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

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), an expansion zone (8) downstream of the junction zone (7), and a sorting zone (18) arranged downstream of the expansion 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 narrowest width x of between 2 and 10 micrometres, each of the two lateral nozzles (22, 23) having a narrowest width x1 of between 3 and 10 micrometres, a fluidic microchannel (24) having a width y of between 2 and 7 micrometres and a length z of between 2 and 10 micrometres, and an expansion zone (8) having a widest width u of between 20 and 75 micrometres and a length V of between 100 and 600 micrometres, the junction zone (7), the fluidic microchannel (24) and the expansion zone (8) having a depth h of between 2 and 10 μm.

Description

Microfluidic chip and method for generating and sorting high-frequency monodisperse microdroplets 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 (fL) and 200 fL.

[0002] It relates in particular to a microfluidic chip capable of generating monodisperse and ordered microdroplets of a defined volume at high frequency, i.e., 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 capable of actively generating and sorting each microdroplet individually from the train of monodisperse microdroplets, with the sorting being performed at high frequency, i.e., at a frequency greater than 10 kHz, for example, 60 kHz. State of the art

[0003] In the field described above, flow cytometers are known to be used to detect, count, and identify cells or micrometer-sized particles suspended in a flowing fluid by passing them, one by one and at high speed, through the beam of one or more lasers. Some sorting cytometers also allow for the sorting of these particles using various sorting methods. Advances in microscopy and biology have made it possible to study increasingly smaller objects, particularly nano-objects smaller than 100 nm. However, traditional flow cytometers generate droplets with diameters ranging from 30 micrometers (pm) to 300 pm, corresponding to volumes between 10 picoliters and 10 nanoliters, and can only sort objects larger than several hundred nanometers.

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

[0005] Indeed, studies involving objects of nanometric size (ranging from a few nanometers to a few tens or even a few hundred nanometers) require the ability to analyze these objects one by one. Given the extremely small size of these objects, it has proven practically advantageous to encapsulate them in fluid droplets, as these droplets are then easier to handle, analyze, and sort. When droplets are extracted from a liquid containing a certain concentration of these particles, Poisson's distribution allows us to calculate the probabilities that a given droplet contains N particles, where N is a positive integer or zero. This distribution is written as:

[0006] in which P(N) is the probability that a drop contains N particles, and A 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 we seek 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 10 9 and 10 1 ° particles per ml. According to the calculations above, spherical drops with a diameter of 30 pm to 300 pm (with a volume between 10 picoliters and 10 nanoliters) are suitable for samples with a concentration between 10 3 and 10 6particles per ml, which is completely insufficient for typical samples of biological nanoparticles. To achieve these concentrations, it would be necessary to dilute the biological samples excessively (for example, a typical 1 ml sample would have to be diluted to a minimum volume of 1 liter, or even 1000 liters). On the contrary, biologists want to concentrate such samples. For an average concentration of 5.10 9 The Poisson distribution allows us to determine the probabilities that a given drop contains 0, 1, or 2 particles per ml, depending on the drop's volume. For the number of drops containing a single particle to be calculated, the probability of a given drop containing 0, 1, or 2 particles is calculated. The particle concentration must be greater than or equal to 10%, while keeping the number of drops containing more than one particle less than or equal to 1%, with a drop volume between 30 fL and 40 fL. At this concentration of 5.10 9For droplets with a volume less than 30 fl oz (particles per ml), the percentage of empty droplets, i.e., those without any particles, is greater than 90%, which lengthens analysis times because all droplets must be analyzed even though most are empty. Conversely, for droplets with a volume greater than 40 fl oz (particles per ml), the percentage of droplets 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] Microdroplets of the sample fluid (i.e., the core fluid) can be generated by emulsification in a sheath fluid, provided the sample and sheath fluids are immiscible, for example, in a water-in-oil or oil-in-water mixture. Microdroplets can also be generated by focusing flow into a microchannel of a microfluidic chip, causing the sample fluid to split into microdroplets dispersed within the sheath fluid. The diameter or volume of the microdroplets generally depends on the size of the microchannels in the microfluidic chip and also varies according to numerous environmental parameters, such as pressure or flow rate variations in the injected fluids. However, hydrodynamic phenomena at the microscopic scale are complex to predict and model. Within a single microfluidic device, droplet generation is highly dependent on the injection conditions.Over time, variations in the volume of microdroplets are observed, which are difficult to control.

[0010] In this document, monodisperse microdroplets are defined as microdroplets all having the same volume + / - 20%, for example. In other words, the volume of monodisperse microdroplets is uniform. Monodisperse microdroplets are generally suspended in a stream of encapsulation fluid or sheath fluid.

[0011] We know of microfluidic devices capable of generating monodisperse microdroplets with a volume on the order of 10 pL, i.e., a diameter greater than 30 pm, which can operate at a generation frequency generally below 5 kHz and sometimes up to 30 kHz. However, the volume of these microdroplets is too large compared to the nano-objects that we wish 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 / s10404-011-0776-7" 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 droplets 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 droplets.

[0014] Microfluidic systems typically include one microfluidic chip for droplet production and another microfluidic chip for droplet sorting, with the droplets stored between production and sorting. This arrangement necessitates reinjecting the droplets into another chip, a particularly complex process that significantly increases both processing time and the risk of errors or other difficulties such as droplet melting, leakage of droplet fluid into the sheath fluid, handling difficulties, and the requirement for relatively large droplet volumes.

[0015] There is a need for a device and method to isolate, detect, analyze and sort individually objects smaller than a micrometer, which are for example encapsulated in microdroplets, at high speed.

[0016] To this end, there is a need for a device and a method for generating monodisperse microdroplets with a defined volume on 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, allowing for individual analysis and / or sorting of each microdroplet at high speed, ideally at the same rate as the generation frequency. There is a need for a device and method for generating such monodisperse microdroplets that is stable, reproducible, insensitive to variations in environmental parameters, and easy to manufacture.

[0017] Furthermore, there is a need for a device and method to generate such monodisperse microdroplets, and then to individually detect and actively sort each microdroplet on the same fluidic chip, at a high sorting rate, above 10 kHz and potentially reaching several tens of kHz or hundreds of kHz. Presentation of the invention

[0018] In this context, the present invention proposes 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, a decompression zone downstream of the junction zone, and a sorting zone disposed downstream of the decompression zone, a fluidic microchannel fluidically connecting the junction zone to the decompression zone, the sorting zone comprising a fluidic channel fluidically connected at the inlet to the decompression 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 disposed 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 disposed on an opposite face of the junction zone with respect to the central nozzle,the central nozzle having a smaller width x between 2 and 10 micrometers, each of the two lateral nozzles having a smaller width x1 between 3 and 10 micrometers, the fluidic microchannel having a width y between 2 and 7 micrometers and a length z between 2 and 10 micrometers, the expansion zone having a larger width u between 20 and 75 micrometers and the, relaxation zone having a length V between 100 and 600 micrometers, and in that the junction zone, the microfluidic channel, the relaxation zone and the fluidic channel of the sorting zone have a depth h between 2 micrometers and 10 micrometers, the microfluidic chip being able to generate via the microfluidic channel monodisperse microdroplets having a determined volume between 1 femtoliter and 200 femtoliters.

[0020] Thus, the microfluidic chip allows the sequential generation, one by one, of monodisperse microdroplets of a central fluid within a sheath fluid at a stable production frequency above 10 kilohertz, for example, 60 kHz. The microdroplets have a diameter determined by the dimensions and geometry of the central and sheath fluid inlet nozzles, and the outlet nozzle (i.e., the fluidic microchannel connecting the junction zone to the expansion zone), for example, a diameter between 4 and 5 micrometers. Therefore, the microdroplets are monodisperse, meaning that all microdroplets have the same volume, and this volume is predetermined. Furthermore, the monodisperse microdroplets thus generated remain ordered in a single column within the expansion zone.

[0021] Other non-limiting and advantageous features of the microfluidic chip according to the invention, taken individually or in all technically possible combinations, are as follows: - the sorting zone includes an active electrode disposed on one side of the fluidic channel and two ground electrodes disposed on both sides of the fluidic channel, the two ground electrodes framing the active electrode in the sorting zone around the fluidic channel, the active electrode being at a minimum distance from the fluidic channel of between 5pm and 40pm; - the sorting zone comprises two active electrodes and two ground electrodes, the two active electrodes being arranged on two opposite sides of the fluidic channel, the two ground electrodes being arranged on two opposite sides of the fluidic channel, each of the two ground electrodes framing one of the two active electrodes in the sorting zone around the fluidic channel, each of the two active electrodes being at a minimum distance from the fluidic channel of between 5pm and 40pm; - the microfluidic chip includes at least one lateral channel fluidically connected to the inlet of the sorting zone, at least one lateral channel being adapted to inject a duct fluid flow; - the microfluidic chip includes 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 a reduced width r at the outlet of the fluidic microchannel, between 5 and 15 micrometers, and the relaxation zone widens downstream of the reduced width r up to the greatest width u; - the relaxation zone is connected to the fluidic channel of the sorting zone by a fluidic channel having a depth equal to the shallow depth h and a width of about 20pm, the width of the relaxation zone narrowing downstream from the greatest width u to the width of about 20pm of the fluidic channel; - the central fluid injection channel, the two duct 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 duct 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 includes 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, the method comprising the following steps: - simultaneous injection of a central fluid into the central fluid injection channel towards the junction zone of the microfluidic chip and of a sheath fluid into the two sheath fluid injection channels towards the junction zone; - generation of monodisperse microdroplets of central fluid within the sheath fluid in the fluidic microchannel fluidically connecting the junction zone to the expansion zone of the microfluidic chip, the microdroplets having a determined volume, between 1 femtoliter and 200 femtoliters, and - orderly propagation of microdroplets towards a sorting zone of the microfluidic chip so as to actively and individually sort each microdroplet.

[0023] Thus, the microfluidic chip of this disclosure allows three functions to be performed on the same chip: creation of monodisperse drops of determined volume, between 1 fL and 200 fl_, orderly propagation of the drops to 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 small distance between the microdroplet production zone and the sorting zone, makes it possible to both generate low-volume monodisperse microdroplets, between 1 fL and 200 fL, at high frequency (e.g. several tens of kHz) and to conduct them in a single ordered column from the generation zone to the sorting zone, and then to actively and individually sort each microdroplet, 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 their alignment in a single column, oriented in the direction of flow of the sheath fluid, and at a stable speed, then the individual sorting of the drops that present themselves sequentially at the measurement and sorting zone.

[0025] Of course, the different features, variants and embodiments of the invention can be combined with each other in various ways as long as they are not incompatible or mutually exclusive. Brief description of the drawings

[0026] Furthermore, various other features of the invention become apparent from the attached description made with reference to the drawings which illustrate non-limiting embodiments of the invention and where:

[0027] Figure 1 is a schematic view of the fluidic circuit of a microfluidic chip according to an example embodiment of this disclosure;

[0028] Figure 2 is an enlarged view of the central part of the microfluidic chip in Figure 1;

[0029] Figure 3 is an enlarged view of the junction area and part of the relaxation area of ​​the microfluidic chip in Figures 1 to 3;

[0030] Figure 4 is an enlarged view of the junction zone, the relaxation zone and the sorting zone of the microfluidic chip of figures 1 and 2, with arrows indicating the direction of the different flows;

[0031] Figure 5 is an enlarged top view of the sorting area of ​​the microfluidic chip shown in Figures 1 to 4;

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

[0033] Figure 7 is an enlarged view of a junction zone and a sorting zone according to a variant of the three-output-channel microfluidic chip;

[0034] Figure 8 is an enlarged view of a microfluidic device in operation, showing the generation of a droplet train and its propagation into 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, the term "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 fabricated in a wafer. Various materials are suitable for the wafer, such as glass or a polymer, for example polydimethylsiloxane (PDMS), with a thickness of a few millimeters, for example 5 mm. The microfluidic chip has a lid 31 which is attached, for example, by gluing or adhesive. The lid 31 is preferably transparent to allow the observation and detection of particles suspended in a fluid. For example, the lid 31 is a microscope slide to allow observation of the microfluidic chip under an optical microscope objective. microfluidic circuit including 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 relation to figures 1 to 8.

[0039] Figure 1 shows an example of a microfluidic chip in a top view, for example, through the blade 31. The microfluidic chip includes a sheath fluid inlet 2 and a central fluid inlet 4. The central fluid and the sheath fluid are immiscible with each other. The central fluid, or sample fluid, is, for example, an aqueous solution containing suspended 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 a surfactant. Inlets 2 and 4, respectively, are fluidically connected to an external reservoir of sheath fluid and an external reservoir of central fluid, respectively. These reservoirs (not shown) are equipped with micropumps or syringe pumps to inject the sheath fluid and central fluid, respectively, into the microfluidic chip.

[0040] Optionally, inlet 2, or 4 respectively, includes a filter consisting, for example, of channels of predetermined dimensions formed between micrometer-sized pillars 43 arranged in concentric circles around an opening 41 that passes through the microfluidic chip to the corresponding reservoir. Advantageously, additional micrometer-sized spacer pillars 42 are arranged around the opening 41 to maintain the depth of the spaces through which the fluid flows between the opening 41 and the channels between the pillars 43. The micrometer-sized pillars 42 and 43 are part of the microfluidic chip plate and are generally attached at their other end to the blade 31 that forms a lid. Such a filter allows the sheath fluid and the central fluid, respectively, to be filtered during their injection into the microfluidic chip, as schematically illustrated in Figures 1-2.

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

[0042] The microfluidic chip includes a junction zone 7 between the two sheath fluid injection channels 1 and the central fluid injection channel 3, illustrated in particular in Figure 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 comprises three inlet nozzles and one outlet nozzle. More specifically, the junction zone 7 includes a central nozzle 21 fluidically connected to the central fluid injection channel 3 and two lateral nozzles 22, 23 arranged on opposite sides of the junction zone 7, each of the two lateral nozzles 22, 23 being fluidically connected to one of the two fluid injection channels of the sheath 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 with respect to the central nozzle 21. We note 28 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 microdroplet production zone is located in the fluidic microchannel 24.

[0044] Advantageously, as illustrated in Figure 4, the two duct fluid injection channels 1 have filters 6 consisting of pads distributed along the duct fluid paths 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 unwanted particles larger than the width of the nozzles 21, 22, 23. Such unwanted particles are likely to clog the nozzles 21, 22, 23 and prevent the formation of microdroplets.

[0045] According to this disclosure, the inlets and outlets of junction zone 7 have specific micrometer dimensions. In this document, the depth of a microfluidic channel is defined as a dimension measured perpendicular to the plane of Figures 1 to 5. The length of a microfluidic chip element, for example, the length of a microchannel, is defined as a dimension measured along the longitudinal axis of that element, i.e., in the direction of fluid flow in that microchannel, in the plane of Figures 1 to 5 and 7-8. The width of a microfluidic chip element is defined as, for example, the width of a microchannel, a dimension taken transversely to the longitudinal axis of this element, that is to say 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 between 4 and 10 micrometers. For example, the central nozzle 21 has a length t less than or equal to 7 micrometers. Each of the two lateral nozzles 22, 23 fluidically connects one of the two fluid injection channels of the duct 1 to the junction zone 7 by progressively reducing its width. At the connection end of the junction zone 7, each of the two lateral nozzles 22, 23 has a smaller width x1 between 4 and 10 micrometers. For example, each of the two lateral nozzles 22, 23 has 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 between 2 and 7 micrometers and a length z between 2 and 10 micrometers.

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

[0048] Advantageously, the expansion zone 8 has a shape that narrows downstream of the microfluidic chip so as to allow the microdroplets to pass one by one into the measurement and sorting zone, hereinafter referred to as the sorting zone 18. A fluidic channel 25 connects the outlet of the expansion zone 8 to the sorting zone 18. The fluidic channel 25 has a width of 20 µm and a length, for example, of 300 µm. The sorting zone 18 includes a fluidic channel 27 forming the inlet of a The junction has at least two outlet channels 13 and 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, for example, retains the selected drops, while the outlet channel 14 acts as a waste bin to collect the unselected drops. The outlet channels 13 and 14 have, for example, a width of 50 µm.

[0049] According to a particular and advantageous embodiment, the microfluidic chip has channels with a uniform depth h, also called shallow depth, throughout the entire 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 microfluidic channel 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 shallow 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 microdroplets are produced have a depth h that limits the diameter of the microdroplets and therefore their volume. This shallow depth h is maintained from the junction zone 7 throughout the droplet propagation zone, particularly in the expansion zone 8, the fluidic channel 25, and the sorting zone 18. Maintaining the shallow depth h continuously along the entire path of the microdroplets from their generation to sorting ensures that the droplet sequencing remains in a single ordered column in the direction of the cladding fluid flow (see, for example, Figure 8). At the outlet of the microfluidic channel 24, the generated microdroplets 32 form a single column that is carried along by the cladding fluid. Counterintuitively, although the width of the expansion zone 8 is approximately one order of magnitude greater than the diameter of the microdroplets 32, the microdroplets 32 remain ordered in a single column from the outlet of the microchannel 24 to the measurement and sorting zone.

[0052] For example, a reduced depth h of 4.5 pm contributes to the formation of microdroplets with a diameter less than or equal to the reduced depth. However, fluid movement in shallow areas is only possible with a strong upstream-downstream pressure gradient. Specifically, in the junction zone 7 and the fluidic microchannel 24 where microdroplets are produced, the channels are narrowest and shallowest. In one embodiment, some channels are deeper (H = 10 pm) in areas that are not critical for microdroplet production, orderly propagation, or detection. Considering a fluidic channel with a rectangular cross-section and a given fluid flow rate, the head loss is inversely proportional to the cube of the fluidic channel depth for a given channel width.Thus, a fluidic channel with a depth H of 10 pm requires an upstream pressure approximately 10 times lower than a channel with a reduced depth of 4.5 pm and the same width. Fabrication of channels with greater depth H on the same microfluidic chip reduces mechanical stress on the chip wherever channel depth is not a critical parameter for microdroplet formation, ordered propagation, or detection.

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

[0054] An important aspect of this disclosure relates 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 this disclosure, the expansion zone 8 has a defined geometry that is neither too long nor too short, in order to maintain a linear sequence of droplets 42. As mentioned above, the expansion zone 8 has a depth equal to the shallow depth h. This expansion zone 8 allows the droplets 42 to be produced in a single column, in other words, it maintains the droplet sequencing. The configuration and operation of this expansion zone 8 are completely counterintuitive to those skilled in the art, and fluidic theory cannot predict why this zone is so important.In contrast, in the prior art, most microfluidic droplet production devices generate droplet sets in which the drops either mix or are distributed into two or more parallel rows, presumably due to velocity variation phenomena between the drops and the sheath fluid.

[0055] As a non-limiting example, the microfluidic chip 30 is manufactured by molding onto a silicon wafer that has been previously textured in relief using micro-etching techniques. A first layer of thin photosensitive resin (e.g., 4.5 µm) is deposited onto an initially flat silicon wafer, for example, by spin coating. This first layer is exposed by photolithography with a first mask corresponding to the shallow channels or microfluidic elements (h). Once this layer is developed, the positive (relief) shape of the future shallow channels or microfluidic elements (h) remains.

[0056] In cases where some channels have a greater depth, a second, thicker layer of resin, of thickness H (for example, thickness H equal to 10) is applied. pm) is deposited on top of the first layer, in a similar manner. The second layer is exposed by photolithography with a second mask corresponding to the deep H microfluidic channels or elements, then developed to reveal the deep H microfluidic channels or elements in positive. The second mask is aligned with the first resin layer so that the shallow h channels (4.5 pm) and the deep H channels (10 pm) communicate fluidly, particularly at the nozzles 22, 23.

[0057] The microstructured silicon mold is used to deposit the PDMS, for example, by casting. The fabrication of the microfluidic chip relies 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 significantly 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, which forms one side of the microfluidic channels. The central fluid is injected through the central fluid inlet 4 and flows through the central fluid injection channel 3 towards the junction zone 7. The sheath fluid is injected through the inlet 2 and flows through 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 lateral nozzles 22 and 23. The junction zone 7 thus forms a constriction between the central fluid and the sheath fluid.At the junction zone, in the microdroplet formation zone 29, the sheath fluid pinches the central fluid, which splits into microdroplets 32 in the fluidic microchannel 24. Microdroplets 32 of central fluid are thus generated one by one within the sheath fluid. Upon exiting the fluidic microchannel 24, these microdroplets 32 are carried by the sheath fluid into the expansion zone 8, where they are transported in an orderly fashion. More precisely, in the expansion zone 8, the microdroplets 32 are aligned with one another, starting from the exit of the microchannel 24. behind the other, in a single column, oriented in the direction of flow of the duct fluid (see figure 8).

[0059] Table I summarizes the ranges of values ​​for the various junction zone dimensions and the ranges of microchannel depth values, as well as all these values ​​for an example embodiment with two channel depths. The combination of these parameter values ​​allows for the formation of monodisperse microdroplets of a specific diameter and their propagation in a single, ordered line.

[0060] [Tables 1]

[0061] Although these parameters are not independent of each other, it is possible to associate certain parameters with the microdroplet formation regime or the dimensional properties of the microdroplets thus obtained. According to this disclosure, the smallest width x and length t of the central nozzle 21 allow the central nozzle to form a constriction of the central fluid. The smallest width x1 of each of the two lateral nozzles 22, 23 is also a critical parameter for microdroplet formation. The ranges of values ​​of the parameters x and x1 of the junction zone 7 allow the formation of droplets with a determined volume at the outlet of the fluidic microchannel 24 and with a stable production of monodisperse droplets. The smallest width x1 and length S of each of the two lateral nozzles 22, 23 allow the formation of a constriction zone of the sheath fluid.The small depth h, the width y and the length z of the fluidic microchannel 24 determine the diameter of the microdroplets 32. Here we have determined a set of critical parameters for the formation of microdroplets and which influence the value of the diameter of the microdroplet 32.

[0062] The greater width u of the expansion zone 8 determines the fluidic regime, enabling the formation and regular, linear flow of microdroplets in a single, ordered column. The length V of the expansion zone 8 stabilizes the microdroplet train by maintaining it in a single, ordered column. The reduced width r of the expansion zone 8 allows for easy fabrication by photolithography of the expansion zone 8, which extends over a greater width u and length V, and also contributes to stabilizing the droplet flow.

[0063] The example values ​​shown in Table I allow for the generation of microdroplets with a stable diameter of approximately 4.5 pm, corresponding to a volume of approximately 45 fL. Remarkably, the microdroplets 32 are monodisperse and all have the same diameter, approximately equal to the shallow depth h of 4.5 pm. Furthermore, the microdroplets 32 are produced at a frequency ranging from 10,000 drops / sec to 60,000 drops / sec. This geometry not only allows for stable control of the droplet volume but also maintains a constant volume over a wide range of cladding and sample fluid flow rates. When the flow rates change, the droplet production frequency changes, but the volume remains constant. This is particularly important for accurately predicting the number of particles contained in each droplet, as discussed above.

[0064] More precisely, the width y and length z of the fluidic microchannel 24 influence the diameter of the microdroplets 32. For example, with a shallow depth h of 4.5 pm, a smallest width x of 5 pm, and a width y of 4 pm, the resulting microdroplets 32 have a diameter between 4 and 5 micrometers, for example, 4.5 pm. Since the depth of the junction zone 7, the fluidic microchannel 24, and the expansion zone 8 is 4.5 pm in this example, the microdroplets 32 are generally nearly spherical. Proportionally modifying the values ​​of length z, width y, and depth h allows for the production of microdroplets 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 13f I approximately, or with y=x=h= 6pm we obtain drops of volume 10Ofl.The microfluidic chip allows the generation of monodisperse microdroplets, of determined volume, between 1 fL and 200 fL, for example between 10 fL and 100 fL, preferably between 20 fL and 50 fL. and preferably between 30 fL and 40 fL. Respecting this relationship between y, x and h (with certain tolerances, on 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 generated droplets have a poorly controlled diameter, most often too large and fluctuating, or much smaller and irregular: this results in polydisperse rather than monodisperse microdroplets. If the length z of the fluidic microchannel 24 is too large, the generated droplets have too large a volume, leading to instabilities in the downstream flow and errors in the number of particles per droplet.

[0066] Certain dimensional parameters of the microfluidic chip are adjusted to facilitate fabrication using photolithography and molding techniques. The length t of the central nozzle 21 is preferably 5 pm. If the length t of the central nozzle 21 is too small, the nozzle may be malformed during fabrication. 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 nozzle exhibits high hydrodynamic resistance, which could impede the advance of the central fluid. To compensate for such resistance, it would be necessary to increase the pressure of the central fluid, which could cause damage to the microfluidic chip, such as delamination between the PDMS wafer and the glass plate. The length t is therefore less than or equal to 7 pm.

[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 region where the linear velocity of the fluids is reduced, which stabilizes droplet formation and prevents droplet aggregation, either chaotically or in multiple columns. Throughout the expansion zone, the drops are accelerated by viscosity 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 length V of the expansion chamber are therefore relatively tolerant, but have limits beyond which the flow is no longer stable in a single column. These values The maximum values ​​are, for example, 50 pm and 500 pm, respectively. Once the flow has stabilized in the expansion zone 8, another chamfer 39 allows a transition to the narrower (e.g., 20 pm) fluidic channel 25 and fluidic channel 27 of the sorting zone 18. This reduced width of the fluidic channel 27 in the sorting zone 18 allows the electrodes 10 and 11 to be positioned close to the droplet row 32, thus increasing the dielectrophoresis effect used for sorting.

[0069] The expansion zone 8 advantageously features a chamfer 38 at the inlet (see Figure 3) which allows the expansion zone to be widened from the reduced width r to the largest width u. Advantageously, the expansion zone 8 also features another chamfer 39 downstream (see Figure 4) which allows the expansion zone 8 to be reduced from the largest width u to the width of the fluidic channel 25 at the outlet of the expansion zone 8. When using the microfluidic chip, this / these chamfer(s) promote(s) fluid flow.

[0070] The microdroplets 32 can be detected individually, for example, by a fluorescence detection system by directing an excitation laser beam towards the fluidic channel 25 at the outlet of the expansion zone 8 or towards the fluidic channel 27 in the sorting zone 18. At the outlet of zone 8, the narrowing causes an acceleration of the flow due to flow rate conservation; since the droplets 32 have a stable diameter, this acceleration has the effect of spacing the droplets, which are then better separated for individual analysis and sorting. The lateral channel 9 provides an additional flow of sheath fluid, which further increases the spacing between the droplets. Particularly advantageously, detection takes place in the shallow depth h fluidic channel 25 or 27. 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 microdroplets, thus maintaining droplet order. Furthermore, the shallow depth h also limits the contribution of the sheath fluid to the detected signal. Following the detection of a microdroplet of interest containing a fluorescent marker, this microdroplet can be selectively extracted from the flow in the sorting zone 18 and directed to a specific collection channel. The detection system generates a signal that triggers the sorting of the detected microdroplet. Various sorting devices and methods are available. can be used, for example based on dielectrophoresis, as in the detailed example below.

[0071] Advantageously, the microfluidic chip also includes a sorting zone 18 integrated on the same support plate. The sorting zone 18 is located 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 includes a fluidic channel 27 fluidically connected at its inlet to the expansion zone 8 via the fluidic channel 25 and at its outlet to at least two output channels 13, 14, for example via a Y-junction denoted 12, one branch of which is the fluidic channel 27. The fluidic channel 25, the fluidic channel 27, and the Y-junction to the two output channels have a depth equal to the shallow depth h. In one embodiment, the two output channels 13, 14 have a depth equal to the deepest depth H. Alternatively, the two output channels 13, 14 have a depth equal to the shallowest depth h.

[0072] Optionally, a lateral channel 9 is fluidically connected to the inlet of the sorting zone 18 via a nozzle 26. The lateral channel 9 conducts a flow of injected sheath fluid through an inlet 19 (see Figure 1). The lateral 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 one embodiment, the lateral channel 9 has a depth equal to the large depth H. Alternatively, the lateral channel 9 has a depth equal to the small depth h. The nozzle 26 has a depth equal to the small depth h.

[0073] Advantageously, as illustrated in Figure 4, the lateral channel 9 includes a filter 6 consisting of pads distributed along the paths of the duct fluid upstream of the nozzle 26. As mentioned above, such a filter makes it possible to retain any undesirable particles larger than the width of the nozzle 26 which could clog the nozzle 26 and impair its operation.

[0074] The lateral channel 9 allows for the delivery of additional sheath fluid to the already formed microdroplet train. The lateral channel 9 also allows for modification of the spacing between the microdroplets. Indeed, at the outlet of the expansion zone 8, the microdroplets 32 can be relatively close to each other. This makes sorting very difficult, as it requires high precision to sort a microdroplet without affecting adjacent microdroplets. The addition of Sheath fluid via lateral channel 9 allows the spacing between microdroplets to be increased to values ​​of several microdroplet diameters, which allows the sorting selectivity to be increased.

[0075] On the other hand, the lateral channel 9 has the effect of laterally diverting the flow of microdroplets, which is directed towards one of the two default output channels, for example, output channel 14, which corresponds, for instance, to a bin collecting unselected microdroplets. Sorting consists of actively extracting a microdroplet selected individually based on a detected signal and directing it towards the other output channel 13, while the unselected microdroplets are passively directed towards the bin outlet.

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

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

[0078] More specifically, an active electrode 10 is disposed on one side of the fluidic channel 27, a ground electrode 11 is disposed on the same side as the active electrode 10, and another ground electrode 11 is disposed on the opposite side of the fluidic channel 27. The active electrode 10 has a U-shape with the base of the U closest to the fluidic channel 27. The two ground electrodes 11 are advantageously symmetrical with respect to the longitudinal direction of the fluidic channel 27 of the sorting zone 18. Each ground electrode 11 has an M-shape, with the tips of the M disposed closest to the fluidic channel 27. The U-shaped active electrode 10 is disposed between the two tips of the M-shaped ground electrode 11. The minimum distance between each of the electrodes 10, 11, and the fluidic channel 27 is between 5 pm and 40 pm, for example Here, approximately 10 pm. The active electrode 10 is at the minimum distance from the fluidic 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 fluidic 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. However, 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 limit the spatial extent of the electric field gradient 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 electrodes 10 and 11 both maximizes the electric field gradient in the central region of the fluidic channel 27, located in the middle of the sorting zone 18, and spatially confines the electric field gradient to the central region containing the microdroplet to be sorted. The two mass electrodes 11 limit the spatial extent of the electric field gradient upstream of the sorting zone 18, where this gradient could potentially influence the next microdroplet. The two mass electrodes 11 also limit the spatial extent of the electric field gradient downstream of the sorting zone 18, where this gradient could potentially affect the already sorted microdroplets located in a wider and deeper area, thus preventing the merging of several already sorted microdroplets.

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

[0081] The sorting process comprises applying 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 10 ps and 200 ps; it may be a positive pulse, a negative pulse, a period of a sinusoid or square wave or any zero-mean waveform, or several periods of sinusoids, square waves, or zero-mean waveforms. This pulse may be of direct current voltage. Preferably, this pulse is of alternating current voltage, at an electrical frequency f chosen, firstly, so that the duration of the pulse T is equal to one or more periods of the alternating frequency 1 / f, in order 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 shorter than, or equal to, or slightly greater than, the time between the passage of two successive microdroplets 32 in the sorting zone 18, in order to ensure the extraction of a single microdroplet 32. The electrical frequency range f is for example between 20 kHz and 200 kHz and the duration range T between 10ps and 50ps to allow sorting for a droplet passage frequency between 10 kHz and 50 kHz.

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

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

[0084] According to this disclosure, continuously along the fluidic path of the microdroplets 32 from the microdroplet formation zone 29 to and including the sorting zone 18, all channels and zones have a depth equal to the shallow depth h, which determines the microdroplet diameter, 4.5 pm in the example of Table I. The junction zone 7, including the inlet nozzles 21, 22, 23, the microfluidic channel 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 pm in the same example), these areas not being critical to the production and detection and sorting of microdroplets.

[0085] For example, in Figure 2, zones 3, 4, 5, 7, and 8 are shallow depth zones (h ~4.5 pm). Between the connection of the central fluid injection channel 3 and the nozzle 21, the linear velocity of the fluid is determined by the volumetric flow rate divided by the channel cross-section. Choosing a shallow channel depth (4.5 pm) at this point ensures a higher linear velocity, thus preventing the particles carried by the central fluid from settling. However, from nozzle 21 to the exit of sorting zone 18, the shallow depth h helps to maintain the volume of microdroplets and to ensure that the microdroplets produced and detected in a certain order arrive in the same order in sorting zone 18. The other zones 1, 2, 9, 19, 13, 14, 16, 17, can have a large depth H of 10 pm, to reduce the constraints on the fluid flow and therefore the pressure required.

[0086] Advantageously, the microfluidic chip includes one or more balancing microchannels 15 fluidically connecting one of the two output channels 13 to the other of the two output channels 14. The microchannel(s) 15 allow pressure to be balanced between the two output channels 14, which may have different flow rates, linked to different pressures, which could impair the efficiency of the The balancing microchannels 15 have a depth equal to the shallow depth h, for example, 4.5 pm, and a narrow width of approximately 5 pm. These dimensions prevent the passage of sorted microdroplets between the two outlet channels 14 via one of the balancing microchannels 15 (see Figures 5-6). As a non-limiting example, the microdroplets are water-based and the sheath fluid is based on an oil used in microfluidics. The microfluidic chip is arranged horizontally, with the shallow balancing microchannels 15 oriented towards the bottom of the chip, so that the microdroplets 32, being lighter than the oil, quickly rise to the top of the outlet channel 13, or 14, which has a greater depth H, for example, 10 pm. Thus, microdroplets 32 with a diameter of 4.5pm have little risk of being captured by the lower equilibration microchannels 15.

[0087] Figure 7 shows a microfluidic chip according to a variant of the microfluidic chip described in connection with Figures 1 to 6. In this variant, the sorting zone 18 has three outlet channels. The outlet channel 14 is located in the center and corresponds to the waste 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 carries a sheath fluid flow. Each lateral channel 9 is adapted to inject a sheath fluid flow 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 major depth H.Furthermore, 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 fluidic channel 27, symmetrically with respect to the longitudinal direction of the fluidic channel 27. Each of the two active electrodes 10, 110 is at a minimum distance from the fluidic channel 27 of between 5 pm and 40 pm, for example, about 10 pm. Preferably, the tips of the M of each ground electrode 11 are arranged as close as possible to the fluidic channel 27, for example, at a minimum distance of between 5 pm and 40 pm, for example, about 10 pm. Each active electrode 10, 110 is framed by the fluidic channel 27 and by a... Tl 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 output channel 14. The application of an electrical pulse on 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 Figure 7 offers several advantages. First, it allows for greater flow symmetry, which improves stability. Indeed, splitting the lateral channel 9 into two lateral channels located on either side of the central fluidic channel 27 results in a symmetrical supply of sheath fluid, which is then used solely to control the spacing of the droplets 32. Finally, an electrode 10, 110 is available to selectively direct the droplets towards one or the other of the outlet arms 13, 131. This configuration allows for the separation and collection of two different populations of sorted droplets into two different collection arms 13 and 131, respectively. In contrast, the device in Figure 4 has only one active electrode to direct the selected droplets towards a single collection arm 13.

[0089] In summary, the droplet formation zone of the microfluidic chip comprises a fluidic microchannel 24 at the outlet of the junction zone 7. The fluidic microchannel 24 has dimensions such that y ~ h approximately, and z > y and h. The constriction zones of the central fluid (e.g., an aqueous fluid) in the sheath fluid flow (e.g., an oily fluid) are such that x ~ x1 ~ y. The expansion zone 8 has a width u of approximately 50 µm and a length V of approximately 500 µm. The outlet of the expansion zone 8, connected to the fluidic channel 25, exhibits a constriction to a width of approximately 20 µm in the measurement and sorting zone 18.In one particular embodiment, the microfluidic chip comprises channels and zones having two distinct depths: a shallow depth h, continuous along the fluidic path of the microdroplets 32 from the microdroplet formation zone 29 to and including the sorting zone 18, and a large depth H outside the zones critical to the production, detection, and sorting of microdroplets, 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 fluidic channel 27, for example, at a distance of approximately 10 pm. The use of ground electrodes 11 on either side of the active electrodes 10, 110 allows... to limit the spatial extent of the electric field in the longitudinal direction of the fluidic channel 27 in the sorting zone 18.

[0090] The microfluidic device and method described in this disclosure enables the selection and sorting of droplets containing a nanoparticle detected, for example, by fluorometry, and directs only the droplets containing the desired type of nanoparticle to a specific outlet. The microfluidic device and method collect at this outlet only the droplets containing the desired type of nanoparticle, excluding empty droplets and droplets containing other types of nanoparticles. The microfluidic device and method thus concentrate the desired nanoparticles within the collected fluid.

[0091] Integrating a monodisperse microdroplet production zone, a decompression zone, and a detection and sorting zone onto a single microfluidic chip, all located close together, allows for the generation of small-volume monodisperse microdroplets (between 1 fL and 200 fL) at high frequencies (e.g., several tens of kHz) and the active, individual sorting of each microdroplet with high efficiency and a high sorting frequency. The decompression zone keeps the droplets arranged in a single column until they reach the detection and sorting zone. This configuration eliminates the need for intermediate storage and transport of microdroplets from one microdroplet generation device to another. It also prevents the microdroplets from sticking to each other or to the walls of a storage container.Overall, the generation and sorting of microdroplets are both faster and more reliable.

[0092] This disclosure has applications for the generation of monodisperse microdroplets with volumes ranging from 1 fL to 200 fL containing chemical or biological particles of nanometric dimensions. Furthermore, this disclosure has applications for the dilution-free sorting of such microdroplets by detecting a signal associated with a property of the chemical or biological particle of nanometric dimensions and triggering the sorting process based on the detected signal. In particular, this disclosure has applications for the generation and sorting of monodisperse microdroplets containing nano-objects with concentrations as high as 10 9 at 10 1 ° nano-objects by milliliters, such as extracellular vesicles or cellular secretions, particularly exosomes, but also viral vectors, or even large individual molecules. The disclosed microfluidic chip enables the encapsulation of fluorescent nanoparticles and their sorting based on measured fluorescence. The disclosed microfluidic chip also enables the measurement and sorting of fluorescently labeled organic particles by encapsulating them in droplets. This disclosure has further applications in chemistry, for example, for the purification of nanometric probes or in the particle size analysis of nanometric objects.In addition, the microfluidic chip integrates drop generation and sorting functions without intermediate storage, which allows not only high-frequency drop generation but also individual drop sorting, on the fly, immediately after their generation, and at high frequencies, for example from a few kHz to over 100 kHz.

Claims

Claims 1. 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, 1), 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 x1 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 relaxation 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., 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 relaxation zone (8) has at the outlet of the fluidic microchannel (24) a reduced width r of between 5 and 15 micrometers and the relaxation 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 sheath fluid injection channels (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 using 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 relaxation zone (8) of the microfluidic chip, the microdrops having a determined volume, between 1 femtoliter and 200 femtoliters, and - 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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