown... Figure 18 As shown in C. A second method of randomizing the segment string is to constantly arrange the first immiscible fluids 21-25 separated by spacers of the second immiscible fluid 31, while randomly changing the volume of the segments of the first immiscible fluids 21-25, such as... Figure 18 As shown in D. The total volume of the segments of the first immiscible fluid 21-25 in a single sequence, i.e., the total volume in a sequence in which each segment containing the first immiscible fluid appears once, is preferably constant, i.e., equal for all sequences. The segments of the first immiscible fluid 21-25 can be randomly distributed, while the volume of the segments of the first immiscible fluid 21-25 is randomly varied. The effect of randomizing the segments is to spatially mix the droplets in the reservoir, such as... Figure 18 As shown on the right side of BD.

[0055] The connection between the first input terminal 51 and at least one first and second tank 61, 62 can be obtained using a tubular connector 12, such as a flexible tube made of rubber, polyvinyl chloride (PVC), etc.

[0056] The suction of at least one first immiscible fluid 21-25 inside the tubular connector can be performed by at least one first flow device 32 or at least one second flow device 33.

[0057] For the purpose of droplet collection modes, such as Figure 2As shown, the shut-off device 26 may be located upstream of the first input terminal 51. The shut-off device 26 may be a valve, plug, or any device configured to block flow through the first input terminal 51. The shut-off device 26 may be made of an elastic material, such as rubber or PDMS, to facilitate rapid opening and / or closing. The shut-off device 26 may be operated manually or electromechanically and is capable of withstanding an internal pressure of at least 10 mbar when in the closed position.

[0058] Second input terminal 52 like Figure 1 and Figure 2 As shown, the second input terminal 52 is configured to connect to at least one first flow device 32, which is configured to supply a flow of at least one second immiscible fluid 31 to the second input terminal 52. The first flow device 32 may also be configured to aspirate a flow from the emulsion junction 50.

[0059] The connection between the second input terminal 52 and at least one first flow device 32 can be achieved using a tubular connector, such as a flexible tube made of rubber, polyvinyl chloride (PVC), etc.

[0060] The first flow device 32 may be a syringe pump, peristaltic pump, pressure-driven flow generator using a flow sensor, pressure-driven flow generator using flow resistance, hydrostatic flow generator, etc., ranging from 0.1 mL to 50 mL.

[0061] In a first embodiment, the first flow device 32 includes a tank configured to contain a second immiscible fluid 31 of a first type. Alternatively, the first flow device 32 may include a plurality of tanks containing different types of second immiscible fluids 31. The first flow device 32 may be configured to optionally provide a specific type of second immiscible fluid 31 by selecting the correct tank. Optionally, the second input terminal 52 may be connected to a plurality of first flow devices 32, each first flow device 32 containing a tank filled with a specific type of second immiscible fluid 31. Depending on the desired second immiscible fluid 31, the correct first flow device 32 can be selected to supply the second input terminal 52 with the second immiscible fluid 31. Furthermore, at least one first flow device 32 may include a tank configured to receive fluid drawn from the emulsion junction 50.

[0062] Output 53 like Figure 1 and Figure 2 As shown, the output terminal 53 is configured to be connected to at least one second flow device 33, which is configured to supply a flow of at least one second immiscible fluid 31 or to draw a flow from the emulsion junction 50.

[0063] The connection between the output end 53 and at least one second flow device 33 can be obtained using a tubular connector 14, which is a flexible tube made of, for example, rubber, silicone, polyvinyl chloride (PVC) or any other elastic material.

[0064] The second flow device 33 can be a 0.1 mL to 50 mL syringe pump, peristaltic pump, pressure-driven flow generator using a flow sensor, pressure-driven flow generator using flow resistance, hydrostatic flow generator, etc. In a first embodiment, the second flow device 33 includes a tank configured to contain a second immiscible fluid 31 of a first type. Alternatively, the second flow device 33 may include several tanks containing different types of second immiscible fluids 31. The second flow device 33 may be configured to optionally provide a specific type of second immiscible fluid 31 by selecting the correct tank. Alternatively, the second input 52 may be connected to a plurality of second flow devices 33, each containing a tank filled with a specific type of second immiscible fluid 31. Depending on the desired second immiscible fluid 31, the correct second flow device 33 can be selected to supply the second immiscible fluid 31. Furthermore, the at least one second flow device 33 may include a tank configured to receive fluid drawn from the emulsion junction 50.

[0065] Accumulator 15 like Figure 1 and Figure 2 As shown, the device 100 according to the invention may advantageously include a reservoir 15, which includes a storage region 17 configured to capture the generated droplets 41-45. The reservoir 15 is located downstream of the output terminal 53, preferably between the output terminal 53 and the second flow device 33.

[0066] The reservoir 15 may have a tapered shape including a base 151 and an apex 154. The apex 154 may have an angle ranging from 15° (e.g., ...). Figure 19 (as shown in A) to 170° (as shown in A) Figure 19 (As shown in B) Angle. The base 151 advantageously includes at least one opening 152 configured to allow the generated droplets 41-45 from the output end 53 to pass through. The reservoir 15 may also include a second opening 153 configured to allow the flow of a second immiscible fluid 31. The reservoir 15 may have a vertical wall 156 connecting the base 151 to a tapered portion including a top end 154, as shown. Figure 19 As shown in B and 19C. The vertical wall 156 can be straight or curved.

[0067] In another embodiment, the reservoir 15 may have a parallelepiped base, the top of which is formed by an inclined reservoir area 17, such as... Figure 19 As shown in Figure C, this is a specific embodiment of the conical accumulator 15, wherein the top end 154 is located at the end of the vertical wall 156. The top end 154 may have an angle ranging from 15° to 170°.

[0068] In another embodiment, the reservoir 15 may have a pointed dome shape, including a base 151 and a tip 154, such as Figure 19 As shown in E. The dome-shaped liquid storage area 17 can be located on the variable cross-section body, such as... Figure 19 As shown in D.

[0069] The reservoir 15 may include multiple reservoir zones 17. Variations in the tilt angle of the reservoir 15 relative to the vertical axis Z allow control over which reservoir zone 17 is filled with droplets, such as... Figure 20 As shown. Alternatively, a rotary valve can be used to select the reservoir 17 in which the droplets are stored. Each reservoir 17 may include a tip 154.

[0070] For the purpose of droplet collection modes, such as Figure 2 As shown, outlet 16 can be located downstream of output terminal 53.

[0071] Outlet 16 is configured to extract the generated droplets 41-45 from device 100, for example, into vial 101. Preferably, outlet 16 is included at the top 154 ​​of reservoir 15. Outlet 16 can be a sealable opening having an open and a closed state. Outlet 16 can be sealed, for example, using a valve, plug, or the like. Outlet 16 may also include a tubular connector 161 configured to deliver the generated droplets 41-45 directly into the vial. Outlet 16 can be closed using a valve made of an elastic material such as rubber or PDMS for easy opening / closing. It can be operated manually or electromechanically and can withstand an internal pressure of at least 10 mbar in the closed position.

[0072] Alternatively, the accumulator can have other shapes, such as asymmetrical conical, spherical, cylindrical, elliptical, and combinations thereof. Alternatively, openings 151 and 152 can be positioned on the sidewalls of the accumulator 15. Other examples of accumulator geometries may include all geometries having a smooth transition from a large cross-section to a small orifice, such that no space is left inside the accumulator, wherein at least one droplet can be retained and captured after droplet extraction.

[0073] Typical dimensions of the accumulator 15 with a conical shape are as follows. The base 151 preferably has a diameter of 1 mm to 50 mm. The height of the accumulator 15 is preferably between 10 mm and 10 cm.

[0074] method like Figure 5 and Figure 6 As shown, according to another aspect, the present invention relates to a method 200 for generating droplets using, for example, an apparatus 100 as described above, and a method 300 for collecting said droplets.

[0075] The first step of the method 200 for generating droplets is to supply an input flow 202 of at least one first immiscible fluid 21-24 to a first input terminal 51.

[0076] According to the preferred embodiment, such as Figures 7 to 15 As shown, the flow of at least one second immiscible fluid 31 includes multiple segments of different first immiscible fluids 21-24, which are separated by segments of at least one second immiscible fluid 31.

[0077] For example, a robotic arm capable of moving in three spatial directions can be used to prepare multiple segments. Figure 7 As shown, the robotic arm can move the tubular connector 12 into a first orifice 64 included in the orifice plate 63. The orifice 64 contains a first immiscible fluid 21 of a first type. A first section of the first immiscible fluid 21 can be aspirated. In this example, the aspiration is generated by a first flow device 33.

[0078] Then, as Figure 8 As shown, the robotic arm can move the tubular connector 12 into a tank 62 containing a second immiscible fluid 31. A first section of the second immiscible fluid 31 can be aspirated.

[0079] In the third movement, the robotic arm can move the tubular connector 12 into a second orifice included in the orifice plate 63 and containing the first immiscible fluid 22 of the second type. In the fourth movement, the robotic arm can move the tubular connector 12 back into the tank 62 to aspirate a second section of the second immiscible fluid 31.

[0080] These movements can be repeated indefinitely to prepare multiple segments of the first immiscible fluid 21-24, thereby generating a string of immiscible fluid segments. Therefore, the segments can be generated sequentially. For example, the first immiscible fluid 21-24, comprising between 2 and 20 segments, can be present in the tubular connector 12, as... Figure 9 As shown. The order of the segments can be periodic, wherein the segments of the first immiscible fluid 21-25 are constantly arranged and the volume of the segments of the first immiscible fluid 21-25 is constant. The segment order can be randomly distributed. In one embodiment, the sample is collected directly from the well plate 63, as shown. Figure 7 As shown.

[0081] During the final movement, the tubular connector 12 is positioned in the tank 62 by a robotic arm to allow the aspiration of the second immiscible fluid 31. Therefore, as the second immiscible fluid 31 is aspirated, the section of the first immiscible fluid 21-24 begins to move upwards from the tubular connector 12 to the emulsion junction 50, as... Figure 10 As shown.

[0082] Typical sizes of the segments of the first immiscible fluid 21-24 range from 1 μL to 1 mL. The sizes of the segments of the first immiscible fluid 21-24 can vary depending on the desired number of droplets generated. The total volume of the segments of the first immiscible fluid 21-25 in a single sequence, i.e., the total volume in a sequence containing each segment of the first immiscible fluid appearing once, is preferably constant, i.e., equal for all sequences. The segments of the first immiscible fluid 21-25 can be randomly distributed, while the volume of the segments of the first immiscible fluid 21-25 can be randomly varied. Typical sizes of the segments of the second immiscible fluid 31 range from 1 μL to 1 mL.

[0083] At least one second immiscible fluid 31 may be an organic solvent combined with a surfactant, such as a fluorohydroether. Alternatively, the at least one second immiscible fluid 31 may be an oil phase selected from fluorinated oils, silicone oils, vegetable oils, mineral oils, and hydrocarbon oils. The droplets may be stabilized in the oil phase with a surfactant (such as PEG-di-Krytox fluorinated surfactant, perfluorooctyl alcohol, perfluorododecyl alcohol, perfluoropolyether (PFPE) derivatives including PFPE-PEG triblock copolymers, span80, Abil EM90, monolein, oleic acid, n-butanol) or in the aqueous phase with a surfactant (phospholipids, Triton-X-100, SDS, Pluronic, Tween 20 / 80). The first immiscible fluids 21-24 are preferably an aqueous phase, comprising at least one of cell culture media, drug solutions, antibody solutions, cell suspensions, organoid or cell tissue suspensions, hydrogel solutions, microsphere or particle suspensions.

[0084] like Figure 11 As shown, once the first section of the first immiscible fluid 21 has entered the first opening 51 of the emulsion junction, in the second step 202, at least one flow of the second immiscible fluid 31 is supplied to the second input end 52 using the first flow device 32, while the second flow device 33 continues to perform suction.

[0085] like Figure 11 and 12As shown, then in the third step 203, droplets 41 of the first type of immiscible fluid 21 are generated at the output end 53 of the emulsion joint 50. Once the first segment of the first immiscible fluid 21 has completely transformed into droplets 41, a segment of the second immiscible fluid 31 passes through the emulsion joint 50 and washes out the remaining droplets 41. Then, a second segment of the first immiscible fluid 22 enters the first opening 51 of the emulsion joint and transforms into droplets 42 of the second type of immiscible fluid 22. This process continues until no segments of the first immiscible fluid 21-24 remain in the tubular connector 12.

[0086] The generated droplets 41-45 may have a size ranging from 2 nL to 2 μL, depending on the geometry of the delivery tube used in the emulsification junction 50.

[0087] Typical flow rates for supplying the emulsion junction 50 in the first immiscible fluid 21-25 and the second immiscible fluid 31 may range from 10 to 1000 μL / min. The number of droplets 41-45 produced may range from 50,000 to 100,000.

[0088] When the first immiscible fluid 21-24 is transformed into droplets 41-44, the resulting droplets 41-45 tend to move towards the reservoir 15, as... Figure 13 As shown, and in step 204, the liquid accumulates in the storage area 17 due to buoyancy, as... Figure 14 As shown.

[0089] Once all the generated droplets 41-44 are captured in the storage area 17, the first flow device 32 and the second flow device 33 stop, as shown. Figure 15 As shown, the droplet generation mode is complete.

[0090] The aspiration of individual segments of the first immiscible fluid 21-25 will result in the accumulation of droplets in a stratified distribution within the reservoir 15. The sequential aspiration of small-volume segments of the first immiscible fluid 21-25 will result in the mixing of droplets on the reservoir. The random distribution of the segments of the first immiscible fluid 21-25 and variations in the typical size of these segments will lead to increased mixing of droplets on the reservoir.

[0091] Advantageously, the device 100 may include a third flow device (not shown in the figure) connected to the outlet 16 of the accumulator 15. The third flow device may be configured to generate a flow that is configured to mix the generated droplets included within the accumulator zone 17. In practice, the flow generated by the third flow device is configured to push the generated droplets against gravity towards the base of the accumulator 15, thereby enabling the mixing of droplets with different properties. This mixing occurs in a manner similar to that of a fluidized bed.

[0092] For the droplet collection mode, in the first step 301, the end of the tubular connector 12 is blocked using the shut-off device 26, while in the second step 302, the outlet 16 is opened. In the third step 303, the second flow device 33 is activated to supply a flow of the second immiscible fluid 31, thereby propelling the generated droplets 41-44 through the outlet 16 and into the vial 101, as shown. Figure 16 As shown. Then create a droplet library. The size of the created library can be from 0.1 mL to 10 mL.

[0093] The apparatus and method of the present invention can be used for drug screening. For this purpose, an aqueous droplet library can be prepared using various compounds such as drugs (including chemotherapy, cells, and biologics) to test cells. Such cells can be cancer cells collected from patients.

[0094] The apparatus and method of the present invention can also be used to test culture conditions. For this purpose, a library of different media compositions can be prepared by changing the viscosity, serum type and concentration, growth factors, amino acids, buffers, adding small amounts of hydrogel molecules or molecules that promote 3D cell aggregation.

[0095] The apparatus and method of the present invention can also be used for cell encapsulation. For this purpose, cells can be encapsulated from a single or multiple sources. This can be used to screen multiple patient samples.

Claims

1. An apparatus (100) for generating droplets (41-45), comprising an emulsification junction (50) configured to generate droplets (41-45) by contacting a flow of at least one first immiscible fluid (21-25) with a flow of at least one second immiscible fluid (31), the emulsification junction (50) comprising a first input (51), a second input (52), an output (53), and an emulsification region (55) in which the generation of the droplets occurs, wherein: - The first input terminal (51) is configured to be supplied with at least one first immiscible fluid (21-25). - The second input terminal (52) is configured to be connected to at least one first flow device (32), the first flow device (32) being arranged to supply a flow of the second immiscible fluid (31) to the second input terminal (52). The output terminal (53) is connected to at least one second flow device (33), the device (100) being configured to operate in a droplet generation mode, wherein the first input terminal (51) is supplied with a flow of the first immiscible fluid (21-25) by the second flow device (33), while the second input terminal (52) is supplied with a flow of the second immiscible fluid (31) by the first flow device (32), such that droplet generation occurs in the emulsification zone (55), and the generated droplets (41-45) are removed from the emulsification junction (50) by the second flow device (33).

2. The apparatus (100) according to claim 1, wherein, The device (100) further includes: - A shut-off device (26) is disposed upstream of the first input terminal (51), the shut-off device (26) having an open state and a closed state, the shut-off device (26) being configured to block the flow through the first input terminal (51). - An outlet (16) is arranged downstream of the output end (53), the outlet (16) having an open state and a closed state, the outlet (16) being configured to allow the extraction of generated droplets (41-45) from the device (100). The device (100) is further configured to operate in a droplet collection mode, wherein the shut-off device (26) is in a closed state and the outlet (16) is in an open state, and a flow of the second immiscible fluid (31) is generated to extract the generated droplets (41-45) from the device (100) through the open outlet (16).

3. The apparatus (100) according to any one of claims 1 to 2, wherein the first input terminal (51) is configured to be supplied with a plurality of segments of the at least one first immiscible fluid (21-25), including at least a first segment and a second segment, the two consecutive segments being separated by a third segment of the at least one second immiscible fluid (31).

4. The apparatus (100) according to any one of claims 1 to 3, wherein, The device (100) further includes a reservoir (15) comprising a storage area (17) configured to capture generated droplets (41-45), the reservoir (15) being located downstream of the output (53).

5. The device (100) according to claim 4, wherein the reservoir (15) is located between the output end (53) and the second flow device (33).

6. The apparatus (100) according to claim 4 or 5, characterized in that, The reservoir (15) is a gravity-based reservoir (15) configured to capture gravity-driven generated droplets (41-45) in the reservoir (15).

7. The device (100) according to any one of claims 4 to 6, wherein the reservoir (15) has a conical shape, the conical shape including a base (151) configured for passing through the generated droplets (41-45) and a top end (154) for positioning the reservoir area (17).

8. The apparatus (100) according to any one of claims 4 to 6, characterized in that, The reservoir (15) has a pointed dome shape, which includes a base (151) configured for passing through the generated droplets (41-45) and a top (154) for positioning the reservoir area (17).

9. The apparatus (100) according to claim 7 or 8, wherein the vertex includes the outlet (16).

10. The apparatus (100) according to any one of claims 1 to 9, wherein the emulsification junction (50) includes a contraction portion (54), the contraction portion (54) including a contraction portion output end, the contraction portion output end being configured to guide the at least one first immiscible fluid (21-25) to the emulsification region (55).

11. The use of the apparatus (100) according to any one of claims 1 to 10 for generating a droplet library comprising at least two sets of droplets (41-45), wherein the first set of droplets (41-45) is generated using a first immiscible fluid (21-25) of a first type, and the second set of droplets (41-45) is generated using a first immiscible fluid (21-25) of a second type.

12. A method (200) for generating droplets using the apparatus (100) according to any one of claims 1 to 10, wherein the method comprises the following steps: - A flow (201) of at least one second immiscible fluid (31) is supplied to the second input terminal (52), the second input terminal (52) being configured to deliver the second immiscible fluid (31) to the emulsification zone (55), and - Simultaneously, at least one flow (202) of the first immiscible fluid (21-25) is supplied to the first input end (51) by drawing the first immiscible fluid (21-25) downstream of the output end (53), the first input end (51) being configured to deliver the first immiscible fluid (21-25) to the emulsification zone (55) such that droplets (41-45) are generated in the emulsification zone (55), and the generated droplets (41-45) are drawn out from the emulsification junction (50) by drawing from the downstream of the output end (53).

13. The method of claim 12, wherein the method further comprises the step of accumulating the generated droplets (41-45) in a reservoir (15) of the device according to any one of claims 5 to 10.

14. The method according to claim 12 or 13, wherein the suction downstream of the output (53) is sequential suction, the first input (51) is configured to be supplied with a plurality of segments of the at least one first immiscible fluid (21-25), including at least one first segment and at least one second segment, the sequential suction being configured to insert at least one third segment of the at least one second immiscible fluid (31) between the at least one first segment and the at least one second segment, thereby generating a segment string including the at least one first segment, the at least one second segment and the at least one third segment.

15. The method according to claim 14, wherein, The at least one first segment, the at least one second segment, and the at least one third segment in the segment string are randomly distributed.

16. The method of claim 14 or 15, wherein the at least one first segment has a first volume, the at least one second segment has a second volume, and the third segment has a third volume, wherein the first volume, the second volume, and the third volume are different.

17. A method for collecting droplets from the apparatus (100) according to any one of claims 1 to 10, wherein the method comprises the following steps: - Use the closing device (26) to close the first input terminal (51) (301). - Open the outlet (16) (302), and - A flow of a second immiscible fluid (31) is supplied, such that the generated droplets (41-45) are extracted (303) from the device (100) through the open outlet (16).

18. The method for collecting droplets according to claim 17, wherein the flow of the second immiscible fluid (31) is supplied from downstream of the output (53).

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