Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6844—Nucleic acid amplification reactions
  • C12Q1/686—Polymerase chain reaction [PCR]
• • 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
  • B01L3/50273—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 the means or forces applied to move the fluids
• • 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
  • B01L3/502761—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 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
• • 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
  • 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
  • B01L3/502792—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 for moving individual droplets on a plate, e.g. by locally altering surface tension
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L7/00—Heating or cooling apparatus; Heat insulating devices
  • B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
• • 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
  • B01L2200/0642—Filling fluids into wells by specific techniques
• • 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
  • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
  • B01L2200/0668—Trapping microscopic beads
• • 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
  • B01L2400/00—Moving or stopping fluids
  • B01L2400/04—Moving fluids with specific forces or mechanical means
  • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
  • B01L2400/0457—Moving fluids with specific forces or mechanical means specific forces passive flow or gravitation
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2400/00—Moving or stopping fluids
  • B01L2400/04—Moving fluids with specific forces or mechanical means
  • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
  • B01L2400/0463—Hydrodynamic forces, venturi nozzles
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2400/00—Moving or stopping fluids
  • B01L2400/08—Regulating or influencing the flow resistance
  • B01L2400/084—Passive control of flow resistance

Definitions

• Microfluidic process for the treatment and analysis of a solution containing a biological material for the treatment and analysis of a solution containing a biological material, and corresponding microfluidic circuit.
• the present invention relates to a process for the treatment and analysis of a solution containing a biological material, implementing a microfluidic method in which the solution is divided into a plurality of drops.
• the invention also relates to a microfluidic circuit, allowing the manipulation of very small amounts of fluids, in particular to implement such a method.
• FR-2873171, FR-2901717, WO-2011/121220 and WO-2011/039475 disclose microfluidic processes for the manufacture and manipulation, in suitable microfluidic circuits, of drip drops.
• a first fluid placed in a second fluid, called a carrier fluid The first fluid is generally an aqueous solution, divided into drops of a volume of the order of 10 to 100 ⁇ 3 .
• the carrier fluid is generally oil, which may be supplemented with a surfactant product to prevent the spontaneous fusion of the manipulated fluid drops, if they come into contact.
• RNA RNA
• Polymerase Chain Reaction that make it possible to copy in large numbers a nucleic acid sequence, such as DNA (acronym for “deoxyribonucleic acid”) or RNA (acronym for "ribonucleic acid”).
• a solution containing a small amount of nucleic acid is prepared, which is subjected to a heat treatment called thermocycling, consisting of cyclic temperature variations. These temperature variations allow for forced duplications of the nucleic acid molecules present in the solution. It is thus possible to increase very strongly the concentration of the nucleic acid in the solution.
• Document WO 2010/036352 discloses such a digital PCR method.
• a flow, or flow, of carrier fluid is used to divide the solution containing the nucleic acid into a large quantity of drops.
• the concentration of the nucleic acid in the solution is chosen so that, statistically, a small number of drops contains a molecule of the desired nucleic acid.
• the drops are placed in a container for thermocycling, allowing for polymerase chain amplification of the nucleic acid. They are then introduced into a channel to be analyzed optically, one after the other, to detect those which contained, before thermocycling, at least one occurrence of the nucleic acid, and which after this thermocycling contain a large amount of this nucleic acid.
• This method also has certain disadvantages. Thus, it imposes to have an initial drop of a well-defined size, which can be divided into drops of size suitable for subsequent processing and measurement.
• the method used for the production of the initial drops by divusion of a solution stream under the action of a carrier fluid flow, assumes a transient phase at the beginning of the production of the drops, during which the flows of the solution containing the nucleic acid and the carrier fluid must equilibrate.
• the drops formed during this transient phase therefore have an unsuitable size.
• the successive divisions of these initial drops lead to the introduction into the channel of a large number of drops of unsuitable size, which can not be validly analyzed. As a result, only a portion of the biological fluid sample can be analyzed, while another portion, representing about 10% of the sample, is lost.
• the drops can be produced and divided only under the action of a carrier fluid flow, a large amount of this carrier fluid is introduced into the channel at the same time as the drops. As a result, the concentration of the drops in the carrier fluid in this channel is not optimal.
• this method requiring the balancing of a solution stream containing the nucleic acid and a flow of biological fluid, is relatively complex to implement and requires special skills. Indeed, without a rigorous implementation of the method, the drops produced may have non-homogeneous sizes, which is detrimental to the analysis.
• the present invention aims to overcome these disadvantages of the prior art.
• the present invention aims to propose a method of treatment and analysis of a solution containing a biological material, implementing a microfluidic method in which the solution is divided into a plurality of drops, which is more rapid to implement that the processes of the prior art, more efficient, simpler and less expensive, which requires less training operators to implement the process, and that allows to process and analyze a useful proportion more important biological material consumed.
• Another object of the present invention is to provide a microfluidic circuit for the implementation of such a method.
• the invention particularly aims, according to at least one of its embodiments, to provide such a method, and the micro fluidic circuit to implement it, which allows for a digital PCR drops easier, more effective and less expensive than the methods of the prior art.
• the process advantageously allows the reaction generated by the treatment to proceed independently in each of the drops. It can be implemented particularly easily, in a single microfluidic circuit in which the different steps are performed. Moreover, the drops can be manufactured and transported independently of the presence or absence of a flow of the carrier fluid. The size of the drops, in particular, does not depend strongly on a movement of the carrier fluid, and is homogeneous from the beginning of their formation. It is thus possible that all, or almost all, of the consumed sample undergoes the treatment and is analyzed. For this, the microchannels of the microfluidic circuit are configured so that the solution circulates between walls that deviate from each other, causing a variation in the confinement of the solution.
• each wall may be progressive (steep walls) or steep (on).
• the surface tension of the solution that is to say the interfacial tension between the solution and the carrier fluid with which it is in contact, imposes on the flow of solution a form taking into account this variable confinement, which results in the separation of drops.
• This drop separation method in which the surface tension of the solution is used to cause the detachment of the drop, is therefore radically different from the methods requiring a carrier fluid flow to create a drop shear solution, in contrasting the surface tension of the solution which tends to bring together the solution. It also has the advantage of not requiring balancing of a carrier fluid flow with the solution stream, which simplifies the process.
• the displacement of the drops is also caused by the separation of the walls coupled with the effects of the surface tension of the drops. It can be caused directly, a drop moving between walls deviating, under the effect of its surface tension, or indirectly, the drop being pushed by another drop, which itself moves between the walls s 'spreading, under the effect of its surface tension.
• the drops are maintained, after their formation and their displacement, in at least one storage zone, which is an area in which it can penetrate, but from which it can not leave without external intervention (for example a flow of fluid carrier giving them sufficient energy to go out). They can thus very easily be subjected to a treatment and be analyzed.
• the carrier fluid in which the drops are detached and displaced is substantially static.
• the manufacture and displacement of the drops are thus more reliable, insofar as they are defined solely by the configuration of the walls of the microchannels, without being disturbed by a flow of carrier fluid.
• the carrier fluid although substantially static, undergoes slight disturbances caused by the displacement of the drops.
• the treatment applied to the drops comprises variations in the temperature of the drops.
• the temperature variations are applied to the entire micro fluidic circuit containing the drops. They can also be applied to sub-regions or to individual drops, for example successively one after the other.
• Temperature variations, or thermocycling may for example allow the realization of a polymerase chain reaction.
• Other treatments may also be applied, such as for example an incubation, of keeping the drops, for a sufficiently long time, at temperature conditions allowing a reaction to occur.
• the analysis of the drops is an optical analysis.
• At least one of the storage zones is constituted by a zone in which the drops have a lower surface energy than in the neighboring zones.
• the configuration of the microchannels microfluidic circuit allows the drops are maintained in the storage area, which can also be called trapping area, under the effect of their surface tension. They are therefore effectively maintained in this storage zone, independently of a possible flow of carrier fluid, as long as this flow of carrier fluid or other external action, for example the thrust of another drop, does not give them a sufficient energy to raise its surface energy through an area surrounding the storage area.
• the biological material contained in the solution comprises at least one nucleic acid
• the treatment applied to the drops is a polymerase chain reaction, making it possible to increase the concentration of at least one sequence of said nucleic acid.
• the method according to the invention thus makes it possible to carry out chain amplification by digital droplet polymerase, which is simpler and more effective than those implemented in the prior art.
• the invention also relates to a microfluidic circuit, in which are defined microchannels that can contain fluids, the circuit comprising at least one device for forming drops of a solution in a carrier fluid, and at least one drop storage zone.
• the drop forming devices comprise microchannel wall portions, spaced apart from each other so as to detach a drop of the solution under the effect of the surface tension of the solution
• the microfluidic circuit comprises means for guiding drops comprising microchannel wall portions, deviating from each other so as to move the drops to the storage area under the effect of the drop voltage.
• This circuit makes it possible in particular to implement the method described above, particularly easily. No flow of carrier fluid is indeed necessary in this circuit, the only introduction of the solution into the circuit automatically causing its division into drops and the displacement of these drops to the storage area where they can be processed and analyzed.
• At least one of the storage zones is constituted by a zone of a microchannel in which the walls of said microchannel are farther apart from one another than in the neighboring zones.
• This storage area may for example be defined in a chamber, in which the drops are confined only by an upper wall and a lower wall. An area of this room in which these two walls are more distant allows the drops to be less confined. This zone then retains the drops and constitutes a storage area.
• the microfluidic circuit contains at least two distinct storage zones.
• the microfluidic circuit comprises at least two drop-forming device, each making it possible to form drops of different volumes.
• the circuit makes it possible to simultaneously process and analyze drops of several sizes.
• the drop guide means are configured to guide the drops of different volumes to separate storage areas.
• At least one of said storage areas is configured so as to be able to receive only one drop.
• each drop can be maintained in its storage area, or individual trapping. This allows a better positioning of drops, in particular to facilitate their analysis.
• the drops being, in this case, not in contact with each other during their treatment and analysis, they are not likely to merge. It is therefore possible, in this case, to reduce the surfactant properties of the carrier fluid without inconvenience.
• At least one of the storage areas is configured to receive drops in the same plane.
• Such an embodiment makes it easier to analyze the drops.
• At least one of the storage areas is configured so as to distribute the drops it contains over at least two superposed planes.
• the microfluidic circuit is constituted, at least in part, a transparent material for seeing at least one of the storage areas from outside the circuit.
• FIG. 1 is a plan, in plan view, of a microfluidic circuit enabling the implementation of a method according to a first embodiment of the invention
• FIG. 2 is a section of the microfluidic circuit of FIG. 1;
• FIGS. 3A, 4A, 5A and 6A are details of the plane of FIG. 1, at different moments of use of the microfluidic circuit;
• FIGS. 3B, 4B, 5B and 6B are sections respectively corresponding to FIGS. 3A, 4A, 5A and 6A;
• FIGS 7, 8 and 9 are respectively a plane and two sections of a microfluidic circuit for carrying out a method according to a second possible embodiment of the invention.
• FIGS. 10 and 11 are respectively a plane and a section of a microfluidic circuit for carrying out a method according to a third possible embodiment of the invention.
• FIG. 12 and 13 are respectively a plane and a section of a microfluidic circuit for carrying out a method according to a fourth possible embodiment of the invention.
• FIG. 1 is a plane, seen from above, of a microfluidic circuit 1 making it possible to implement a method according to a preferred embodiment of the invention.
• This plan shows the different microfluidic channels that are formed within this microfluidic circuit.
• a section of this circuit micro fluidic 1 is also shown in Figure 2.
• this microfluidic circuit 1 may be composed of two superimposed plates, glued to each other.
• the circuit 1 is composed of a plate 102, which may for example be a transparent microscope slide, and a plate 101 whose faces in contact with the plate 102 are etched so as to define microchannels between the plates. two plates that are superimposed and glued to each other.
• the plate 101 may be made of a polymeric material.
• the material constituting at least one of the two plates is transparent, in order to facilitate the observation of the fluids in the microchannels. In this case, the observation of the circuit 1 makes it possible to see the microchannels by transparency, as shown in FIG.
• the dimensions of these microchannels can be chosen freely by adapting the width and the depth of the engravings in the etched plate.
• the microchannels may have a width of about 100 ⁇ and a depth of about 50 ⁇ .
• These microchannels may also have larger dimensions, or on the contrary lower, so as to adapt to the characteristics of different fluids, or the sizes of the drops to handle.
• microfluidic circuits manufactured according to other methods known to those skilled in the art can obviously be used to implement the invention.
• microchannels are normally dimensioned so that their walls exert a constraint confining the solution or on the drops which circulate there. In most microchannels, the drops are thus confined by the upper, lower, right and left walls. Some microchannels, called “chambers" thereafter, are however dimensioned so as to exert a constraint only in one dimension, two of their substantially parallel walls (generally the upper wall and the lower wall) being close to one another. another to confine the drops, and the other walls being sufficiently distant not to confine the drops.
• the microfluidic circuit 1 must, prior to its use, be filled with an inert fluid, subsequently called carrier fluid, which is immiscible with the fluids that one wishes to manipulate in the circuit.
• This carrier fluid is generally oil, which may be supplemented with a surfactant additive product that makes it possible to avoid the spontaneous fusion of manipulated solution drops, if they come into contact. This surfactant additive may sometimes be unnecessary, depending on the characteristics of the oil used as the carrier fluid and the solution to be treated and analyzed.
• the microfluidic circuit 1 shown comprises a microchannel 1 1 supply, dividing into two supply branches 1 10 and 1 1 1 extending perpendicular to each other.
• This microchannel 11 is connected to a feed hole 10 which is pierced in one of the plates making up the microfluidic circuit 1, and into which the needle of a syringe or the end of a pipette can be inserted in order to injecting a fluid into the supply channel 11.
• the chamber 13 also has an evacuation opening connected to a hole 14 pierced through one of the plates of the circuit 1. This opening allows in particular the evacuation of a part of the carrier fluid, when the total volume of fluid inserted in the microchannels is greater than the volume of these microchannels.
• the two feed branches 110 and 111 are each connected to a plurality of drop forming nozzles 12.
• the nozzles have been shown in the figure with dimensions larger than their normal dimensions. Moreover, only some of the nozzles 12 are referenced in FIG.
• drop forming nozzles 12 are microchannels, or small section conduits that can be supplied with fluid at their first end and passing a small flow of this fluid to a second end.
• Figures 3A, 4A, 5A and 6A show in detail the plane of a drop forming nozzle 12 and the chamber into which it opens, at several moments of the formation of a drop of fluid.
• This nozzle and this chamber are also represented in detail by the sections of FIGS. 3B, 4B, B and 6B, which respectively correspond to the views of FIGS. 3A, 4A, 5A and 6A.
• the carrier fluid that fills the channels of circuit 1 is not shown in these figures.
• the second end of the nozzle 12 opens on a central chamber 13, which has an upper surface etched in the plate 101 and a lower surface formed by the plate 102.
• the upper surface of the chamber 13 has an inclined zone 131, so that the two surfaces of the chamber 13 move apart as they move away from the second end of the nozzle 12. This separation of the walls allows the confinement suffered by the solution decreases during its journey, after passing through the nozzle 12.
• the inclined zone may be replaced by a zone forming a succession of several steps in the surface of the chamber, without departing from the scope of the invention.
• the person skilled in the art knows that such a succession of steps has the same technical effect as an inclined zone.
• the walls deviate in width rather than deviate in height.
• the shape of the microchannels of the microfluidic circuit 1, and more precisely the succession of a drop forming nozzle 12 and a chamber 13 in which the surfaces deviate from each other away from the nozzle 12, allows the formation of drops 40 of fluid 4, without any flow of the carrier fluid is necessary.
• the only action necessary to form these drops is indeed the introduction of the fluid 4 into the hole 10 with sufficient pressure.
• the formation of the drops can also be performed by applying suction (or negative pressure) to the outlet 14 of the microfluidic circuit, after introducing the fluid 4 into the hole 10. The drops are then formed in the same manner.
• the fluid introduction pressure 4 in the microfluidic circuit 1 has only a very slight effect on the size of the drops 40 formed. It has thus been shown by the inventors that a multiplication per thousand of the fluid introduction pressure 4 only multiplies by two the size of the drop produced.
• the microfluidic circuit 1 thus makes it possible to produce drops
• Each nozzle 12 can thus, when it is powered by upstream by a continuous flow of fluid, here by the fluid from the supply branches 1 10 and January 1, provide downstream drops of homogeneous size of the same fluid.
• nozzles 12 are shown on the microfluidic circuit 1 of FIG. 1. However, it is obvious that more numerous, and smaller, nozzles can be used in other microfluidic circuits for carrying out the process. 'invention. For example, a microfluidic circuit comprising 256 nozzles of height 50 ⁇ and width 100 ⁇ each, makes it possible to decompose a sample of approximately 20 ⁇ of solution in approximately 100,000 drops in two minutes.
• the nozzles may be distributed around three sides, or four sides of a rectangular chamber, or be distributed around a part or the whole of the periphery a chamber having a different shape, for example round, hexagonal, etc.
• storage area or “trapping area”, in the present description means an area of the microfluidic circuit in which a drop can penetrate, but it can not leave without outside intervention.
• an area is etched in the upper surface of this chamber 13, so as to form a drop storage area 130, located in the middle of the chamber 13.
• the chamber 13 Around the storage area 130, the chamber 13 has upper and lower surfaces which are preferably parallel and which are sufficiently close so that the drops placed in the chamber are confined between these two surfaces, without being able to take the spherical shape which corresponds to a minimum surface energy.
• the distance between the upper surface of the chamber and its lower surface is greater (for example about 50 ⁇ ) in the storage area than in neighboring areas.
• a drop placed in this storage area may therefore take a more compact shape than a drop confined between the upper and lower surfaces of the chamber 13, around the Storage area 130.
• a drop in the storage area has less surface energy than a drop outside that area.
• a drop placed in this storage area can not leave without energy to increase its surface energy.
• the storage area 130 thus forms a space in which the drops are held, and is preferably dimensioned such that the drops are disposed in a plane, in two dimensions. All its drops contained in this area are thus directly visible from outside the microfluidic circuit, because of the transparency of at least one of the surfaces of the chamber.
• the storage area 130 is located near the place where the drops are formed.
• the drops are introduced into this storage area 130 as soon as they are formed, without any external means being necessary to move them towards this zone.
• the conformation of the walls of the chamber 13, and in particular the separation of the walls at the inclined zone 131 and the edges of the storage zone 130, makes it possible for each drop to move under the effect of its surface tension. up to this storage area. It is also possible that the drops move in the chamber 13 towards the storage area, being pushed by other drops.
• the microfluidic circuit 1 is, before use, filled with a carrier fluid.
• a carrier fluid To perform a treatment and analysis of a solution containing a biological material, an operator introduces this solution through the hole This introduction is simply done by adjusting the end of a pipette or the needle of a syringe in the hole 10 before ejecting this fluid by pressing the syringe or the pipette.
• the fluid then flows into the feed channel 11, then into its branches 110 and 111. It then passes through the various nozzles 12, at the exit of which it breaks down into drops that flow into the chamber 13. In this way, the large number of nozzles 12 distributed along the branches 110 and 111 of the feed channel, a large number of drops can be created simultaneously. These drops are captured and retained in the storage area 130, and quickly fill the entire storage area.
• the operator can monitor the filling of the chamber 13 and stop injecting the solution when the storage area 130 is completely filled, to prevent drops of the solution from escaping through the discharge opening connected to the hole 14.
• the sample volume to create enough drops to fill the storage area is known, it is also possible to inject precisely this volume of the solution, to avoid losing part of the sample. In this case, it may be useful to inject a small amount of carrier fluid into the hole 10 after the solution injection, in order to repel the solution. remaining in the feed channel 11 and its branches 110 and 111 to the chamber 13.
• the operator can withdraw the pipette or the syringe from the hole 10. Due to the retention of the drops in the storage area 130, the microfluidic circuit 1 can then be handled by the operator without the risk of escape of the drops.
• the entire microfluidic circuit 1 may for example be placed in a heating device for its thermocycling, or any other heat treatment, without risk of losing a portion of the solution sample divided into drops. It is also possible to perform other types of treatment, in addition to or instead of a heat treatment.
• an optical analysis of the drops can be performed very easily, all the drops contained in the storage zone 130 of the chamber 13 being advantageously visible by a transparent face of the microfluidic circuit 1. This analysis can advantageously be carried out automated way.
• Figure 7 is a plan, in top view, of a microfluidic circuit 7 for implementing a method according to a second possible embodiment of the invention. Sections of this microfluidic circuit 7 are also represented by FIGS. 8 and 9. Like the microfluidic circuit 1, the microfluidic circuit 7 is composed of a transparent plate 702 and a plate 701 engraved so as to define microchannels between the microfluidic circuits. two plates, when they are superimposed and glued to each other.
• This microfluidic circuit 7 comprises a feed hole 70 connected to a feed microchannel 71. Twelve drop forming nozzles 72 are connected to this microchannel supply 71, and open on a chamber 73. In the embodiment shown, all the nozzles 72 (which, for the sake of clarity, are not all referenced in Figure 7) are identical. They are preferably of the same type as the nozzles 12 of the microfluidic circuit 1.
• the upper surface of the chamber 73 has several inclined zones 731, 732 and 733, respectively, with different slopes. Each of these inclined zones is located near the end of some of the nozzles 72.
• the inclined zone 731 visible in particular in the section of FIG. 8, has a relatively small slope, so that the upper and lower surfaces
• the sloped zone 733 visible in particular in the section of FIG. 9, has a relatively steep slope, from the lower part of the chamber 73, to a small distance from each other. such that the lower and upper surfaces of the chamber 73 move sharply away from the nozzles 72.
• the inclined zone 732 has an intermediate slope.
• the drops that are produced by the nozzles 72 and the surfaces of the chamber 73 are of different sizes for each of the inclined zones.
• the drops produced at the inclined zone 731 are larger than those produced at the inclined zone 732, itself greater than that produced at the inclined zone 733.
• the storage area 734 is located near the inclined area 731 to collect the drops formed at that inclined area.
• the storage areas 735 and 736 are placed, respectively, close to the inclined zones 732 and 733.
• the dimensions of each of these storage areas are adapted to the dimensions and the quantity of the drops they are intended to receive.
• partitions 737 and 738 rising over the entire height of the chamber 73, make it possible to partially partition the latter to prevent some of the drops from moving towards a zone of storage that is not intended for them.
• the microfluidic circuit 7 makes it possible to prepare, simultaneously, samples of drops of different sizes of the same solution. These samples can then undergo the same treatments, before being analyzed. Such a method may be useful, for example, for the analysis of a solution for which the size of drops making it possible to obtain an optimal result is not known.
• FIG. 10 is a plan, in plan view, of a microfluidic circuit 8 making it possible to implement a method according to a third possible embodiment of the invention.
• a section of this microfluidic circuit 8 is also represented by FIG. 11.
• This circuit 8 is largely identical to the microfluidic circuit 1. It comprises in particular the same feed hole 10, the same feed microchannel 11 dividing in two supply branches 110 and 111, and the same drop-forming nozzles 12.
• the central chamber 83, into which the drop-forming nozzles 12 open, has inclined zones 831 identical to the inclined zones 131 of the microfluidic circuit 1.
• the upper wall of the chamber 83 is etched to define, not one, but four storage areas of the separate drops.
• These four storage areas 832, 833, 834 and 835 are, in the embodiment shown, identical. They may, however, for the purposes of an experiment, have different dimensions, for example to contain drops distributed in a different number of layers. During the production of the drops, they fill the different storage areas, if necessary by being pushed to these storage areas by other drops.
• FIG. 12 is a plane, seen from above, of a microfluidic circuit 9 making it possible to implement a method according to a fourth possible embodiment of the invention.
• a section of this microfluidic circuit 9 is also shown in FIG. 13.
• This circuit 9 is also largely identical to the microfluidic circuit 1. It comprises in particular the same feed hole 10, the same feed microchannel 1 1 dividing in two supply branches 110 and 1 1 1, and the same nozzles for forming drops 12.
• the central chamber 93, into which the drop-forming nozzles 12 open, has inclined zones 931 identical to the inclined zones 131 of the circuit microfluidic 1.
• This chamber 93 is etched to define a plurality of small holes 932.
• These holes 932 (which are not all referenced in FIGS. 12 and 13, for the sake of clarity) may have a small diameter, for example example between about 10% and 120% of the diameter of a drop.
• Each of these holes 932 constitutes a storage area, or a "trap", capable of receiving a single drop.
• the forming drops fill the chamber 93, they are placed on each of these storage areas 932, where appropriate by being pushed from one storage area to another by another drop. It is also possible, according to a variant of this embodiment, that the upper wall of the chamber 93 is not perfectly parallel to its lower wall, in order to form a slight slope favoring the displacement of the drops towards the storage areas 932 which are the furthest from the drop forming nozzles 12.
• Each of the storage areas 932 is thus quickly occupied by a single drop.
• the microfluidic circuit 9 thus makes it possible to produce, process and analyze a plurality of drops which each occupy a well-defined position. precise, known in advance. Such an arrangement of the drops can greatly facilitate the optical analysis of the results of a treatment performed on the drops.
• the drops produced do not remain in prolonged contact with each other.
• the positions of the various storage areas 932 are advantageously chosen so that the trapped drops do not touch. This absence of prolonged contact between the drops considerably reduces the risk of merging several drops into one.
• the use of a surfactant additive (surfactants used to prevent the coalescence of drops between them), added to the carrier fluid, may be unnecessary. In other cases, a low-performance surfactant additive may be sufficient.
• This embodiment is therefore particularly advantageous in that it makes it possible to avoid the use of the most effective surfactant additives, which can be expensive.
• the method according to the invention therefore makes it possible to make the treatment and the analysis of a solution containing a biological material divided into drops faster, more efficient, simpler and less expensive.
• the process according to the invention allows almost all of the solution consumed to be divided into droplets that can be processed and analyzed. which is advantageous over the solutions of the prior art which induce the loss of a significant part of the treated solution.
• the drop forming nozzles may be distributed over several sides of the chamber intended to collect the drops. It is thus possible to distribute them on two sides of a square chamber, as represented for example in the embodiment of FIG. 1. It is also possible to distribute them on three or four sides of such a chamber. It is also possible to distribute them around a chamber of different shape, for example around almost the entire diameter of a circular chamber.
• This method also makes it possible to perform other types of treatment and analysis of solutions containing biological material.
• it is for example possible to introduce into the microfluidic circuit a solution containing a small amount of enzymes and a substrate capable of reacting with the enzyme.
• Some time after the formation of the drops it is possible to analyze them optically (either automatically, or by visual observation and counting) to determine the proportion of the drops in which an enzymatic reaction has occurred, and thus to quantify the presence of enzyme.
• the treatment applied to the drops is an incubation, consisting only in maintaining them for a sufficiently long time at temperature conditions allowing the enzymatic reaction.
• the microfluidic circuit It is also possible, for example, to introduce into the microfluidic circuit a solution containing cells and markers capable of to interact with some of these cells. Some time after the formation of the drops, it is possible to analyze them optically (either automatically, or by visual observation and counting) to determine the proportion of the drops in which cells have interacted with the markers, and thus to quantify the presence of the cells to be characterized.
• the treatment applied to the drops is a simple incubation.
• microfluidic circuit object of the invention making it possible to implement the method according to the invention, is itself particularly simple and inexpensive to manufacture. Many variants of this circuit can be implemented easily. It is thus possible, for example, that the central chamber of the circuit itself constitutes the drop storage zone, provided that suitable means prevent the drops from leaving without external intervention.

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