Classifications

• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
  • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
  • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
  • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
  • B01L3/502769—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
  • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
  • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
  • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
  • B01L3/502753—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
  • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
  • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
  • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
  • B01L3/502769—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements
  • B01L3/502784—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements specially adapted for droplet or plug flow, e.g. digital microfluidics
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
  • B01L2200/06—Fluid handling related problems
  • B01L2200/0636—Focussing flows, e.g. to laminate flows
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
  • B01L2200/06—Fluid handling related problems
  • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
  • B01L2200/0652—Sorting or classification of particles or molecules
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
  • B01L2200/06—Fluid handling related problems
  • B01L2200/0673—Handling of plugs of fluid surrounded by immiscible fluid
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/08—Geometry, shape and general structure
  • B01L2300/0809—Geometry, shape and general structure rectangular shaped
  • B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/08—Geometry, shape and general structure
  • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
  • B01L2300/0864—Configuration of multiple channels and/or chambers in a single devices comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/08—Geometry, shape and general structure
  • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
  • B01L2300/0867—Multiple inlets and one sample wells, e.g. mixing, dilution
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/08—Geometry, shape and general structure
  • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
  • B01L2300/0874—Three dimensional network
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/08—Geometry, shape and general structure
  • B01L2300/0896—Nanoscaled
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
  • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
  • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
  • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
  • B01L3/502769—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements
  • B01L3/502776—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements specially adapted for focusing or laminating flows

Definitions

• 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.
• microfluidic chip for generating microdroplets of a determined volume, monodisperse 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 for generating and actively sorting one by one each microdroplet of the train of monodisperse microdroplets, 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.
• kHz kilohertz
• P(N) is the probability that a drop contains N particles
• ⁇ is the average number of particles per drop, for a drop of given volume and a given volume concentration of particles.
• Typical concentrations of liquids containing organic nanoparticles are generally between 10 9 and 10 1 ° particles per ml. According to the above calculations, spherical drops of 30 pm to 300 pm in diameter (with a volume of between 10 picoliters and 10 nanoliters) are suitable for samples having a concentration of between 10 3 and 10 6 particles per ml, which is completely insufficient for usual samples of biological nanometric particles. To achieve these concentrations, it would be necessary to dilute 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 want to concentrate such samples.
• Poisson's distribution allows us to determine the probabilities that a given drop contains 0, 1 or 2 particles depending on the volume of the drop.
• the volume of the drops is between 30 fL and 40 fL.
• the percentage of empty drops, i.e. without any particles is greater than 90%, which lengthens the analysis times, since all the drops must be analyzed while most are empty.
• 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.
• the microdroplets of sample fluid can 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.
• the 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 very dependent on the injection conditions. Over time, variations in the volume of microdrops are observed which are difficult to control.
• monodisperse microdroplets are understood to mean microdroplets all having the same volume +/- 20% for example. In other words, the volume of the monodisperse microdroplets is uniform.
• the monodisperse microdroplets are generally suspended in a flow of encapsulation fluid or sheath fluid.
• 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.
• the volume of these microdroplets are too large compared to nano-objects that we want to analyze individually.
• 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 an arrangement requires the reinjection of the drops into another chip, which is particularly complex, and significantly increases both the processing time and the risks of errors or other difficulties such as drop fusion, leakage of the liquid from the drops to the sheath liquid, handling difficulties and requires drops of relatively large volume.
• a device and a method for generating monodisperse microdroplets 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 microdroplet also at high speed, ideally at the same speed as the generation frequency.
• a device and a method for generating such monodisperse microdroplets that is stable, reproducible, insensitive to variations in environmental parameters and easy to manufacture.
• 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.
• 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 relative to the central nozzle, the central nozzle having a smaller width x of between 2 and 10 micrometers, each of the two nozzles lateral having a smaller width x1 between 3 and 10 micrometers, the fluidic microchannel having a width
• the microfluidic chip makes it possible to sequentially generate, one by one, monodisperse microdroplets of a central fluid in a sheath fluid at a stable production frequency greater than 10 kilohertz, for example 60 kHz.
• the microdroplets 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.
• the microdroplets are monodisperse, that is to say that all the microdroplets have the same volume and this volume is predetermined.
• the monodisperse microdroplets thus generated remain ordered in a single column in the expansion zone.
• microfluidic chip according to the invention taken individually or in all technically possible combinations, are as follows:
• 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 sheath fluid flow;
• the microfluidic chip comprises at least one balancing microchannel fluidically 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 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.
• 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:
• microdroplets having a determined volume, between 1 femtoliter and 200 femtoliters, and - ordered propagation of microdroplets towards a sorting zone of the microfluidic chip so as to actively and individually sort each microdroplet.
• the microfluidic chip of the present disclosure makes it possible to perform three functions on the same chip: creation of monodisperse drops of a determined volume, between 1 fL and 200 fL, ordered 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 measurement result.
• 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 1 fL 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.
• high frequency for example several tens of kHz
• 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 in 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.
• Figure 1 is a schematic view of the fluidic circuit of a microfluidic chip according to an exemplary embodiment of the present disclosure
• Figure 2 is an enlarged view of the central portion of the microfluidic chip of Figure 1;
• Figure 3 is an enlarged view of the junction area and a portion of the relaxation area of the microfluidic chip of Figures 1 to 3;
• Figure 4 is an enlarged view of the junction area, the relaxation area and the sorting area of the microfluidic chip of Figures 1 and 2, with arrows indicating the direction of the different flows;
• Figure 5 is an enlarged top view of the sorting area of the microfluidic chip of Figures 1 to 4;
• Figure 6 is a sectional view AA of the sorting area of the microfluidic chip illustrated in Figure 5;
• Figure 7 is an enlarged view of a junction area and a sorting area according to a variant of the three-output channel microfluidic chip
• Figure 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 expansion zone.
• fluid means a pure liquid or a mixture, or an emulsion.
• 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, for example 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.
• 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.
• 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 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.
• 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.
• 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 FIGS. 1-2.
• 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.
• the central fluid injection channel 3 comprises a meandering portion 5, for example in the shape of an S, which makes it possible to stabilize the production of microdroplets.
• 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 towards the junction zone 7 and the central fluid injection channel 3 conducts the central fluid towards the junction zone 7.
• the fluidic junction zone 7 here comprises three inlet nozzles and an outlet nozzle. More precisely, the junction zone 7 comprises a central nozzle 21 fluidically 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 fluidically 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.
• 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.
• the inlets and the outlet of the junction zone 7 have particular micrometric dimensions.
• the depth of a microfluidic channel is understood to mean a dimension taken perpendicular to the plane of FIGS. 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 FIGS. 1 to 5 and 7-8.
• the width of an element of the microfluidic chip 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.
• 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 of 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 x1 of between 4 and 10 micrometers.
• 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.
• 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.
• the lateral channel has a depth equal to the shallow depth h.
• the outlet channels 13, 14 have a depth equal to the large depth H.
• the inlet 4 and the central fluid injection channel 3 have a depth equal to the small depth h.
• the inlet 4 and the central fluid injection channel 3 have a depth equal to the large depth H.
• the junction zone 7 and the fluidic microchannel 24 where the microdroplets are produced have a depth h which makes it possible to limit the diameter of the microdroplets and therefore their volume.
• This low depth h is maintained from the junction zone 7 throughout the drop propagation zone, in particular in the relaxation zone 8, the fluidic channel 25 and in the sorting zone 18. Maintaining the low depth h continuously over the entire path of the microdroplets 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).
• the microdroplets 32 generated form a single column which is entrained by the sheath fluid.
• 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 measurement and sorting zone.
• a reduced depth value h of 4.5 pm contributes to the formation of microdroplets having a diameter less than or equal to the reduced depth.
• the movement of fluids in the shallow areas is only possible by means of a strong pressure gradient between upstream and downstream.
• the channels are the narrowest and shallowest.
• the pressure drop is inversely proportional to the cube of the depth of the fluidic channel, for the same channel width.
• the microfluidic chip 30 is manufactured by molding on a silicon wafer previously structured in relief by microengraving techniques.
• a first layer of photosensitive resin of low thickness h (for example of thickness h equal to 4.5 ⁇ m) 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 low depth h. Once this layer has been developed, the positive shape (relief) of the future channels or microfluidic elements of low depth h remains.
• a second thicker layer of resin of thickness H (for example of thickness H equal to 10 pm) 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 and 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 pm) and the channels of great depth H (10 pm) communicate fluidically, in particular at the nozzles 22, 23.
• the microstructure silicon mold is used to deposit the PDMS therein, for example by casting.
• the manufacture 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 manufacture of the microfluidic chip does not require the implementation of nanophotolithography and nanoetching technologies, which are technically more demanding and much more expensive.
• the microfluidic chip thus formed for the generation of monodisperse microdroplets.
• the microfluidic chip is fixed 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.
• 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.
• these microdrops 32 are entrained 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 with each other behind the other, in a single column, oriented in the direction of flow of the sheath fluid (see figure 8).
• 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 microdroplets of determined diameter and their propagation in a single ordered file.
• 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 x1 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 x1 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 x1 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 microdroplets 32.
• a set of critical parameters for the formation of microdroplets and which influence the value of the diameter of the microdroplet 32 have been determined.
• 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.
• microdroplets 32 having a stable diameter of approximately 4.5 pm, i.e. a volume of approximately 45 fL.
• the microdroplets 32 are monodisperse and all have the same diameter approximately equal to the low depth h of 4.5 pm.
• the microdroplets 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.
• the width y and the length z of the fluidic microchannel 24 influence the diameter of the microdroplets 32.
• the small depth h being equal to 4.5 pm
• the smallest width x being equal to 5 pm
• the width y being equal to 4 pm
• 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 microdroplets 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 microdroplets of different diameters.
• the microfluidic chip makes it possible to generate 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 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.
• the drops generated have a poorly controlled diameter, most often too large and fluctuating, or much smaller and irregular: polydisperse microdrops are then obtained and not monodisperse ones. If the length z of the fluidic microchannel 24 is too large, the drops generated have too large a volume which leads to instabilities in the downstream flow and errors in the number of particles per drop.
• the length t of the central nozzle 21 is preferably 5 ⁇ m. If the length t of the central nozzle 21 is too small, the central nozzle 21 may be 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 may cause 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 ⁇ m.
• the expansion zone 8 is a zone where the linear speed 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 reaching a speed 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 values maximum are respectively 50pm and 500pm for example.
• another chamfer 39 allows a transition to the fluidic channel 25 and the fluidic channel 27 of the sorting zone 18 of narrower width (for example 20pm). 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.
• the relaxation zone 8 advantageously has a chamfer 38 at the inlet (see FIG. 3) which makes it possible to widen the relaxation zone from the reduced width r to the greater width u.
• the relaxation zone 8 also has another chamfer 39 downstream (see FIG. 4) which makes it possible to reduce the relaxation zone 8 from the greater width u to the width of the fluid channel 25 at the outlet of the relaxation zone 8.
• this (these) chamfer(s) promote(s) the flow of fluid.
• 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.
• the narrowing causes an acceleration of the flow by preserving the flow rate; the drops 32 having a stable diameter, this acceleration has the effect of spacing out the drops, which are better separated to analyze and sort them individually.
• the lateral channel 9 provides an additional flow rate of sheath fluid, which makes it possible to space out the drops even more.
• the detection is done in the fluidic channel 25 or 27 of shallow depth h.
• 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, 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.
• this microdroplet can be selectively extracted from the flow in the sorting zone 18 to be oriented towards a specific collection channel.
• the detection system generates a signal which triggers the sorting of the microdroplet thus detected.
• Different sorting devices and methods can be used. be used, for example based on dielectrophoresis, as in the detailed example below.
• 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 small depth h.
• the two output channels 13, 14 have a depth equal to the large depth H.
• the two output channels 13, 14 have a depth equal to the small depth h.
• 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 sheath fluid injected via an inlet 19 (see FIG. 1 ).
• 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.
• the side channel 9 has a depth equal to the large depth H.
• the side channel 9 has a depth equal to the small depth h.
• the nozzle 26 has a depth equal to the small depth h.
• the lateral channel 9 comprises a filter 6 consisting of pads distributed along the paths of the sheath fluid upstream of the nozzle 26.
• a filter 6 consisting of pads distributed along the paths of the sheath fluid upstream of the nozzle 26.
• such a filter makes it possible to retain any undesirable particles of dimensions greater than the width of the nozzle 26 likely to block the nozzle 26 and impair its operation.
• the lateral channel 9 channel makes it possible to supply additional sheath fluid to the microdroplet train already formed.
• the lateral channel 9 channel makes it possible, on the one hand, to modify 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, since it requires great precision of action to sort a microdroplet without influencing the adjacent microdroplets.
• the addition of sheath fluid via the side channel 9 allows the spacing between microdroplets to be increased to values of several microdroplet diameters, thereby increasing the sorting selectivity.
• 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.
• the flow rate of sheath fluid in the side channel 9 also influences the production frequency by modifying the hydrodynamic resistance of the fluids circulating in the expansion zone 8.
• the sorting zone 18 is a dielectrophoresis sorting zone.
• 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 identically to the fluidic channels with inlets at their ends.
• These channels are then filled with a conductive material which may be hot-injected indium, for example at approximately 80°C.
• Other materials may be considered for manufacturing the electrodes, such as ionic liquid, charged resin or conductive gel.
• 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 symmetrical in shape relative to the longitudinal direction of the fluid channel 27 of the sorting zone 18.
• Each ground electrode 11 has an M shape, the tips of the M being arranged closest 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 ⁇ m and 10 ⁇ m.
• 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 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.
• the two ground electrodes 11 move away from the fluidic channel 27 so as to avoid interfering with the active electrode 10.
• 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 upstream and downstream of the sorting zone 18.
• the active electrode 10 is framed by the fluid channel 27 and by one of the ground electrodes 11.
• 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 fusion of several already sorted microdrops.
• the arrangement of the electrodes 10, 11 allows greater efficiency of the electric field, in particular because the fluid 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 fluid channel 27. For example, the distance between the electrodes and the fluid channel 27 is 10 pm, the channel has a width of 20 pm and the drops 32 circulate in the middle of the fluid channel 27. distance between electrodes 10, 11 and drops 32 is in this example approximately 20pm.
• 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 10 ps and 200 ps; it may be a positive pulse, a negative pulse, a period of a sinusoid or square or any waveform with zero average, or several periods of sinusoids, squares or waveforms with zero average.
• This pulse may be of direct voltage.
• 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.
• 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 10ps and 50ps to allow sorting for a drop passage frequency of between 10 kHz and 50 kHz.
• 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 output channels 13. Conversely, in the absence of an electrical pulse, a microdrop is directed towards the other of the two output channels 14.
• the fractions sorted in the two output channels 13, 14 can be collected in separate collectors connected respectively to the outputs 16 and 17.
• 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.
• the two active electrodes 10 may be controlled alternately: one of the electrodes being used to direct a microdrop 32 towards the outlet 14 (trash bin) and the other electrode 10 being used to direct another microdrop 32 towards the channel 13 of collection.
• the two active electrodes 10 can be driven simultaneously with different voltages, so as to reduce the voltage required to obtain the dielectrophoresis effect.
• the microfluidic chip comprises channels or zones having two distinct depths.
• 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 microdrops.
• zones 3, 4, 5, 7 and 8 are the zones of low depth h ⁇ 4.5 pm.
• the linear velocity of the fluid is determined by the volume flow divided by the channel section. Choosing a low channel depth (4.5 pm) at this location ensures a higher linear velocity, and thus prevents the particles transported by the central fluid from settling.
• 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 large depth H of 10 pm, to make it possible to reduce the constraints on the flow of the fluid and therefore the necessary pressure.
• 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 efficiency of the sorting.
• the balancing microchannels 15 have a depth equal to the shallow depth h, of 4.5 pm for example, and a narrow width of the order of 5 pm. These dimensions make it possible to avoid the passage of sorted microdroplets between the two outlet channels 14 via one of the balancing microchannels 15 (see figures 5-6).
• the microdroplets 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 microdroplets 32 lighter than the oil, quickly rise to the top of the outlet channel 13, or 14 of great depth H, for example equal to 10 pm.
• the microdroplets 32 having a diameter of 4.5 pm have little risk of being captured by the balancing microchannels 15 located lower down.
• FIG. 7 shows a microfluidic chip according to a variant of the microfluidic chip described in connection with Figures 1 to 6.
• the sorting zone 18 comprises 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.
• 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 side channel 9 is adapted to inject a flow of sheath fluid onto one side of the fluid channel 27 of the sorting zone 18.
• the configuration illustrated in FIG. 7 has several advantages. First, 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 the 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.
• 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 flow of sheath fluid (for example an oily fluid) are such that x ⁇ x1 ⁇ y.
• the relaxation 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 a width of approximately 20 ⁇ m in the measuring and sorting zone 18.
• the microfluidic chip comprises channels and zones having two distinct depths: a low depth h, continuously on the fluidic path of the microdroplets 32 from the microdroplet formation zone 29 to the sorting zone 18 inclusive, 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.
• the active electrodes 10, 110 are arranged as close as possible to the fluidic channel 27, for example at a distance of approximately 10 ⁇ m.
• the use of ground electrodes 11 on either side of the active electrodes 10, 110 allows to limit the spatial extension of the electric field in the longitudinal direction of the fluid channel 27 in the sorting zone 18.
• the microfluidic device and method of the present disclosure make it possible to select and sort the drops that 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 on 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.

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