Microfluidic chip and method for generating and sorting monodisperse microdroplets at high frequency

Abstract

The present invention relates to a microfluidic chip (30) for generating and sorting monodisperse microdroplets (32).According to the invention, the microfluidic chip (30) comprises a junction zone (7), a relaxation zone (8) downstream of the junction zone (7), and a sorting zone (18) arranged downstream of the relaxation zone (8), the junction zone (7) comprising a central nozzle (21) for injecting central fluid (3), two lateral nozzles (22, 23) for injecting sheath fluid (1), the central nozzle (21) having a smaller width x of between 2 and 10 micrometers, each of the two lateral nozzles (22, 23) having a smaller width x1 of between 3 and 10 micrometers, a fluidic microchannel (24) having a width y of between 2 and 7 micrometers and a length z of between 2 and 10 micrometers, and a relaxation zone (8) having a larger width u of between 20 and 75 micrometers and a length V of between 100 and 600 micrometers, the junction zone (7), the fluidic microchannel (24) and the relaxation zone (8) having a depth h between 2 and 10 µm.Figure for abstract: Fig. 2.

Description

Title of the invention: Microfluidic chip and method for generating and sorting monodisperse microdroplets at high frequency Technical field of the invention

[0001] The present invention relates to the technical field of microfluidic chips and methods for generating and sorting at high frequency monodisperse and ordered microdroplets having a volume in the range between 1 femtoliter (IL) and 200 IL.

[0002] It relates in particular to a microfluidic chip making it possible to generate microdrops of a determined volume, monodispersed and ordered, at high frequency, that is to say at a frequency greater than or equal to 1 kilohertz (kHz), preferably greater than or equal to 10 kHz, for example 60 kHz. It also relates to a microfluidic chip making it possible to generate and actively sort one by one each microdrop of the train of monodispersed microdrops, the sorting being able to be carried out at high frequency, that is to say at a frequency greater than 10 kHz, for example 60 kHz. State of the art

[0003] In the above field, it is known to use flow cytometers to detect, count, identify cells or particles of micrometric dimensions suspended in a flowing fluid by passing them, one by one and at high speed, in the beam of one or more lasers. Certain sorting cytometers also make it possible to sort these particles by implementing sorting methods based on various techniques. Advances in microscopy and biology have made it possible to focus on objects of ever smaller size, in particular nano-objects of size less than 100 nm. However, traditional flow cytometers generate drops of diameter between 30 micrometers (pm) and 300 pm, i.e. volumes of between 10 picoliters and 10 nanoliters and can only sort objects of size greater than several hundred nanometers.

[0004] For objects smaller than 100 nm, cytometers are currently limited by their detection capacity. In addition, regarding the operation of sorting nano-objects, the fluid volumes generally used by known cell sorters and the size of the drops used to encapsulate the objects to be sorted are not compatible with the sorting of nanometric-sized objects.

[0005] Indeed, studies involving objects of nanometric size (between a few nanometers and a few tens or even a few hundreds of nanometers) require the ability to analyze these objects one by one. Given the very low size of these objects, it has proven interesting from a practical point of view to encapsulate these objects in drops of fluid, these drops then being easier to handle, analyze, sort. When we extract drops of a liquid containing a certain concentration of these particles, the Poisson law allows us to very classically calculate the probabilities that a given drop contains N particles, N being a positive integer or zero. This law is written:

[0006] in which P(N) is the probability that a drop contains N particles, and / . is the average number of particles per drop, for a drop of given volume and a given volume concentration of particles.

[0007] In particular, if one seeks to encapsulate individual objects, this law makes it possible to determine, for a given dilution or volume concentration, the optimal volume of drops for which the number of drops containing more than one particle is minimized, while maximizing the number of non-empty drops.

[0008] Typical concentrations of liquids containing organic nanoparticles are generally between 109 and 1010 particles per ml. According to the above calculations, spherical drops of 30 pm to 300 pm in diameter (with a volume between 10 picoliters and 10 nanoliters) are suitable for samples with a concentration between 103 and 106 particles per ml, which is completely insufficient for usual samples of biological nanometric particles. To achieve these concentrations, it would be necessary to dilute the biological samples quite excessively (for example, a typical 1 ml sample would have to be diluted to a volume of at least 1 liter, or even 1000 liters). On the contrary, biologists wish to concentrate such samples. For an average concentration of 5.109 particles per ml, the Poisson distribution allows to determine the probabilities that a given drop contains 0, 1 or 2 particles according to the volume of the drop. To ensure that the number of drops containing a single particle is greater than or equal to 10%, while keeping the number of drops containing more than one particle less than or equal to 1%, the volume of the drops is between 30 fL and 40 fL. At this concentration of 5.109 particles per ml, for drops with a volume less than 30 fL, the percentage of empty drops, i.e. without any particles, is greater than 90%, which lengthens the analysis times, as all the drops must be analyzed while most are empty. Conversely, for drops with volumes greater than 40 fl, the percentage of drops containing more than one particle is greater than 1%, which is a significant source of error, thus reducing the quality of the analysis and sorting.

[0009] The microdrops of sample fluid (i.e. the core fluid) may be generated by emulsification in a sheath fluid, the sample fluid and the sheath fluid being immiscible with each other, for example based on water in oil or oil in water. Microdroplets can be generated by focusing flow in a microchannel of a microfluidic chip, so that the sample fluid splits into microdroplets dispersed in the sheath fluid. The diameter or volume of the microdroplets generally depends on the size of the microchannels of the microfluidic chip and also varies according to many environmental parameters, such as variations in pressure or flow rate of the injected fluids. However, hydrodynamic phenomena at the microscopic scale are complex to predict and model. In the same microfluidic device, the generation of drops is highly dependent on the injection conditions. Variations in the volume of the microdroplets are observed over time, which are difficult to control.

[0010] In this document, monodisperse microdrops are understood to mean microdrops all having the same volume + / - 20% for example. In other words, the volume of the monodisperse microdrops is uniform. The monodisperse microdrops are generally suspended in a flow of encapsulation fluid or sheath fluid.

[0011] Microfluidic devices are known for generating monodisperse microdroplets having a volume of the order of 10 pL, i.e. a diameter greater than 30 pm, which can operate at a generation frequency of less than 5 kHz in general and sometimes up to 30 kHz. However, the volume of these microdroplets is too large compared to nano-objects that one wishes to analyze individually.

[0012] The publication “Shui, L., van den Berg, A. & Eijkel, JCT Scalable attoliter monodisperse droplet formation using multiphase nano-microfluidics. Microfluid Nanofluid 11, 87-92 (2011). https: / / doi.org / 10.1007 / sl0404-011-0776-7” describes a device comprising a nanochannel-microchannel interface for generating monodisperse droplets with a diameter between 0.4 and 3.5 pm (with a minimum volume of 30 aL). However, the generation frequency of these microdroplets is limited to a few hundred hertz. The generated drops are stored in a reservoir and form a compact 2D stack.

[0013] The publication Shim JU, Ranasinghe RT, Smith CA, Ibrahim SM, Hollfelder F, Huck WT, Klenerman D, Abell C. Ultrarapid generation of femtoliter microfluidic droplets for single-molecule-counting immunoassays. ACS Nano. 2013 Jul 23;7(7):5955-64. doi: 10.1021 / nn401661d. Epub 2013 Jul 8. PMID: 23805985, describes a droplet generation device with a volume of 32 fL. However, this device does not allow for on-the-fly sorting of individual drops.

[0014] Microfluidic systems generally comprise a microfluidic chip for producing drops and another microfluidic chip for sorting the drops, the drops being stored in the interval between their production and their sorting. Such a This arrangement requires re-injection of the drops into another chip, which is particularly complex, and significantly increases both processing time and the risk of errors or other difficulties such as droplet fusion, leakage of droplet liquid into the sheath liquid, handling difficulties, and requires relatively large volume drops.

[0015] There is a need for a device and a method for isolating, detecting, analyzing and individually sorting objects of size less than a micrometer, which are for example encapsulated in microdrops, at high speed.

[0016] To this end, there is a need for a device and a method for generating monodisperse microdrops having a determined volume of the order of a femtoliter, for example between 1 fL and 200 fL, at a generation frequency greater than 1 kHz, preferably greater than or equal to 10 kHz and capable of reaching several tens of kHz or hundreds of kHz, and allowing individual analysis and / or sorting of each microdrop also at a high rate, ideally at the same rate as the generation frequency. There is a need for a device and a method for generating such monodisperse microdrops which is stable, reproducible, insensitive to variations in environmental parameters and easy to manufacture.

[0017] Furthermore, there is a need for a device and a method for generating such monodisperse microdrops, then individually detecting and actively sorting each microdrop on the same fluidic chip, at a high sorting rate, greater than 10 kHz and capable of reaching several tens of kHz or hundreds of kHz. Presentation of the invention

[0018] In this context, the present invention provides a microfluidic chip for generating and sorting monodisperse microdroplets based on a central fluid in a sheath fluid, the central fluid and the sheath fluid being immiscible with each other.

[0019] According to the invention, the microfluidic chip comprises a central fluid injection channel, two sheath fluid injection channels and in that the microfluidic chip comprises a junction zone, an expansion zone downstream of the junction zone and a sorting zone arranged downstream of the expansion zone, a fluidic microchannel fluidly connecting the junction zone to the expansion zone, the sorting zone comprising a fluidic channel fluidly connected at the inlet to the expansion zone and downstream to at least two outlet channels, the junction zone comprising a central nozzle fluidly connected to the central fluid injection channel, two lateral nozzles arranged on two opposite sides of the junction zone, each of the two lateral nozzles being fluidly connected to one of the two sheath fluid injection channels, the fluidic microchannel being arranged on an opposite face of the junction zone with respect to the central nozzle, the central nozzle having a smaller width x of between 2 and 10 micrometers, each of the two lateral nozzles having a smaller width xl of between 3 and 10 micrometers, the fluidic microchannel having a width y of between 2 and 7 micrometers and a length z of between 2 and 10 micrometers, the relaxation zone having a greater width u of between 20 and 75 micrometers and the relaxation zone having a length V of between 100 and 600 micrometers, and in that the junction zone, the fluidic microchannel, the relaxation zone and the fluidic channel of the sorting zone have a depth h of between 2 micrometers and 10 micrometers, the microfluidic chip being capable of generating via the fluidic microchannel monodisperse microdrops having a determined volume of between 1 femtoliter and 200 femtoliters.

[0020] Thus, the microfluidic chip makes it possible to sequentially generate, one by one, microdrops of a central fluid monodispersed in a sheath fluid at a stable production frequency greater than 10 kilohertz, for example 60 kHz. The microdrops have a diameter determined by the dimensions and geometry of the central fluid and sheath fluid inlet nozzles, and of the outlet nozzle (i.e. the fluidic microchannel connecting the junction zone to the expansion zone), for example a diameter of between 4 and 5 micrometers. Thus, the microdrops are monodispersed, that is to say that all the microdrops have the same volume and this volume is predetermined. In addition, the monodispersed microdrops thus generated remain ordered in a single column in the expansion zone.

[0021] Other non-limiting and advantageous characteristics of the microfluidic chip according to the invention, taken individually or in all technically possible combinations, are the following: - the sorting zone comprises an active electrode arranged on one side of the fluid channel and two ground electrodes arranged on both sides of the fluid channel, the two ground electrodes framing the active electrode in the sorting zone around the fluid channel, the active electrode being at a minimum distance from the fluid channel of between 5 pm and 40 pm; - the sorting zone comprises two active electrodes and two ground electrodes, the two active electrodes being arranged on two opposite sides of the fluid channel, the two ground electrodes being arranged on two opposite sides of the fluid channel, each of the two ground electrodes framing one of the two active electrodes in the sorting zone around the fluid channel, each of the two active electrodes being at a minimum distance from the fluid channel of between 5 pm and 40 pm; - the microfluidic chip comprises at least one lateral channel fluidically connected to the inlet of the sorting zone, the at least one lateral channel being adapted to inject a flow of sheath fluid; - the microfluidic chip comprises at least one balancing microchannel fluidly connecting one of the output channels to another output channel, said at least one balancing microchannel having a depth equal to the shallow depth h; - the relaxation zone has at the outlet of the fluidic microchannel a reduced width r of between 5 and 15 micrometers and the relaxation zone widens downstream from the reduced width r to the greatest width u; - the relaxation zone is connected to the fluid channel of the sorting zone by a fluid channel having a depth equal to the shallow depth h and a width of approximately 20 pm, the width of the relaxation zone narrowing downstream from the greatest width u to the width of approximately 20 pm of the fluid channel; - the central fluid injection channel, the two sheath fluid injection channels and the at least two outlet channels have a depth equal to the shallow depth h; - the central fluid injection channel, the two sheath fluid injection channels and the at least two outlet channels have a depth H greater than the depth h of the junction zone, the depth H being between 6 micrometers and 20 micrometers; - the microfluidic chip comprises a device based on micro-pumps or syringe pumps adapted to inject the sheath fluid and respectively the central fluid into the microfluidic chip.

[0022] The invention also relates to a method for generating and sorting monodisperse microdroplets using a microfluidic chip according to the present disclosure.

[0023] Thus, the microfluidic chip of the present disclosure makes it possible to perform three functions on the same chip: creation of monodisperse drops of determined volume, between 1 fL and 200 fL, orderly propagation of the drops towards a measurement and sorting zone, the drops being arranged in a single column, then measurement and deflection of the individual drops according to the result of the measurement.The configuration of the microfluidic chip, in particular the dimensions of the junction zone, the relaxation zone and the sorting zone as well as the short distance between the microdrop production zone and the sorting zone make it possible both to generate monodisperse microdrops of low volume, between IfL and 200 fL, at high frequency (for example several tens of kHz) and to conduct them in a single ordered column from the generation zone to the sorting zone, then to actively and individually sort each microdrop, with high efficiency and with a high sorting frequency.

[0024] Thus the present disclosure presents the conditions for producing a train of drops of a known diameter, then the propagation of the drops while maintaining the alignment of these into a single column, oriented in the direction of flow of the sheath fluid, and at stable speed, then the individual sorting of the drops which appear sequentially at the measurement and sorting zone.

[0025] Of course, the various features, variants and embodiments of the invention may be combined with each other in various combinations provided that they are not incompatible or mutually exclusive. Brief description of the drawings

[0026] Furthermore, various other characteristics of the invention emerge from the appended description given with reference to the drawings which illustrate non-limiting forms of embodiment of the invention and where:

[0027] [Fig-1] is a schematic view of the fluidic circuit of a microfluidic chip according to an exemplary embodiment of the present disclosure;

[0028] [Fig.2] is an enlarged view of the central part of the microfluidic chip of the [Fig.l];

[0029] [Fig.3] is an enlarged view of the junction area and part of the area of relaxation of the microfluidic chip of figures 1 to 3;

[0030] [Fig.4] is an enlarged view of the junction area, the relaxation area and the area of sorting of the microfluidic chip in Figures 1 and 2, with arrows indicating the direction of the different flows;

[0031] [Fig.5]] is an enlarged top view of the sorting area of ​​the microfluidic chip figures 1 to 4;

[0032] [Fig.6] is a sectional view AA of the sorting area of ​​the illustrated microfluidic chip in [Fig.5];

[0033] [Fig.7] is an enlarged view of a junction area and a sorting area according to a three-channel output microfluidic chip variant;

[0034] [Fig.8] is an enlarged view of a microfluidic device in operation, showing the generation of a train of drops and its propagation in a single column in the relaxation zone.

[0035] It should be noted that in these figures the structural and / or functional elements common to the different variants may have the same references. Detailed description

[0036] In this document, fluid means a pure liquid or a mixture, or an emulsion.

[0037] The microfluidic chip 30 is generally planar in shape. The microfluidic chip 30 is manufactured in a plate. Different materials are suitable for the plate, such as glass or a polymer, for example polydimethylsiloxane (PDMS), having a thickness of a few millimeters per example of 5 mm. The microfluidic chip comprises a blade 31 forming a cover which is fixed for example by gluing or adhesion. The blade 31 is preferably transparent to allow the observation and detection of particles suspended in a fluid. For example, the blade 31 is a microscope slide to allow the observation of the microfluidic chip under an optical microscope objective. A microfluidic circuit comprising in particular microfluidic channels is formed on the face of the microfluidic chip 30 arranged opposite the blade 31.

[0038] We will describe in detail an example of a microfluidic chip in connection with figures 1 to 8.

[0039] In [Fig. 1], an example of a microfluidic chip is shown in a top view, for example through the blade 31. The microfluidic chip comprises an inlet 2 for sheath fluid and an inlet 4 for central fluid. The central fluid and the sheath fluid are immiscible with each other. The central fluid or sample fluid is for example an aqueous solution comprising in suspension micro- or nanoparticles to be analyzed. The sheath fluid is a fluid immiscible with the central fluid, for example an oil or a mixture of oil and surfactant. The inlet 2, respectively 4, is fluidically connected to an external reservoir of sheath fluid, respectively to an external reservoir of central fluid, the reservoirs (not shown) being equipped with micropumps or syringe pumps for injecting the sheath fluid and respectively the central fluid into the microfluidic chip.

[0040] Optionally, the inlet 2, respectively 4, comprises a filter consisting for example of channels of predetermined dimensions formed between micrometric pillars 43 arranged in concentric circles around an opening 41 passing through the microfluidic chip towards the corresponding reservoir. Advantageously, other micrometric spacer pillars 42 are for example arranged around the opening 41, to maintain the depth of the spaces where the fluid circulates between the opening 41 and the channels between the pillars 43. The micrometric pillars 42 and 43 are part of the plate of the microfluidic chip and are generally fixed at their other end to the blade 31 forming a cover. Such a filter makes it possible to filter the sheath fluid and respectively the central fluid during their injection into the microfluidic chip, as illustrated schematically in Figures 1-2.

[0041] The inlet 2 of the sheath fluid is connected to two sheath fluid injection channels 1 by a T-junction with one inlet and two outlets. The inlet 4 of the central fluid is connected to a central fluid injection channel 3. Optionally, the central fluid injection channel 3 has a meandering portion 5, for example in the shape of an S, which makes it possible to stabilize the production of microdrops.

[0042] The microfluidic chip comprises a junction zone 7 between the two sheath fluid injection channels 1 and the central fluid injection channel 3, illustrated in particular in [Fig.6]. The two sheath fluid injection channels 1 conduct the sheath fluid to the junction zone 7 and the central fluid injection channel 3 conducts the central fluid to the junction zone 7.

[0043] The fluidic junction zone 7 here comprises three inlet nozzles and one outlet nozzle. More specifically, the junction zone 7 comprises a central nozzle 21 fluidly connected to the central fluid injection channel 3 and two lateral nozzles 22, 23 arranged on two opposite sides of the junction zone 7, each of the two lateral nozzles 22, 23 being fluidly connected to one of the two sheath fluid injection channels 1. The outlet nozzle of the junction zone is formed by a fluidic microchannel 24 arranged on an opposite face of the junction zone 7 relative to the central nozzle 21. 28 denotes a longitudinal direction of the central nozzle 21. Advantageously, the lateral nozzles 22, 23 are arranged transversely to the longitudinal direction 28 and the fluidic microchannel 24 is in the axis of the longitudinal direction 28. The microdrop production zone is located in the fluidic microchannel 24.

[0044] Advantageously, as illustrated in [Fig. 4], the two sheath fluid injection channels 1 comprise filters 6 consisting of pads distributed over the paths of the sheath fluid upstream of the junction zone 7. Optionally, a similar filter is arranged on the central fluid injection channel 3 upstream of the junction zone 7. These filters 6 serve to retain any undesirable particles of dimensions greater than the width of the nozzles 21, 22, 23. Such undesirable particles are likely to block the nozzles 21, 22, 23 and prevent the formation of microdrops.

[0045] According to the present disclosure, the inlets and the outlet of the junction zone 7 have particular micrometric dimensions. In the present document, the depth of a microfluidic channel is understood to mean a dimension taken perpendicular to the plane of Figures 1 to 5. The length of an element of the microfluidic chip, for example the length of a microchannel, is understood to mean a dimension taken along the longitudinal axis of this element, i.e. in the direction of flow of the fluid in this microchannel, in the plane of Figures 1 to 5 and 7-8. The width of an element of the microfluidic chip, for example the width of a microchannel, is understood to mean a dimension taken transversely to the longitudinal axis of this element, i.e. perpendicular to the direction of flow of the fluid in this microchannel, in the plane of Figures 1 to 5 and 7-8.

[0046] The central nozzle 21 fluidically connects the central fluid injection channel 3 to the junction zone 7 by progressively reducing its width. At the connection end of the junction zone 7, the central nozzle 21 has a smaller width x of between 4 and 10 micrometers. The central nozzle 21 here has, for example a length t less than or equal to 7 micrometers. Each of the two lateral nozzles 22, 23 fluidically connects one of the two sheath fluid injection channels 1 to the junction zone 7 by progressively reducing their width. At the connection end of the junction zone 7, each of the two lateral nozzles 22, 23 has a smaller width xl of between 4 and 10 micrometers. Each of the two lateral nozzles 22, 23 here has, for example, a length S less than or equal to 7 micrometers. Advantageously, the two lateral nozzles 22, 23 are symmetrical with respect to the longitudinal direction 28. The fluidic microchannel 24 has a width y of between 2 and 7 micrometers and a length z of between 2 and 10 micrometers

[0047] The fluidic microchannel 24 connects the outlet of the junction zone 7 to an expansion zone 8. The expansion zone 8 has a flared shape from the outlet of the fluidic microchannel 24 towards the downstream of the microfluidic chip. The expansion zone 8 has a reduced width r of between 5 and 15 micrometers at the junction with the fluidic microchannel 24. The expansion zone 8 widens downstream to a greater width u of between 20 and 75 micrometers. The expansion zone 8 has a length v of between 100 and 600 micrometers. For example, as illustrated in [Fig. 3], the expansion zone 8 comprises a chamfer 38 adapted to linearly widen the expansion zone 8 from the reduced width r to the greater width u.

[0048] Advantageously, the relaxation zone 8 has a shape that narrows towards the downstream of the microfluidic chip so as to pass the microdrops one by one into the measurement and sorting zone, hereinafter called the sorting zone 18. A fluidic channel 25 connects the outlet of the relaxation zone 8 to the sorting zone 18. The fluidic channel 25 has a width of 20 μm and a length of, for example, 300 μm. The sorting zone 18 comprises a fluidic channel 27 forming the inlet of a junction with at least two outlet channels 13, 14. The fluidic channel 27 is connected downstream of the fluidic channel 25. At the outlet of the sorting zone 18, the outlet channel 13 makes it possible, for example, to keep the selected drops while the outlet channel 14 serves as a bin to collect the unselected drops. Output channels 13, 14 have for example a width of 50pm.

[0049] According to a particular and advantageous embodiment, the microfluidic chip has channels having a depth h, also called shallow depth, uniform over the entire microfluidic chip. The depth h is between 2 micrometers and 10 micrometers. Thus, the junction zone 7, the central nozzle 21, the lateral nozzles 22, 23, the fluidic microchannel 24, the expansion zone 8, the fluidic channel 25 and the fluidic channels in the sorting zone 18 have a depth equal to the depth h. The inlet 4 and the central fluid injection channel 3 have a depth equal to the depth h. Similarly, the lateral channel has a depth equal to the shallow depth h.

[0050] According to another particular and advantageous embodiment, the microfluidic chip comprises two channel depths: a large depth H and a small depth h, such that h <H. La faible profondeur h est comprise entre 2 micromètres et 10 micromètres. La grande profondeur H est par exemple comprise entre 6 micromètres et 20 micromètres. Plus précisément dans ce cas, la zone de jonction 7, la buse centrale 21, les buses latérales 22, 23, le microcanal fluidique 24 et la zone de détente 8 ont une profondeur égale à la faible profondeur h. Le canal fluidique 25 et les canaux fluidiques dans la zone de tri 18 ont aussi une profondeur égale à la faible profondeur h. Au contraire, l’entrée 2 du fluide de gaine et les deux canaux d’injection de fluide de gaine 1 ont une profondeur égale à la grande profondeur H. De manière analogue, en sortie de la zone de tri 18, les canaux de sortie 13, 14 ont une profondeur égale à la grande profondeur H.Advantageously, but not necessarily, the inlet 4 and the central fluid injection channel 3 have a depth equal to the shallow depth h. Alternatively, the inlet 4 and the central fluid injection channel 3 have a depth equal to the deep depth H.

[0051] In all embodiments, the junction zone 7 and the fluidic microchannel 24 where the microdrops are produced have a depth h which makes it possible to limit the diameter of the microdrops and therefore their volume. This low depth h is maintained from the junction zone 7 throughout the drop propagation zone, in particular in the expansion zone 8, the fluidic channel 25 and in the sorting zone 18. Maintaining the low depth h continuously over the entire path of the microdrops from their generation to sorting makes it possible to ensure that the sequencing of the drops is maintained in a single ordered column in the direction of flow of the sheath fluid (see for example [Fig. 8]). At the outlet of the fluidic microchannel 24, the generated microdrops 32 form a single column which is entrained by the sheath fluid.Non-intuitively, although the width of the relaxation zone 8 is greater than the diameter of the microdroplets 32 by approximately one order of magnitude, the microdroplets 32 remain ordered in a single column from the outlet of the microchannel 24 to the measuring and sorting zone.

[0052] For example, a reduced depth value h of 4.5 pm contributes to the formation of microdrops having a diameter less than or equal to the reduced depth. However, the movement of fluids in shallow areas is only possible by means of a strong pressure gradient between upstream and downstream. In particular in the junction zone 7 and the fluidic microchannel 24 where the microdrops are produced, the channels are the narrowest and shallowest. In one embodiment, some channels are deeper (H=10 pm) in areas that are not critical to the production, orderly propagation, or detection of microdrops. Consider a fluidic channel of rectangular cross-section and a given fluid flow rate, the pressure drop is inversely proportional to the cube of the depth of the fluidic channel, for the same channel width. Thus, a fluidic channel with a depth H equal to 10 qm requires an upstream pressure ~10 times lower than a channel with a reduced depth of 4.5 qm and the same width. The fabrication, on the same microfluidic chip, of channels with a large depth H makes it possible to reduce the mechanical constraints on the microfluidic chip wherever the channel depth is not a critical parameter for the formation, ordered propagation or detection of microdrops.

[0053] In all embodiments, the geometry of the outlet nozzle, i.e. the fluidic microchannel 24, makes it possible to generate drops in a controlled manner, in a predictable manner. The volume of the drops is determined by the geometry of the junction 7, and in particular of the fluidic microchannel 24 forming the outlet nozzle, and does not depend on other factors, in particular is practically independent of the pressures of the sheath fluid and the central fluid. The frequency of production of the drops can be controlled by varying the sample fluid flow rate: by conservation of mass, the flow rate of the sample fluid at the inlet, denoted Q, is equal to the product of the volume of the drops V by the production frequency f: Q=V . f. The volume of the drops being constant, the variation in the flow rate of the sample fluid results in a variation in the frequency of production of the drops.

[0054] An important aspect of the present disclosure is related to the configuration and operation of the expansion zone 8 which is coupled to the fluidic microchannel 24, i.e. the outlet nozzle of the junction zone 7. Experimentally, according to the present disclosure, the expansion zone 8 has a defined geometry that is neither too long nor too short, to maintain a linear sequence of drops 42. As indicated above, the expansion zone 8 has a depth equal to the shallow depth h. This expansion zone 8 makes it possible to maintain the drops 42 produced in a single column, in other words makes it possible to maintain the sequencing of the drops. The configuration and operation of this expansion zone 8 are totally counter-intuitive for those skilled in the art and fluidic theory cannot predict why this zone is so important.On the contrary, in the prior art, most microfluidic drop production devices generate sets of drops in which the drops either mix or are distributed in two or more parallel rows, probably due to the phenomena of variation in speed between the drops and the sheath fluid.

[0055] By way of non-limiting example, the microfluidic chip 30 is manufactured by molding on a silicon wafer previously structured in relief by micro-engraving techniques. A first layer of photosensitive resin of low thickness h (for example of thickness h equal to 4.5 μm) is deposited on an initially flat silicon wafer, for example by centrifugal coating (or “spin coating” in English terminology). This first layer is exposed by photolithography with a first mask corresponding to the channels or microfluidic elements of shallow depth h. Once this layer is developed, the positive shape (relief) of the future channels or microfluidic elements of shallow depth h remains.

[0056] In the case where certain channels have a greater depth, a second thicker layer of resin, of thickness H (for example of thickness H equal to 10 qm) is deposited over the first layer, in a similar manner. The second layer is exposed by photolithography with a second mask corresponding to the channels or microfluidic elements of great depth H then developed to reveal in positive the channels or microfluidic elements of great depth H. The second mask is aligned with respect to the first layer of resin so that the channels of low depth h (4.5 qm) and the channels of great depth H (10 qm) communicate fluidly, in particular at the nozzles 22, 23.

[0057] The micro-structured silicon mold is used to deposit the PDMS therein, for example by casting. The fabrication of the microfluidic chip is based on photolithography and micro-etching techniques with critical dimensions greater than one micrometer, the smallest depth being greater than or equal to 2 micrometers and the smallest width being greater than or equal to 2 micrometers. The fabrication of the microfluidic chip does not require the implementation of nanophotolithography and nano-etching technologies, which are technically more demanding and much more expensive.

[0058] We will now explain the operation of the microfluidic chip thus formed for the generation of monodisperse microdroplets. The microfluidic chip is attached to the blade 31 forming one side of the microfluidic channels. The central fluid is injected through the central fluid inlet 4 and flows in the central fluid injection channel 3 towards the junction zone 7. The sheath fluid is injected through the inlet 2 and flows in the two sheath fluid injection channels 1 towards the junction zone 7. The central fluid enters the junction zone 7 through the central nozzle 21 while the sheath fluid enters the junction zone 7 simultaneously through the two side nozzles 22, 23. The junction zone 7 thus forms a constriction of the central fluid and the sheath fluid.At the intersection of the junction zone, in the microdrop formation zone 29, the sheath fluid pinches the central fluid which splits into microdrops 32 in the fluidic microchannel 24. Microdrops 32 of central fluid are thus generated one by one within the sheath fluid. At the outlet of the fluidic microchannel 24, these microdrops 32 are carried by the sheath fluid into the expansion zone 8 while being transported in an orderly manner. More precisely, in the expansion zone 8, from the outlet of the microchannel. 24, the microdrops 32 are aligned one behind the other, in a single column, oriented in the direction of flow of the sheath fluid (see [Fig.8]).

[0059] Table I summarizes the ranges of values ​​of the different dimension parameters of the junction zone and the ranges of depth values ​​of the microchannels, as well as all of these values ​​for an exemplary embodiment having two channel depths. The combination of the values ​​of these different parameters makes it possible to obtain the formation of monodisperse microdrops of determined diameter and their propagation in a single ordered file.

[0060] [Tables 1] Parameter Min value (pm) Max value (pm) Example value (pm) x 2 10 5 y 2 7 5 Z 2 10 7 xl 3 10 5 U 20 75 50 V 100 600 300 - 400 t 0 7 5 S 0 7 5 r 5 15 7 h 2 10 4.5 H 6 20 10

[0061] Although these parameters are not independent of each other, it is possible to associate certain parameters with the microdrop formation regime or with the dimensional properties of the microdrops thus obtained. According to the present disclosure, the smallest width x and the length t of the central nozzle 21 allow the central nozzle to form a constriction of the central fluid. The smallest width xl of each of the two lateral nozzles 22, 23 is also a critical parameter for the formation of microdrops. The ranges of values ​​of the parameters x and xl of the junction zone 7 allow the formation of drops having a determined volume at the outlet of the fluidic microchannel 24 and with a stable production of monodisperse drops. The smallest width xl and the length S of each of the two lateral nozzles 22, 23 allow the formation of a constriction zone of the sheath fluid.The shallow depth h, the width y and the length z of the fluidic microchannel 24 determine the diameter of the microdrops 32. Here we have determined a set of critical parameters for the . formation of microdrops and which influence the value of the diameter of the microdrop 32.

[0062] The greatest width u of the relaxation zone 8 determines the fluidic regime allowing the formation and the regular and linear flow of the microdrops on a single ordered column. The length V of the relaxation zone 8 makes it possible to stabilize the train of microdrops by maintaining it on a single ordered column. The reduced width r of the relaxation zone 8 allows the easy manufacture by photolithography of the relaxation zone 8 which extends over a greater width u and the length V and also contributes to the stabilization of the flow of the drops.

[0063] The values ​​of the example indicated in Table I make it possible to generate microdrops having a stable diameter of approximately 4.5 μm, i.e. a volume of approximately 45 fL. Remarkably, the microdrops 32 are monodisperse and all have the same diameter approximately equal to the small depth h of 4.5 μm. In addition, the microdrops 32 are produced at a frequency of between 10,000 drops / sec and 60,000 drops / sec. This geometry not only makes it possible to control the volume of the drops in a stable manner, but also to keep the volume constant over a wide range of flow rates of the sheath fluids and sample fluids. When the flow rates are modified, the frequency of production of the drops is modified, but their volume remains constant. This is particularly important to allow a good prediction of the number of particles contained in each drop as indicated above.

[0064] More precisely, the width y and the length z of the fluidic microchannel 24 influence the diameter of the microdrops 32. For example, the small depth h being equal to 4.5 pm, the smallest width x being equal to 5 pm and the width y being equal to 4 pm, microdrops 32 having a diameter of between 4 and 5 micrometers, for example 4.5 pm, are obtained. The depth of the junction zone 7, of the fluidic microchannel 24 and of the expansion zone 8 being in this example 4.5 pm, the microdrops 32 are generally of quasi-spherical shape. The proportional modification of the values ​​of the length z, of the width y and of the depth h makes it possible to produce microdrops of different diameters. Advantageously, the values ​​of x, y and h are equal: x=y=h. For example, with y=x=h = 3pm we obtain drops of diameter 3pm and volume approximately 13fl, or with y=x=h= 6pm we obtain drops of volume 100fl.The microfluidic chip makes it possible to generate monodisperse microdroplets of a determined volume, between 1 fL and 200 fL, for example between 10 fL and 100 fL, preferably between 20 fL and 50 fL, and even more preferably between 30 fL and 40 fL. Respecting this relationship between y, x and h (with certain tolerances, of the order of 20% to 30%) ensures the stability of the downstream flow.

[0065] If the length z of the fluidic microchannel 24 is too small, the drops generated have a poorly controlled diameter, most often too large and fluctuating, or much smaller and irregular: we then obtain polydisperse microdrops and not monodisperse ones. If the length z of the fluidic microchannel 24 is too large, the generated drops have too large a volume which leads to instabilities in the downstream flow and errors in the number of particles per drop.

[0066] Certain dimensional parameters of the microfluidic chip are adjusted to allow easy manufacturing by photolithography and molding techniques. The length t of the central nozzle 21 is preferably 5 μm. If the length t of the central nozzle 21 is too small, the central nozzle 21 risks being poorly formed during manufacturing. Advantageously, the length t is greater than or equal to the smallest width x of the central nozzle 21. If the length t of the central nozzle 21 is too large, the central nozzle 21 has a high hydrodynamic resistance likely to slow the advance of the central fluid. To compensate for such resistance, it would be necessary to increase the pressure of the central fluid, which risks inducing damage to the microfluidic chip, for example detachment between the PDMS plate and the glass slide by delamination. The length t is here less than or equal to 7 μm.

[0067] The same constraints as for the length t of the central nozzle 21 apply to the length S of the two lateral nozzles 22, 23.

[0068] The expansion zone 8 is a zone where the linear velocity of the fluids is reduced, which makes it possible to stabilize the formation of the drops and prevents the grouping of the drops, either chaotically or in several columns. Throughout the expansion zone, the drops are accelerated by viscosity effect with the sheath fluid, for example oil, until they reach a velocity almost equal to that of the oil. The values ​​of the width u and the length V of the expansion chamber are thus relatively tolerant, but have limits beyond which the flow is no longer stable in a single column. These maximum values ​​are respectively 50 pm and 500 pm for example. Once the flow has stabilized in the expansion zone 8, another chamfer 39 allows a transition to the fluidic channel 25 and the fluidic channel 27 of the sorting zone 18 of smaller width (for example 20 pm).This reduced width of the fluidic channel 27 in the sorting zone 18 allows the positioning of the electrodes 10, 11 at a short distance from the row of drops 32, which increases the dielectrophoresis effect used for sorting.

[0069] The expansion zone 8 advantageously has a chamfer 38 at the inlet (see [Fig. 3]) which makes it possible to widen the expansion zone from the reduced width r to the greater width u. Advantageously, the expansion zone 8 also has another chamfer 39 towards the downstream (see [Fig. 4]) which makes it possible to reduce the expansion zone 8 from the greater width u to the width of the fluid channel 25 at the outlet of the expansion zone. 8. When using the microfluidic chip, this chamfer(s) promotes fluid flow.

[0070] The microdrops 32 can be detected individually, for example, by a fluorescence detection system by directing an excitation laser beam toward the fluidic channel 25 at the outlet of the expansion zone 8 or toward the fluidic channel 27 in the sorting zone 18. At the outlet of the zone 8, the narrowing causes an acceleration of the flow by conservation of the flow rate; the drops 32 having a stable diameter, this acceleration has the effect of spacing the drops, which are better separated for analyzing and sorting them individually. The lateral channel 9 provides an additional flow of sheath fluid, which makes it possible to space the drops even further. Particularly advantageously, the detection is carried out in the fluidic channel 25 or 27 of shallow depth h. For example, an excitation laser beam is directed in a plane transverse to the plane of the microfluidic chip and a fluorescence signal is detected in this same transverse plane.The shallow depth h is approximately equal to the diameter of the microdrops, which makes it possible to maintain the order of the drops. In addition, the shallow depth h also makes it possible to limit the contribution of the sheath fluid to the detected signal. Following the detection of a microdrop of interest comprising a fluorescent marker, this microdrop can be selectively extracted from the flow in the sorting zone 18 to be directed towards a specific collection channel. The detection system generates a signal which triggers the sorting of the microdrop thus detected. Different sorting devices and methods can be used, for example based on dielectrophoresis, as in the example detailed below.

[0071] Particularly advantageously, the microfluidic chip also comprises a sorting zone 18 integrated on the same support plate. The sorting zone 18 is arranged downstream of the expansion zone 8. The fluidic channel 25 connects, for example, the expansion zone 8 to the sorting zone 18. The sorting zone 18 comprises a fluidic channel 27 fluidly connected at the inlet to the expansion zone 8 via the fluidic channel 25 and at the outlet to at least two outlet channels 13, 14, for example via a Y junction denoted 12, the one common branch of which is the fluidic channel 27. The fluidic channel 25, the fluidic channel 27 and the Y junction towards the two outlet channels have a depth equal to the shallow depth h. In an exemplary embodiment, the two output channels 13, 14 have a depth equal to the large depth H. Alternatively, the two output channels 13, 14 have a depth equal to the small depth h.

[0072] Optionally, a side channel 9 is fluidically connected to the inlet of the sorting zone 18 via a nozzle 26. The side channel 9 conducts a flow of injected sheath fluid via an inlet 19 (see [Fig.l]). The side channel 9 is adapted to inject a flow of sheath fluid onto one side of the fluidic channel 27 at the inlet of the sorting zone 18. In an exemplary embodiment, the side channel 9 has a depth equal to the large depth H. In alternatively, the side channel 9 has a depth equal to the shallow depth h. The nozzle 26 has a depth equal to the shallow depth h.

[0073] Advantageously, as illustrated in [Fig. 4], the lateral channel 9 comprises a filter 6 consisting of studs distributed over the paths of the sheath fluid upstream of the nozzle 26. As indicated above, such a filter makes it possible to retain any undesirable particles of dimensions greater than the width of the nozzle 26 which are likely to block the nozzle 26 and impair its operation.

[0074] The lateral channel 9 channel makes it possible to supply additional sheath fluid to the already formed train of microdrops. The lateral channel 9 channel makes it possible, on the one hand, to modify the spacing between the microdrops. Indeed, at the outlet of the expansion zone 8, the microdrops 32 can be relatively close to each other. This makes sorting very difficult, since it requires great precision of action to sort a microdrop without influencing the adjacent microdrops. The addition of sheath fluid via the lateral channel 9 makes it possible to increase the spacing between microdrops to values ​​of several microdrop diameters, which makes it possible to increase the selectivity of the sorting.

[0075] On the other hand, the lateral channel 9 has the effect of laterally diverting the flow of microdrops, which is oriented towards one of the two default outlet channels, for example the outlet channel 14 which corresponds for example to a bin collecting the unselected microdrops. The sorting consists of actively extracting a microdrop selected individually according to a detected signal to orient it towards the other outlet channel 13, the unselected microdrops being passively oriented towards the bin outlet.

[0076] To a lesser extent, the flow rate of sheath fluid in the lateral channel 9 also influences the production frequency by modifying the hydrodynamic resistance of the fluids circulating in the expansion zone 8.

[0077] Advantageously, the sorting zone 18 is a dielectrophoresis sorting zone. For this purpose, the microfluidic chip comprises electrodes 10, 11 arranged on either side of the fluidic channel 27 in the sorting zone 18 (see FIGS. 2 and 4). The channels of the electrodes are formed in an identical manner to the fluidic channels with inlets at their ends. These channels are then filled with a conductive material which may be indium injected hot, for example at approximately 80°C. Other materials may be considered for manufacturing the electrodes, such as ionic liquid, charged resin or conductive gel.

[0078] More specifically, an active electrode 10 is arranged on one side of the fluid channel 27, a ground electrode 11 is arranged on the same side as the active electrode 10 and another ground electrode 11 is arranged on an opposite side of the fluid channel 27. The active electrode 10 has a U shape with the base of the U closest to the fluid channel 27. The two ground electrodes 11 are advantageously shaped symmetrical with respect to the longitudinal direction of the fluid channel 27 of the sorting zone 18. Each ground electrode has an M shape, the tips of the M being arranged as close as possible to the fluid channel 27. The U-shaped active electrode 10 is arranged between the two tips of the M-shaped ground electrode 11. The minimum distance between each of the electrodes 10, 11 and the fluid channel 27 is between 5 μm and 40 μm, for example here about 10 μm. The active electrode 10 is at the minimum distance from the fluid channel 27 approximately in the middle of the sorting zone 18 in the direction of fluid flow. Each of the two ground electrodes 11 is at the minimum distance from the fluid channel 27 towards the inlet of the sorting zone 18, for example just downstream of the nozzle 26 of the lateral channel 9.Each of the two ground electrodes 11 is also at the minimum distance from the fluidic channel 27 towards the outlet of the sorting zone 18, upstream of the junction 12 between the two outlet channels 13, 14. On the other hand, in the middle of the sorting zone 18, the two ground electrodes 11 move away from the fluidic channel 27 so as to avoid interfering with the active electrode 10. In this way, the two ground electrodes 11 surround the active electrode 10 in the plane of the microfluidic device and make it possible to limit the spatial extension of the electric field gradient towards the upstream and downstream of the sorting zone 18. The active electrode 10 is framed by the fluidic channel 27 and by one of the ground electrodes 11.

[0079] The arrangement of the electrodes 10, 11 makes it possible both to maximize the electric field gradient on the central zone of the fluidic channel 27 in the middle of the sorting zone 18, and to spatially confine the electric field gradient to the central zone in which the microdrop to be sorted is located. The two ground electrodes 11 make it possible to limit the spatial extension of the electric field gradient upstream of the sorting zone 18, where this field gradient would be likely to influence the next microdrop. The two ground electrodes 11 make it possible to limit the spatial extension of the electric field gradient downstream of the sorting zone 18, where this field gradient would be likely to affect the already sorted microdrops which are located in a wider and deeper zone and thus avoid the merging of several already sorted microdrops.

[0080] The arrangement of the electrodes 10, 11 allows greater efficiency of the electric field, in particular because the fluidic channel 27 has a reduced width, for example a width of 20 pm, compared to the relaxation zone 8. Indeed, the dielectrophoresis effect is all the more effective and spatially constrained as the drops 32 are close to the electrodes 10, 11. Thus, the electrodes are placed as close as possible technologically to the fluidic channel 27. For example, the distance between the electrodes and the fluidic channel 27 is 10 pm, the channel has a width of 20 pm and the drops 32 circulate in the middle of the fluidic channel 27. The distance between the electrodes 10, 11 and the drops 32 is in this example approximately 20 pm.

[0081] The sorting method comprises the application of a high-voltage electrical pulse, for example between 500 V and 1000 V peak (or 1000 V and 2000 V peak to peak for alternating signals), and of short duration T to the active electrode 10. The duration T is for example between 10ps and 200ps; it may be a positive pulse, a negative pulse, a sinusoid or square period or any waveform with zero average, or several periods of sinusoids, squares or waves with zero average. This pulse may be of direct voltage. Preferably, this pulse is of alternating voltage, at an electrical frequency f chosen, on the one hand, so that the duration of the pulse T is equal to one or more periods of the alternating frequency 1 / f, so as to avoid polarizing the contents of the fluidic channel 27 in the sorting zone 18.And, on the other hand, the duration of the electrical pulse is chosen to be shorter than, or equal to or slightly greater than, the time between the passage of two successive microdrops 32 in the sorting zone 18, in order to ensure the extraction of a single microdrop 32. The electrical frequency range f is for example between 20 kHz and 200 kHz and the duration range T between 100s and 50ps to allow sorting for a drop passage frequency of between 10 kHz and 50 kHz.

[0082] The electrodes 10, 11 create a spatially variable electric field (field gradient), which acts on a microdrop by means of a force generated by a dielectrophoresis effect. The application of an electrical pulse to a microdrop deflects the microdrop to selectively direct it towards one of the two outlet channels 13. On the contrary, in the absence of an electrical pulse, a microdrop is directed towards the other of the two outlet channels 14. The fractions sorted in the two outlet channels 13, 14 can be collected in separate collectors connected respectively to the outlets 16 and 17.

[0083] Alternatively, the microfluidic chip with two output channels 13, 14 may comprise two active electrodes 10 arranged symmetrically on either side of the fluidic channel 27, as illustrated in [Fig. 7]. For example, the two active electrodes 10 may be driven alternately: one of the electrodes being used to direct a microdrop 32 towards the outlet 14 (waste bin) and the other electrode 10 being used to direct another microdrop 32 towards the collection channel 13. In addition, the two active electrodes 10 may be driven simultaneously with different voltages, so as to reduce the voltage required to obtain the dielectrophoresis effect.

[0084] According to the present disclosure, continuously on the fluid path of the microdrops 32 from the microdrop formation zone 29 to the sorting zone 18 inclusive, all the channels and all the zones have a depth equal to the shallow depth h, which determines the diameter of the microdrops, of 4.5 qm in the example of Table I. The junction zone 7, including the inlet nozzles 21, 22, 23, the fluidic microchannel 24, the expansion zone 8, the fluidic channel 25, the nozzle 26 and the sorting zone 18, including the fluidic channel 27 and the Y-junction 12, have a depth equal to the shallow depth h. In a particular embodiment, the microfluidic chip comprises channels or zones having two distinct depths. In this embodiment, the sheath fluid injection channels 1 and 9, and the outlet channels 13, 14 are deeper (depth H ~10 qm in the same example), these zones not being critical to the production and detection and sorting of microdrops.

[0085] For example in [Fig.2], zones 3, 4, 5, 7 and 8 are the zones of shallow depth h ~4.5 qm. Between the connection of the central fluid injection channel 3 and the nozzle 21, the linear velocity of the fluid is determined by the volume flow rate divided by the channel section. Choosing a shallow channel depth at this location (4.5 qm) ensures a higher linear velocity, and thus prevents the particles transported by the central fluid from settling. On the other hand, from the nozzle 21 to the exit of the sorting zone 18, the low depth h makes it possible to maintain the volume of the microdrops and to guarantee that the microdrops produced and detected in a certain order arrive in the same order in the sorting zone 18. The other zones 1, 2, 9, 19, 13, 14, 16, 17, can have a great depth H of 10 qm, to make it possible to reduce the constraints on the flow of the fluid and therefore the necessary pressure.

[0086] Advantageously, the microfluidic chip comprises one or more balancing microchannels 15 fluidically connecting one of the two outlet channels 13 to the other of the two outlet channels 14. The microchannel(s) 15 make it possible to balance the pressure between the two outlet channels 14, which may have different flow rates, linked to different pressures, which may be detrimental to the sorting efficiency. The balancing microchannels 15 have a depth equal to the shallow depth h, of 4.5 μm for example, and a narrow width of the order of 5 μm. These dimensions make it possible to avoid the passage of sorted microdrops between the two outlet channels 14 via one of the balancing microchannels 15 (see Figures 5-6). As a non-limiting example, the microdrops are water-based and the sheath fluid is based on an oil used in microfluidics.The microfluidic chip is arranged horizontally, the shallow balancing microchannels 15 being oriented towards the bottom of the chip, so that the microdrops 32 lighter than the oil, rise quickly to the top of the outlet channel 13, or 14 of great depth H, for example equal to 10 qm. Thus, the microdrops 32 having a diameter of 4.5 qm have little risk of being captured by the balancing microchannels 15 located lower down.

[0087] [Fig.7] represents a microfluidic chip according to a variant of the chip microfluidics described in connection with figures 1 to 6. In this variant, the area of sorting zone 18 has three outlet channels. The outlet channel 14 is arranged in the center and corresponds to the trash channel. Two outlet channels 13, 131 or collection arms are arranged laterally, for example symmetrically with respect to the outlet channel 14, to allow sorting according to two distinct criteria. Advantageously, two lateral channels 9 are fluidically connected via a nozzle 26 to the inlet of the sorting zone 18, on two opposite sides of the fluidic channel 27. Each lateral channel 9 conducts a flow of sheath fluid. Each lateral channel 9 is adapted to inject a flow of sheath fluid onto one side of the fluidic channel 27 of the sorting zone 18. In one embodiment, each lateral channel 9 has a depth equal to the large depth H. In addition, the electrode system here comprises two active electrodes 10, 110 and two ground electrodes 11.A U-shaped active electrode 10 or 110 and an M-shaped ground electrode 11 are arranged on each side of the fluid channel 27, symmetrically with respect to the longitudinal direction of the fluid channel 27. Each of the two active electrodes 10, 110 is at a minimum distance from the fluid channel 27 of between 5 pm and 40 pm, for example approximately 10 pm. Preferably, the tips of the M of each ground electrode 11 are arranged as close as possible to the fluid channel 27, for example at a minimum distance of between 5 pm and 40 pm, for example approximately 10 pm. Each active electrode 10, 110 is framed by the fluidic channel 27 and by one of the ground electrodes 11. In the absence of electrical voltage on the active electrodes 10 and 110, all the drops 32 are sent into the outlet channel 14.The application of an electrical pulse to one of the two electrodes 10 or 110 has the effect of deflecting a drop 32 propagating in the sorting zone 18 between the two electrodes 10 and 110 towards one of the two output channels 13 or 131.

[0088] The configuration illustrated in [Fig.7] has several advantages. First of all, it allows for greater symmetry of the flow which improves stability. Indeed, the splitting of the lateral channel 9 into two lateral channels located on either side of the central fluidic channel 27 has the effect of making the supply of sheath fluid symmetrical, which is then used only to control the spacing of the drops 32. Finally, an electrode 10, 110 is provided for selectively directing the drops towards one or other of the outlet arms 13, 131. This configuration makes it possible to separate and collect two different populations of sorted drops in two different collection arms 13 and 131 respectively. In contrast, the device of [Fig.4] comprises a single active electrode for directing the selected drops towards a single collection arm 13.

[0089] In summary, the droplet creation zone of the microfluidic chip comprises a fluidic microchannel 24 at the outlet of the junction zone 7, the fluidic microchannel 24 having dimensions such that y~h approximately, and z > y and h. The constriction zone of the central fluid (for example an aqueous fluid) in the fluid flow of sheath (for example an oily fluid) are such that x~xl~y. The expansion zone 8 has a width u of approximately 50 pm and a length V of approximately 500 pm. The outlet of the expansion zone 8 connected to the fluidic channel 25 has a narrowing to the width of approximately 20 pm in the measurement and sorting zone 18. In a particular embodiment, the microfluidic chip comprises channels and zones having two distinct depths: a low depth h, continuously on the fluidic path of the microdrops 32 from the microdrop formation zone 29 up to and including the sorting zone 18, and a large depth H outside the zones critical to the production, detection and sorting of microdrops, such as in particular the sheath fluid injection channels and the outlet channels. In the sorting zone 18, the active electrodes 10, 110 are arranged as close as possible to the fluid channel 27, for example at a distance of approximately 10 qm.The use of ground electrodes 11 on either side of the active electrodes 10, 110 makes it possible to limit the spatial extension of the electric field in the longitudinal direction of the fluid channel 27 in the sorting zone 18.

[0090] The microfluidic device and method of the present disclosure make it possible to select and sort the drops which contain a nanoparticle detected for example by fluorometry, and to direct only the drops containing the type of nanoparticle sought towards a determined outlet. The microfluidic device and method make it possible to collect at this outlet only the drops containing the type of nanoparticle sought, excluding empty drops and drops containing other types of nanoparticles. The microfluidic device and method thus make it possible to concentrate the nanoparticles sought in the collected fluid.

[0091] The integration on the same microfluidic chip of a monodisperse microdrop production zone, a relaxation zone, a detection and sorting zone, at a short distance from each other makes it possible both to generate monodisperse microdrops of low volume, between IfL and 200 fL, at high frequency (for example several tens of kHz) and to actively and individually sort each microdrop, with high efficiency and also with a high sorting frequency. The relaxation zone makes it possible to maintain the drops ordered in a single column up to the detection and sorting zone. Such a configuration makes it possible to avoid intermediate storage and transport of microdrops from a microdrop generation device to another sorting device. This configuration makes it possible to prevent the microdrops from sticking together or to the wall of a storage container.Overall, microdrop generation and sorting are both faster and more reliable.

[0092] The present disclosure finds applications for the generation of monodisperse microdroplets of volume between 1 fL and 200 fL containing chemical or biological particles of nanometric dimensions. In addition, the present The disclosure finds applications in the dilution-free sorting of microdroplets thus generated, via the detection of a signal associated with a property of the chemical or biological particle of nanometric dimensions, and the triggering of the sorting according to the detected signal. In particular, the present disclosure finds applications for the generation and sorting of monodisperse microdroplets containing nanoobjects with a concentration as high as 109 to 1010 nano-objects per milliliter, such as for example extracellular vesicles or cellular secretions, in particular exosomes, but also viral vectors, or even large individual molecules. The disclosed microfluidic chip allows the encapsulation of fluorescent nanoparticles and their sorting according to the measured fluorescence. The disclosed microfluidic chip also allows the measurement and sorting of fluorescently labeled organic particles, by encapsulating them in drops.The present disclosure finds further applications in chemistry, for example for the purification of nanometric probes or in the granulometry of nanometric objects. In addition, the microfluidic chip integrates the functions of generation and sorting of drops without intermediate storage, which allows not only the generation of drops at high frequency but also the individual sorting of drops, on the fly, immediately after their generation, and at high frequency, for example from a few kHz to more than 100 kHz.

Claims

1. Claims Microfluidic chip (30) for generating and sorting monodispersed microdroplets (32) based on a central fluid in a sheath fluid, the central fluid and the sheath fluid being immiscible with each other, characterized in that: the microfluidic chip (30) comprises a central fluid injection channel (3), two sheath fluid injection channels (1) and in that the microfluidic chip (30) comprises a junction zone (7), an expansion zone (8) downstream of the junction zone (7) and a sorting zone (18) arranged downstream of the expansion zone (8), a fluidic microchannel (24) fluidically connecting the junction zone (7) to the expansion zone (8), the sorting zone (18) comprising a fluidic channel (27) fluidically connected at the inlet to the expansion zone (8) and downstream to at least two outlet channels (13, 131, 14), the junction zone (7) comprising a central nozzle (21) fluidically connected to the central fluid injection channel (3), two lateral nozzles (22, 23) arranged on two opposite sides of the junction zone (7), each of the two lateral nozzles (22, 23) being fluidically connected to one of the two sheath fluid injection channels (1),the fluidic microchannel (24) being arranged on an opposite face of the junction zone (7) relative to the central nozzle (21), the central nozzle (21) having a smaller width x of between 2 and 10 micrometers, each of the two lateral nozzles (22, 23) having a smaller width xl of between 3 and 10 micrometers, the fluidic microchannel (24) having a width y of between 2 and 7 micrometers and a length z of between 2 and 10 micrometers, the expansion zone (8) having a greater width u of between 20 and 75 micrometers and the expansion zone (8) having a length V of between 100 and 600 micrometers, and in that the junction zone (7), the fluidic microchannel (24), the expansion zone (8) and the fluidic channel (27) of the sorting zone (18) have a depth h of between 2 micrometers and 10 micrometers,the microfluidic chip being capable of generating via the fluidic microchannel (24) monodisperse microdrops (32) having a determined volume of between 1 femtoliter and 200 femtoliters, the monodisperse microdrops propagating in an orderly manner in a single column in the relaxation zone up to the sorting zone.,

2. Microfluidic chip according to claim 1 wherein the sorting zone (18) comprises an active electrode (10) arranged on one side of the fluidic channel (27) and two ground electrodes (11) arranged on both sides of the fluidic channel (27), the two ground electrodes (11) framing the active electrode (10) in the sorting zone (18) around the fluidic channel (27), the active electrode (10) being at a minimum distance from the fluidic channel (27) of between 5 pm and 40 pm.

3. Microfluidic chip according to claim 1 wherein the sorting zone (18) comprises two active electrodes (10, 110) and two ground electrodes (11), the two active electrodes (10, 110) being arranged on two opposite sides of the fluidic channel (27), the two ground electrodes (11) being arranged on two opposite sides of the fluidic channel (27), each of the two ground electrodes (11) framing one of the two active electrodes (10, 110) in the sorting zone (18) around the fluidic channel (27), each of the two active electrodes (10, 110) being at a minimum distance from the fluidic channel (27) of between 5 pm and 40 pm.

4. Microfluidic chip according to one of claims 1 to 3 comprising at least one lateral channel (9) fluidically connected to the inlet of the sorting zone (18), the at least one lateral channel (9) being adapted to inject a flow of sheath fluid.

5. Microfluidic chip according to one of claims 1 to 4 in which the expansion zone (8) has at the outlet of the fluidic microchannel (24) a reduced width r of between 5 and 15 micrometers and the expansion zone (8) widens downstream from the reduced width r to the greatest width u.

6. Microfluidic chip according to one of claims 1 to 5 in which the relaxation zone (8) is connected to the fluidic channel (27) of the sorting zone (18) by a fluidic channel (25) having a depth equal to the depth h and a width of approximately 20 pm, the width of the relaxation zone (8) narrowing downstream from the greatest width u to the width of approximately 20 pm of the fluidic channel (25).

7. Microfluidic chip according to one of claims 1 to 6 wherein the central fluid injection channel (3), the two sheath fluid injection channels (1) and the at least two outlet channels (13, 131, 14) have a depth equal to the depth h.

8. Microfluidic chip according to one of claims 1 to 6 in which the central fluid injection channel (3), the two channels sheath fluid injection (1) and the at least two outlet channels (13, 131, 14) have a depth H greater than the depth h of the junction zone (7), the depth H being between 6 micrometers and 20 micrometers.

9. Microfluidic chip according to one of claims 1 to 8 comprising a device based on micro-pumps or syringe pumps adapted to inject the sheath fluid and respectively the central fluid into the microfluidic chip.

10. Method for generating and sorting monodisperse microdrops by means of a microfluidic chip according to one of claims 1 to 9 comprising the following steps: - simultaneous injection of a central fluid into the central fluid injection channel (3) towards the junction zone (7) of the microfluidic chip and of a sheath fluid into the two sheath fluid injection channels towards the junction zone (7); - generation of monodisperse microdrops (32) of central fluid within the sheath fluid in the fluidic microchannel (24) fluidically connecting the junction zone (7) to the expansion zone (8) of the microfluidic chip, the microdrops having a determined volume, between 1 femtoliter and 200 femtoliters, - orderly propagation of microdrops towards a sorting area of ​​the microfluidic chip so as to actively and individually sort each microdrop.

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