EP3085444A1 - Microfluidic device for controlling the flow of a fluid - Google Patents

Abstract

The invention relates to a microfluidic device for controlling the flow of at least one fluid of interest, comprising a microfluidic network (3) comprising at least one series of microfluidic chambers (5) interconnected sequentially, each chamber microfluidic being switchable between a first closed state blocking the flow of the fluid of interest and a second open state allocating a predetermined volume to said chamber while pumping the fluid of interest into said chamber.

Description

TECHNICAL AREA

The present invention relates to microfluidic devices of the biochip type, and more particularly, to a microfluidic device for controlling and sequencing a plurality of fluid flow stages and to an analysis method implemented by such a device.

The invention has applications in many fields, such as among others the fields of medical research, biology and pharmaceuticals.

STATE OF THE PRIOR ART

A microfluidic device that can chain several stages of flow of a fluid is known in the field of microsystems for medical or chemical analysis.

Indeed, the patent application FR 2897282 discloses a method of controlling the advance of a liquid in a microfluidic component comprising a plurality of reaction zones as well as a plurality of passive valves operating capillary forces for blocking the fluid between the reaction zones. The control of the advance of the liquid is carried out by the control of the pressure upstream and downstream of the component. A pressure pulse can unlock a valve and allow the advance of the liquid.

However, by exploiting capillary forces in order to advance the liquid, the success of the blocking / unblocking is dependent on the wetting properties of the walls with the liquid used. More precisely, the parameters such as the geometries of the valves and the reaction zones, the surface states or the wetting properties must be finely determined, which complicates the realization of the device. In particular, the addition or removal of wetting agent required for a given biological protocol may be incompatible with the basic principle of the blocking valves.

The object of the present invention is, therefore, to overcome the aforementioned drawbacks by proposing a microfluidic device for controlling the flow of a fluid perfectly adapted to a chemical or biological protocol and very simple to achieve.

STATEMENT OF THE INVENTION

• un premier substrat (11) ayant une première surface microfluidique (111),
• un deuxième substrat (13) ayant une deuxième surface microfluidique (131),
• une membrane déformable (15) disposée entre lesdites première et deuxième surfaces microfluidiques,
• au moins une suite de cavités (51) formée sur l'une ou l'autre des première et deuxième surfaces microfluidiques et délimitant avec ladite membrane (15) une suite de chambres microfluidiques, ladite membrane déformable étant apte à être déformée au niveau de chaque chambre microfluidique pour être plaquée contre une partie de la première surface microfluidique (111) ou contre une partie de la deuxième surface microfluidique (131) permettant ainsi de commuter l'état de chaque chambre microfluidique (5) entre un premier état fermé bloquant l'écoulement du fluide d'intérêt et un second état ouvert allouant un volume prédéterminé à ladite chambre, la membrane étant adaptée pour ouvrir ou bloquer le passage du fluide ainsi que pour pomper le fluide depuis une chambre vers au moins une autre chambre, les volumes des différentes chambres à l'état ouvert étant identiques, et
• au moins une suite de canaux microfluidiques de communication (25) formée sur l'une ou l'autre des première et deuxième surfaces microfluidiques, ladite suite de canaux microfluidiques reliant de manière séquentielle ladite suite de chambres microfluidiques, ladite au moins une suite de canaux microfluidiques de communication (25) présentant un volume plus petit que celui de ladite au moins une suite de cavités (51).

The subject of the invention is a microfluidic device for controlling the flow of at least one fluid of interest, comprising

• a first substrate (11) having a first microfluidic surface (111),
• a second substrate (13) having a second microfluidic surface (131),
• a deformable membrane (15) disposed between said first and second microfluidic surfaces,
• at least one series of cavities (51) formed on one or the other of the first and second microfluidic surfaces and delimiting with said membrane (15) a succession of microfluidic chambers, said deformable membrane being able to be deformed at each microfluidic chamber for being pressed against a part of the first microfluidic surface (111) or against a part of the second microfluidic surface (131) thus making it possible to switch the state of each microfluidic chamber (5) between a first closed state blocking the flow of the fluid of interest and a second open state allocating a predetermined volume to said chamber, the membrane being adapted to open or block the passage of the fluid as well as to pump the fluid from one chamber to at least one other chamber, the volumes of different rooms in the open state being identical, and
• at least one suite of microfluidic communication channels (25) formed on one or the other of the first and second microfluidic surfaces, said sequence of microfluidic channels sequentially connecting said plurality of microfluidic chambers, said at least one sequence of channels communication microfluidics (25) having a volume smaller than that of said at least one series of cavities (51).

Thus, the microfluidic chambers themselves allow to move a fluid in a sequence of well controlled operations without resorting to subsidiary valves or pumps and without depending on the state of the surfaces or wetting properties of the walls of said chambers . Moreover, this device makes it possible to have a large number of reaction chambers with identical volumes that are well calibrated. requiring no means of measurement while minimizing dead volumes. This makes it possible to easily and accurately transport calibrated volume (s) of fluid (s) in order to integrate a fluidic protocol. This device is very simple to build and use while being very accurate. In particular, the deformable membrane has a dual function, namely a valve and pump function. Indeed, it serves both to block the fluid in the microfluidic chambers and to pump (or suck) the fluid from one chamber to another. It allows a simple and controlled actuation of the state of each chamber and then allows the displacement of a chamber liquid chamber by successive actuation of the chambers between closed state and open state.

Advantageously, at least one of the microfluidic chambers contains an embedded reagent (preferably dried or freeze-dried) adapted to react with the fluid of interest.

Thus, from chamber to chamber, the fluid of interest mixes with the reagent (s) which is (are) already present in the device making it possible in a simple manner to carry out a chemical or biological analysis protocol. many stages.

Advantageously, the volume of a microfluidic communication channel is about ten times smaller than the volume of a cavity. This makes it possible to minimize the appearance of air bubbles and to avoid dilution of the mixture (fluid and onboard reagents) during transport from room to room.

Advantageously, the microfluidic network comprises an inlet orifice (or reservoir) formed in the first substrate and connected to an inlet of said at least one series of microfluidic communication channels, said orifice being adapted to receive the fluid of interest. Thus, a sample of the fluid of interest can be easily injected into the device.

Advantageously, the microfluidic network comprises an outlet orifice (or reservoir) formed in the first substrate or the second substrate and connected to an output of said at least one series of microfluidic communication channels. Thus, the mixture can be easily recovered or evacuated.

Advantageously, the first substrate, the membrane and the second substrate are assembled so as to ensure a tight contact between the membrane and the first and second surfaces of the first and second substrates while providing a space of flow at cavities and microfluidic communication channels.

Thus, good contact and good sealing are maintained between the membrane and the two substrates.

Advantageously, the assembly is carried out by gluing, by plasma, or by mechanical plating.

Thus, the assembly can be achieved by different methods with or without glue or by a simple mechanical plating that can be provided by a flange system.

Advantageously, the device comprises an actuating mechanism adapted to act on the membrane at each microfluidic chamber in order to switch the state of the selected microfluidic chamber.

This allows for a precise and automatic analysis protocol with a simple operating mode.

Advantageously, the actuating mechanism is selected from the following mechanisms: pneumatic, mechanical, electrostatic, piezoelectric, and magnetic.

Thus, there is a large choice of mechanisms for actuating the change of state of the different microfluidic chambers.

Advantageously, the actuating mechanism is a pneumatic mechanism adapted to deform the membrane by exerting pressure via actuating holes formed in the second substrate and opening at the levels of the microfluidic chambers, the change of state of any microfluidic chamber being effected by a modification of the pressure value exerted on the membrane via the actuating hole corresponding to said microfluidic chamber.

This makes it possible to control the actuation of the various microfluidic chambers in a simple, precise, robust, space-saving manner and without resorting to mechanical parts passing through the substrate.

According to a first preferred embodiment of the present invention, said at least one series of cavities and said at least one series of microfluidic communication channels are formed on the first microfluidic surface, a microfluidic chamber being in an open state when the deformable membrane is tackled on the second microfluidic surface so that the membrane and the corresponding cavity delimit a reservoir of predetermined volume equal to that of the cavity thus allowing the flow of the fluid of interest into said reservoir, and a microfluidic chamber being in a closed state when the membrane is plated on the first microfluidic surface while conforming to the shape of the corresponding cavity so that the volume between the membrane and the cavity is virtually zero thus blocking the flow of the fluid of interest through said chamber.

According to this first embodiment, the cavities and the communication channels are advantageously machined on the same substrate and the deformation of the membrane makes it possible to create microfluidic chambers while pumping the fluid of interest from chamber to chamber.

According to a second preferred embodiment of the present invention, said at least one series of cavities is formed on the second microfluidic surface and said at least one sequence of microfluidic communication channels is formed on the first microfluidic surface, a microfluidic chamber being an open state when the membrane is plated on the second microfluidic surface while conforming to the shape of the corresponding cavity so that the membrane and the first microfluidic surface delimit a reservoir of predetermined volume equal to that of the cavity thus allowing the flow of the fluid of interest in said reservoir, and a microfluidic chamber being in a closed state when the membrane is plated on the first microfluidic surface so that the volume between the membrane and the first surface is virtually zero thereby preventing the flow of fluid from interest through said chamber.

According to this second embodiment, the cavities and the communication channels are machined on different substrates.

Advantageously, the first substrate and / or the second substrate are made of a transparent material.

Thus, it is possible to use a simple optical detection to measure the result of a chemical or biological analysis carried out by the device.

Advantageously, the cavities have a spherical cap shape whose base has a diameter between about 1 mm and 1 cm and whose height is between about 100 microns and 1 mm.

Thus, during a chemical or biological analysis, it is advantageous to target reaction volumes of a few microliters (100nl to 100 .mu.l).

Advantageously, each channel of said at least one suite of microfluidic communication channels has a length between about 1 mm and 5 mm and a section between about 50 microns and 500 microns side.

Thus, the volume of each microfluidic channel is at most ten times smaller than that of a microfluidic chamber.

Advantageously, each of said first and second substrates has a thickness between about 200 microns and 4 mm and a surface of the order of several square centimeters.

Advantageously, the material of the first substrate and / or the second substrate is selected from the following materials: polycarbonate polymer, PMMA, COC, silicon, and paper.

According to a preferred embodiment of the present invention, the microfluidic network comprises a set of microfluidic branches connected to the same input port and each comprising a series of microfluidic communication channels connecting sequentially a corresponding sequence of microfluidic chambers.

This makes it possible to perform several analyzes in parallel.

Advantageously, the branches are arranged in comb or star from said inlet port.

By placing different reagents in the different branches, it is thus possible to carry out several analyzes on the same sample placed in the inlet orifice.

• injection d'un liquide correspondant au fluide d'intérêt dans le dispositif microfluidique, et
• commutation de l'état des chambres microfluidiques (entre état fermé et état ouvert) selon une séquence d'opérations prédéterminée adaptée pour contrôler le passage du liquide entre les différentes chambres.

The invention also relates to an analysis method implemented by means of a microfluidic device according to any one of the preceding characteristics, all the microfluidic chambers being initially in the closed state and at least one of the microfluidic chambers containing a reagent embedded system, said method comprising the following steps :

• injecting a liquid corresponding to the fluid of interest into the microfluidic device, and
• switching the state of the microfluidic chambers (between closed state and open state) according to a predetermined sequence of operations adapted to control the passage of the liquid between the different chambers.

Thus, from chamber to chamber, the liquid is mixed with the reagents making, for example, a multi-step biological analysis protocol.

Advantageously, the process comprises stirring steps by flipping the liquid between two microfluidic chambers a specified number of times.

This makes it possible to accelerate the mixing between the reaction liquid and the reagent (s).

Advantageously, the method comprises a step of optical measurement (fluorescence or colorimetric) of the liquid (reaction) in at least one (last) detection chamber.

Thus, one can easily analyze (or read) the result of the analysis protocol.

BRIEF DESCRIPTION OF THE DRAWINGS

• La Fig. 1 illustre de manière très schématique un dispositif microfluidique de contrôle d'écoulement d'un fluide d'intérêt, selon l'invention ;
• La Fig. 2 illustre de manière très schématique un dispositif microfluidique de contrôle d'écoulement d'un fluide d'intérêt, selon un mode de réalisation de l'invention ;
• La Fig. 3 illustre de manière très schématique un dispositif microfluidique de contrôle d'écoulement d'un fluide d'intérêt, selon un premier mode de réalisation préféré de l'invention ;
• Les Figs. 4A et 4B illustrent de manière schématique un assemblage du dispositif microfluidique par un plaquage mécanique ;
• Les Figs. 5A et 5B illustrent de manière schématique les états d'une chambre microfluidique selon le mode de réalisation de la Fig. 3 ;
• La Fig. 6A illustre de manière schématique un procédé d'analyse mis en oeuvre au moyen d'un dispositif microfluidique selon l'invention ;
• La Fig. 6B illustre une première chambre microfluidique dans un état fermé et une deuxième chambre microfluidique dans un état ouvert selon le procédé de la Fig. 6A;
• La Fig. 7 illustre de manière très schématique un dispositif microfluidique de contrôle d'écoulement d'un fluide d'intérêt, selon un deuxième mode de réalisation préféré de l'invention ;
• Les Figs. 8A et 8B illustrent de manière schématique les états d'une chambre microfluidique selon le mode de réalisation de la Fig. 7 ; et
• Les Figs. 9A à 9C illustrent de manière très schématique différentes configurations d'un dispositif microfluidique, selon d'autres modes de réalisation de l'invention.

Embodiments of the invention will now be described, by way of nonlimiting examples, with reference to the accompanying drawings, in which:

• The Fig. 1 illustrates very schematically a microfluidic device for controlling the flow of a fluid of interest, according to the invention;
• The Fig. 2 very schematically illustrates a microfluidic device for controlling the flow of a fluid of interest, according to one embodiment of the invention;
• The Fig. 3 illustrates very schematically a microfluidic device for controlling the flow of a fluid of interest, according to a first preferred embodiment of the invention;
• The Figs. 4A and 4B schematically illustrate an assembly of the microfluidic device by mechanical plating;
• The Figs. 5A and 5B schematically illustrate the states of a room microfluidic according to the embodiment of the Fig. 3 ;
• The Fig. 6A schematically illustrates a method of analysis implemented by means of a microfluidic device according to the invention;
• The Fig. 6B illustrates a first microfluidic chamber in a closed state and a second microfluidic chamber in an open state according to the method of Fig. 6A ;
• The Fig. 7 very schematically illustrates a microfluidic device for controlling the flow of a fluid of interest, according to a second preferred embodiment of the invention;
• The Figs. 8A and 8B schematically illustrate the states of a microfluidic chamber according to the embodiment of the Fig. 7 ; and
• The Figs. 9A to 9C illustrate very schematically different configurations of a microfluidic device, according to other embodiments of the invention.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

The concept underlying the invention consists in controlling the flow of a fluid through a series of tanks without using valves and without resorting to capillary forces.

The Fig. 1 illustrates very schematically a microfluidic device for controlling the flow of a fluid of interest, according to the invention.

The microfluidic device 1 comprises a microfluidic network 3 comprising at least one series of microfluidic chambers 5 interconnected sequentially. The microfluidic chambers 5 are able to be deformed in a retracted or deployed manner. More particularly, each microfluidic chamber 5 is switchable between a first closed state preventing or blocking the flow of the fluid of interest through the chamber 5 and a second open state allocating to the chamber in the open state a predetermined volume while pumping. (aspirant) the fluid of interest in this chamber. The open state thus allows the flow of the fluid of interest while calibrating the volume of the chamber. More particularly, the volume of each chamber 5 is adjustable or inflatable between an almost zero volume and a calibrated or non-zero predetermined volume. When the chamber 5 is in a closed state, its volume is zero and therefore, the advance of the fluid is blocked. On the other hand, when the chamber is in an open state, its volume is calibrated and the fluid can flow to fill the chamber. The action of switching the state of a chamber from a closed state to an open state allows the fluid to be pumped into this chamber. Thus, the successive actuation of the microfluidic chambers 5 between a closed state and an open state makes it possible to move a reaction volume from chamber to chamber without using auxiliary valves and without resorting to pumps or capillary forces. Note that the volumes of the different chambers 5 in the open state are identical. Advantageously, the volume of a chamber 5 in the open state is of the order of a few hundred nanoliters to a few hundred microliters.

The microfluidic device 1 also comprises a reservoir or inlet orifice 7 adapted to receive a sample of a fluid of interest. The inlet orifice 7 is connected to an inlet of the microfluidic network and more particularly to a first chamber of the suite of microfluidic chambers 5. A single chamber suite is shown in FIG. Fig. 1 but of course, the microfluidic device may comprise a plurality of chamber suites (see Figs. 9A and 9B ) and possibly a plurality of inlet ports.

The microfluidic device 1 can be used to perform a chemical or biological analysis on a sample of a fluid of interest consisting for example of a biological fluid. Indeed, reagents can be embedded in dried form in at least one of the microfluidic chambers 5. Thus, and chamber to chamber, the fluid of interest placed in the inlet port 7 mixes with the reagents for the purpose of perform a multi-step analysis protocol.

The Fig. 2 illustrates very schematically a microfluidic device for controlling the flow of a fluid of interest, according to one embodiment of the invention.

The microfluidic device 1 comprises two substrates 11, 13 and a deformable membrane 15 disposed between the two substrates.

The first substrate 11 comprises a first microfluidic surface 111 and the second substrate 13 comprises a second microfluidic surface 131. The first and second surfaces 111, 131 are parallel to each other and separated from each other by the deformable membrane 15. first and second surfaces 111, 131 may be of rectangular or circular shape or any other shape.

Throughout the following description, by convention, a direct orthonormal coordinate system in Cartesian coordinates (X, Y, Z) presented on the Fig. 2A . The plane (X, Y) is parallel to said first and second surfaces 111, 131 and the Z direction is oriented from the second surface 131 to the first surface 111.

Each of the first 11 and second 13 substrates has a thickness in the Z direction between about 200 μm and 4 mm and a surface (ie, area of the first or second microfluidic surface in a (X, Y) plane) of order of several square centimeters, typically a surface equivalent to a microscope slide or a credit card. The deformable membrane has a thickness (in the Z direction) of the order of one hundred microns (10 μm to 1 mm), for example 300 μm.

The material of the first substrate 11 and / or the second substrate 13 is selected from the following materials: polycarbonate polymer, PMMA, COC, silicon, and paper. The membrane 15 is formed of a highly deformable material such as an elastomer of the silicone family (example PDMS polydimethylsiloxane, Ecoflex ^®).

At least one series of cavities 51 is formed on one or the other of the first and second microfluidic surfaces 111, 131. For example, the Fig. 3 shows that the cavities 51 are formed on the first microfluidic surface 111 of the first substrate 11 and the Fig. 7 shows that the cavities 51 are formed on the second microfluidic surface 131 of the second substrate 13.

Advantageously, the cavities 51 have a shape of spherical cap whose base has a diameter in a plane (X, Y) between about 1 mm and 1 cm and whose height in the Z direction is between about 100 microns and 1 mm.

The succession of cavities 51 defines with the deformable membrane 15 the following microfluidic chambers 5. The deformable membrane 15 is able to be deformed at each microfluidic chamber 5 to be pressed against a part of the first microfluidic surface 111 or against a part of the second microfluidic surface 131 thus making it possible to switch the state of each microfluidic chamber 5 between the first and second states. The membrane is adapted to open or block the passage of the fluid and to pump the fluid from a reservoir or room to at least one other room.

Indeed, the microfluidic device 1 comprises an actuating mechanism 21 adapted to act on the membrane 15 at each microfluidic chamber 5 in order to switch the state of the selected microfluidic chamber. The actuating mechanism 21 may be of the pneumatic or mechanical type.

In addition, at least one suite of microfluidic communication channels 25 is formed on either one of the first and second microfluidic surfaces 111, 131. Each suite of microfluidic communication channels 25 sequentially connects the suite of microfluidic chambers. 5 (here, only one suite of microfluidic communication channels 25 formed on the first surface 111 and a single suite of microfluidic chambers 5 are shown). More particularly, the various channels of the suite of microfluidic communication channels 25 connect in series the different microfluidic chambers 5, each communication channel 25 having a length of between approximately 1 mm and 5 mm and a cross section between approximately 50 μm and 500 μm. side. The suite of microfluidic communication channels 25 has a smaller volume than that of the series of cavities 51.

According to a preferred embodiment of the invention, the volume of a microfluidic communication channel 25 is at most ten times smaller than the volume of a microfluidic chamber 5. Let V1 be the volume of a chamber 5 and V2 the volume of a microfluidic communication channel 25. The actuation of two consecutive chambers generates a liquid displacement of V1-V2, because the volume V2 remains lost in the channel 25. For N displacements, a fluid volume of N × V 2 is lost. . Thus, if the system is initially filled with air (more precisely, microfluidic channels 25 are filled with air and all microfluidic chambers 5 in the closed / blocked position), then, after fluidic movement from the first to the second chamber , the experiment shows the appearance of a bubble of a volume approximately equivalent to the volume V2. After N displacements, the volume of the air bubble is N times larger. Similarly, if the system is initially filled with a buffer liquid, for example water, (more precisely the channels 25 are under water and all the microfluidic chambers 5 in the closed position), then after a displacement, the experience shows that the transported solution is slightly diluted. Thus, with each new displacement we will observe a new slight dilution.

It is therefore advantageous to minimize the V2 / V1 ratio in order to minimize the appearance of air bubbles (or at least reduce their volume) or to avoid diluting the reagents / samples transported from room to room. This is all the more advantageous as the number of chambers required for the analysis protocol is large.

By way of example, each cavity 51 has a base of 3 mm and a height of 300 μm defining an open chamber volume of 1.1 μl, whereas each microfluidic communication channel 25 measures 3 mm * 100 μm * 300 μm. represents a volume of 0.1μl. Thus, during a chemical or biological analysis, it is advantageous to target reaction volumes of a few microliters.

Further, the microfluidic device 1 has a reservoir or inlet port 7 formed in the first substrate 11 and connected to an inlet 251 of the suite of microfluidic communication channels 25 (i.e. at the first channel of the channel sequence). Optionally, the microfluidic device 1 comprises an orifice or outlet reservoir (not shown) formed in the first substrate 11 or the second substrate 13 and connected to an outlet 253 of the suite of microfluidic communication channels 25 (ie to the last channel of the following channels).

Moreover, holes (or channels) for actuating 27 (see Figs. 3 and 7 ) are advantageously formed in the first 11 or second 13 substrate in the direction Z to lead to the levels of the microfluidic chambers 5. These actuating holes 27 are used to actuate the deformable membrane 15 by pneumatic or mechanical actuating mechanisms. For example by a suction plating action (suction effect).

The fluidic network 2, that is to say the inlet orifices 7 and possibly the outlet, the cavities 51, the microfluidic communication channels 25 connecting the cavities 51, and the holes 27 or openings for the pneumatic actuation or mechanical machining are machined according to methods known by the plastics industry such as mechanical machining with a CNC machine, by 3D printing, or preferably by injection. The first substrate 11, the membrane 15 and the second substrate 13 are assembled so as to ensure a sealing contact between the membrane 15 and the first and second surfaces 111, 131 of the first and second substrates while providing a flow space at the cavities 51 and microfluidic communication channels 25. The assembly can be made by gluing, by plasma, or by mechanical plating.

Note that a reagent is advantageously deposited in at least one of the microfluidic chambers 5 either on the membrane 15 or on the walls of the cavity 51 before the assembly of the microfluidic device 1. Thus, at least one of the microfluidic chambers 5 contains embedded reagent in dried or lyophilized form and adapted to react with the fluid of interest.

The Fig. 3 illustrates very schematically a microfluidic device for controlling the flow of a fluid of interest, according to a first preferred embodiment of the invention. In particular, this figure illustrates a section of the microfluidic device in a plane (Y, Z).

According to this first embodiment, the cavity continuation (s) 51 (in the form of a spherical cap) and the continuation (s) of microfluidic communication channels 25 are formed on the first microfluidic surface 111 of the first substrate 11. More particularly, each suite of microfluidic communication channels 25 sequentially connects the succession of cavities 51, each channel 25 connecting two consecutive cavities 51. Moreover, the inlet orifice 7 is formed in the first substrate 11.

Actuating holes 27 are formed in the second substrate 13 opening after assembly of the device 1 facing the cavities 51. In addition, the deformable membrane 15 is disposed between the first substrate 11 (here corresponding in orientation Z to a plate upper) and the second substrate 13 (corresponding to a lower plate). Thus, the suite of microfluidic chambers 5 is formed by the deformable membrane 15 and the succession of cavities 51. In other words, each microfluidic chamber 5 is delimited by a corresponding cavity 51 and a corresponding part of the membrane 15 so that the state (open or closed) of the chamber 5 is defined by the actuation of this corresponding part of the membrane 15.

By way of example, the two substrates 11 and 13 can be assembled with the deformable membrane 15 by gluing, the coating of which can be performed by screen printing so as not to locally stick the membrane at the cavities 51 or microfluidic communication channels 25 on the first 11 or second substrates 13.

An adhesive-free assembly can also be envisaged by exposing the first and second microfluidic surfaces 111, 131 to an oxygen plasma prior to assembly. In this case, it is also possible to perform localized treatment so as not to stick the deformable membrane 15 at the cavities 51 or microfluidic communication channels 25.

According to yet another example, one can simply assemble the three elements (ie the first substrate 11, the membrane 15, and the second substrate 13) by a mechanical plating provided by a flange system (enclosed greenhouse, screws etc ... ) or any known mechanical system (clips, rivets etc ...) to maintain a good contact and a good seal between the membrane 15 and the two substrates 11 and 13 except at the cavities 51 and microfluidic channels 25.

Indeed, Figs. 4A and 4B schematically illustrate an assembly of the microfluidic device by mechanical plating. In particular, the Fig. 4A illustrates an exploded view before assembly and Fig. 4B illustrates a view of the microfluidic device after assembly.

According to this example, the first substrate 11 (corresponding to an upper plate) comprises the microfluidic communication channels and cavity cavities 51 in the form of a spherical cap. Reagents 31 were dispensed and dried on the walls of the cavities 51.

The second substrate 13 (corresponding to a lower plate) has actuating holes 27 and lugs 135. The number of lugs depends on the surface of the microfluidic device 1. The assembly is carried out simply by putting into contact the first substrate 11 and the deformable membrane 15 on the second substrate 13 and the holding is obtained by the lugs 135. The lower face of the deformable membrane 15 is in contact with the second surface 131 of the second substrate 13 (lower cover) and the face upper deformable membrane 15 is in contact with the first surface 111 of the first substrate 111 (top cover). By way of example, for a microfluidic device 1 having the size of a microscope slide, ten or so pins will be placed distributed around the device to properly distribute the plating force.

The Figs. 5A and 5B schematically illustrate the states of a microfluidic chamber according to the embodiment of the Fig. 3 .

In particular, the Fig. 5A illustrates a view of a section (Y, Z) of a microfluidic chamber 5 in a closed state where the corresponding portion of the membrane is in a retracted form. Indeed, according to this state, the portion of the membrane 15 corresponding to the chamber 5 is plated on a portion of the first microfluidic surface 111 of the upper plate 11 while perfectly matching the shape of the corresponding cavity 51 so that the volume between the membrane 15 and the cavity 51 is virtually zero thus blocking the flow of the fluid of interest through the chamber 5. Here the cavity 51 has the shape of a spherical cap and the membrane 15 locally complies with the shape of the spherical cap 51 thus closing the access to the microfluidic chamber 5.

The Fig. 5B illustrates a view of a microfluidic chamber 5 in an open state where the corresponding portion of the deformable membrane 15 is put into an expanded form. Indeed, according to this state, the portion of the membrane corresponding to the chamber 5 is plated on a portion of the second microfluidic surface 131 of the lower plate 13 so that the membrane 15 and the corresponding cavity 51 delimit a predetermined volume reservoir. equal to that of the cavity 51 thus allowing the flow of the fluid of interest in the chamber 5.

According to this first embodiment, the microfluidic device 1 comprises a pneumatic actuation mechanism 21 adapted to act on the deformable membrane 15 at each microfluidic chamber 5 in order to switch the state of the selected microfluidic chamber.

Indeed, the pneumatic actuating mechanism 21 is adapted to deform at least a portion of the membrane 15 by exerting a pressure or a pressure pulse by means of a pressure fluid, and in particular a pressure gas via the actuating holes 27 formed in the second substrate 13 and opening at the levels of the microfluidic chambers 5. Thus, the change of state of any microfluidic chamber 5 is achieved by a modification of the pressure value exerted locally on the membrane 15 (here under the membrane) via the actuating hole 27 corresponding to the microfluidic chamber 5.

The pneumatic actuating mechanism 21 comprises an automaton 211 making it possible to program the pressure in each operating hole 27. A positive pressure makes it possible to lift (or retract) the deformable membrane 15 at the local level thus closing the microfluidic chamber 5 while a negative pressure ensures the plating of the deformable membrane 15 locally on the second microfluidic surface 131 of the lower plate 13 and thus the opening of the microfluidic chamber 5. Alternatively, the return to the open state of the microfluidic chamber 5 can be simply ensured by the stiffness of the membrane 15 if it is sufficiently rigid, and the open state can be obtained with a zero pressure (ie a setting to atmospheric pressure).

Alternatively, the actuating mechanism 21 may be a mechanical mechanism adapted to deform the deformable membrane 15 by actuating pistons (not shown) via the actuating holes 27 formed in the second substrate 131.

The Fig. 6A schematically illustrates a method of analysis implemented by means of a microfluidic device according to the invention.

Thereafter, any microfluidic chamber 5 "R" of rank "i" will be designated in a state closed by "Ri = 0" and in an open state by "Ri = 1".

Initially, all the microfluidic chambers 5 are in the closed state (Ri = 0 for all i) and at least one of the microfluidic chambers 5 contains an onboard reagent 31.

In step E0, a sample of a reaction liquid 33 (corresponding to the fluid of interest) is injected into the inlet orifice 7 of the microfluidic device 1. The reaction liquid 33 is for example a biological fluid (blood, saliva, urine, etc.) or a chemical fluid that must be analyzed according to a multi-step protocol.

In steps E1-E2, the state of the microfluidic chambers 5 is switched from the closed state to the open state according to a predetermined sequence of operations adapted to control the passage of the reaction liquid 33 between the various microfluidic chambers 5.

More particularly, in step E1, the deformable membrane 15 is actuated at level of the first chamber R1 which then goes from the closed state to the open state (R1 = 0 → R1 = 1) allowing the aspiration of a calibrated sample volume in the first chamber. This chamber contains the onboard reagents 31 which are dissolved in contact with the liquid 33. This first step makes it possible to carry out the first operation of the biological or chemical protocol by mixing the sample 33 with the first reagent 31.

In the next step E2, actuation of the deformable membrane 15 at the second chamber R2 opens the chamber and pump the reaction volume into the second chamber (R2 = 0 → R2 = 1). Simultaneously, or with a delay (of the order of a few seconds), the first chamber retracts (R1 = 0 → R1 = 1). Similarly, the liquid dissolves the reagents embedded in the second chamber R2 in order to perform the second operation of the analysis protocol.

In particular, Fig. 6B illustrates a top view of a portion of a microfluidic device 1 according to the invention showing a first microfluidic chamber 5 in a closed state "R1 = 0" containing no fluid and a second microfluidic chamber 5 in an open state "R2 = 0 "containing the reaction liquid 33 (optionally mixed with the reagent).

Thus, and step by step, for each operation on a chamber Ri: one has the opening of the room (Ri = 0 → Ri = 1) while the other rooms (ie for all j greater than i and for all j less than i) remain closed.

The succession of states of the process of the Fig. 6A for a four-step protocol performed in four successive microfluidic chambers can be summarized in Table T1 as follows: Table: T1 Step N ° R1 R2 R3 R4 Initial 0 0 0 0 E1 1 0 0 0 E2 0 1 0 0 E3 0 0 1 0 E4 0 0 0 1

According to another example, the four-step protocol can be defined for each operation (or step Ei) by opening the chamber of rank "i" (Ri = 0 → Ri = 1) while the chambers remain closed for all j greater than i and open for all j less than i according to the steps of table T2 as follows: Table: T2 Step N ° R1 R2 R3 R4 Initial 0 0 0 0 E1 1 0 0 0 E2 1 1 0 0 E3 1 1 1 0 E4 1 1 1 1

Other variants are possible by adding for example a buffer chamber "RT" to initiate the protocol and promote the filling of the first microfluidic chamber R1 and isolate the reservoir of the reaction liquid 33 located upstream in the inlet port 7 according to table T3 as follows: Table: T3 Step N ° RT R1 R2 R3 R4 Initial 0 0 0 0 0 E11 1 0 0 0 0 E12 1 1 0 0 0 E13 0 1 0 0 0 E14 0 0 1 0 0 E15 0 0 0 1 0 E16 0 0 0 0 1

The example of Table T3 makes it possible to define a discrete volume of fluid having a volume perfectly determined by the volume of chamber R1. This volume of samples is transported step by step on the tanks R2 to R4 by the changes of states of different chambers.

Moreover, as indicated in the table T4, it is also possible to generate (step E24) then move (step E25) a duplicate with two consecutive tanks placed in state 1. It can also "cut in two the duplicate" ( step E26) and then reassemble it (step E27), etc.

Thus, on the same device can be realized a wide variety of protocols including steps of volume formation, transport, mixing, division, etc. by simply changing the programming of the succession of states of each tank. Table: T4 Step N ° RT R1 R2 R3 R4 Initial 0 0 0 0 0 E31 1 0 0 0 0 E32 1 1 0 0 0 E33 1 0 1 0 0 E34 0 1 1 0 0 E35 0 0 1 1 0 E36 0 0 1 0 1 E37 0 0 1 1 0 E38 0 0 1 0 1

Advantageously, the method comprises stirring steps by flipping the liquid 33 between two microfluidic chambers 5 a given number of times. Indeed, to assist and accelerate the mixing between the sample 33 and the reagents 31 in a chamber 5 of rank "i", it is possible to perform stirring steps by circulating the liquid between the chamber of rank "i" and that rank "i-1". Table T5 below explains the reagent mixture between chambers R2 and R3 by flipping the liquid between these two microfluidic chambers for seven operations. Advantageously, a dozen tilts makes it possible to well homogenize the reaction mixture by stirring. Table: T5 Step N ° T R1 R2 R3 R4 Melange R3 0 0 0 1 0 0 0 1 0 0 0 0 0 1 0 0 0 1 0 0 0 0 0 1 0 0 0 1 0 0 0 0 0 1 0 Etc ...

Finally, the analysis method includes a final step E4 ( Fig. 6A ) measuring the liquid in at least one detection chamber. Indeed, the result of the analysis protocol can be performed for example by a fluorescence measurement or by colorimetry according to the protocol used. Generally this operation is carried out at the end of the protocol therefore in the last microfluidic chamber 5.

Advantageously, the first substrate 11 and / or the second substrate 13 and / or the membrane 15 are made of a transparent material thus making it possible to see the content of the microfluidic chambers 5 and to facilitate the optical measurement of the result.

It will be noted that other detection means known to those skilled in the art, such as, for example, electrical detection (conductimetry, electrochemical measurement, etc.) are also possible.

The Fig. 7 illustrates very schematically a microfluidic device for controlling the flow of a fluid of interest, according to a second preferred embodiment of the invention.

According to this second embodiment, each series of cavities 51 (spherical cap-shaped) is formed on the second microfluidic surface 131 of the second substrate 13. In addition, each suite of microfluidic communication channels 25 is formed on the first microfluidic surface 111 of the first substrate 11. The continuity between the different channels of a sequence of microfluidic channels 25 formed on the first substrate 11 is provided by the cavities 51 of the corresponding cavity sequence formed on the second substrate 13. In other words, the cavities 51 formed on the second microfluidic surface 131 have positions and diameters complementary to the positions and lengths of the channels 25 formed on the first microfluidic surface 111.

Furthermore, the inlet orifice 7 is formed in the first substrate 11 and the actuating holes 27 are formed in the second substrate 13. It will be noted that here, the actuating holes 27 open into the corresponding cavities 51 .

Like the first embodiment, the deformable membrane 15 is disposed between the first substrate 11 (here corresponding in Z orientation to an upper plate) and the second substrate 13 (corresponding to a lower plate). Thus, the suite of microfluidic chambers 5 is formed by the deformable membrane 15 and the series of cavities 51.

However, according to this second embodiment, a microfluidic chamber 5 is in an open state when the portion of the corresponding membrane is deformed and is in a closed state when the portion of the corresponding membrane is released.

Indeed, Figs. 8A and 8B schematically illustrate the states of a microfluidic chamber according to the embodiment of the Fig. 7 .

In particular, the Fig. 8A illustrates a view of a microfluidic chamber in a closed state where the corresponding part of the deformable membrane 15 is put into a relaxed form, for example by maintaining an atmospheric pressure via the actuating hole 27 formed in the second substrate 13. in this state, the portion of the membrane 15 corresponding to the chamber 5 is plated on the corresponding portion of the first microfluidic surface 111 (ie on the upper plate) so that the volume between the membrane 15 and this first surface 111 is almost zero blocking the flow of the fluid of interest through the chamber.

The Fig. 8B illustrates a view of a microfluidic chamber 5 in an open state where the corresponding portion of the membrane is put into a deformed shape. The deformation of the membrane 15 at the chamber 5 can be achieved by exerting a negative pressure via the actuating hole 27 formed in the second substrate 13. Thus, the portion of the membrane 15 corresponding to the chamber 5 is sucked in. to be plated on the corresponding portion of the second microfluidic surface 131 (ie, the lower plate) while conforming to the shape of the corresponding cavity 51 so that the deformed membrane and the corresponding portion of the first microfluidic surface 111 delimit a reservoir of predetermined volume equal to that of the cavity 51 thus allowing the flow of the fluid of interest in this reservoir.

The Figs. 9A, 9B and 9C illustrate very schematically different configurations of a microfluidic device, according to other embodiments of the invention.

The microfluidic network 3 comprises a set of microfluidic branches 37 connected to the same inlet port 7. Each microfluidic branch 37 comprises a series of microfluidic communication channels 25 sequentially connecting a corresponding sequence of microfluidic chambers 5. Thus, several reactions in parallel can be performed on the same sample volume. By placing different reagents in the different branches 37, it is thus possible to carry out several analyzes on the same sample placed in the inlet orifice.

The Fig. 9A shows that the branches 37 are arranged in comb while the Fig. 9B shows that the branches 37 are arranged in a star shape from the inlet orifice 7.

The Fig. 9C shows an example of a more complex network with intersections or crossings between several fluidic paths. Each intersection makes it possible to perform a mixing operation between different liquids, or to transport different samples or reagents on a part of the microfluidic network. Note that the size of the rooms of the different paths may be different. For example, the arrows on the Figure 9C indicate the possible directions of displacement of the fluidic volumes.

Claims (14)

Microfluidic device for controlling the flow of at least one fluid of interest, characterized in that it comprises: a first substrate (11) having a first microfluidic surface (111), a second substrate (13) having a second microfluidic surface (131), a deformable membrane (15) disposed between said first and second microfluidic surfaces, at least one series of cavities (51) formed on one or the other of the first and second microfluidic surfaces and delimiting with said membrane (15) a succession of microfluidic chambers (5), said deformable membrane being able to be deformed at each microfluidic chamber (5) to be pressed against a portion of the first microfluidic surface (111) or against a portion of the second microfluidic surface (131) thereby to switch the state of each microfluidic chamber (5) between a first closed state blocking the flow of the fluid of interest and a second open state allocating a predetermined volume to said chamber, the membrane being adapted to open or block the passage of the fluid as well as to pump the fluid from a chamber to at least another room, and at least one suite of microfluidic communication channels (25) formed on one or the other of the first and second microfluidic surfaces, said sequence of microfluidic channels sequentially connecting said series of microfluidic chambers, said at least one sequence of microfluidic communication channels (25) having a volume smaller than that of said at least one series of cavities (51). Microfluidic device according to claim 1, characterized in that at least one of the microfluidic chambers contains an on-board reagent (31) adapted to react with the fluid of interest. Microfluidic device according to claim 1 or 2, characterized in that the volume of a communication microfluidic channel (25) is about ten times smaller than the volume of a cavity. Microfluidic device according to any one of the preceding claims, characterized in that the microfluidic network comprises an inlet orifice (7) formed in the first substrate (11) and connected to an inlet of said at least one sequence of microfluidic channels of communication (25), said orifice being adapted to receive the fluid of interest. Microfluidic device according to any one of the preceding claims, characterized in that the first substrate, the membrane (15) and the second substrate (13) are assembled so as to ensure a tight contact between the membrane and the first and second surfaces. microfluidics (111, 131) of the first and second substrates (11, 13) while providing a flow space at the cavities and microfluidic communication channels. Microfluidic device according to claim 5, characterized in that the assembly is carried out by gluing, by plasma, or by mechanical plating. Microfluidic device according to any one of the preceding claims, characterized in that it comprises an actuating mechanism (21) adapted to act on the deformable membrane (15) at each microfluidic chamber (5) in order to switch the state of the selected microfluidic chamber. Microfluidic device according to claim 7, characterized in that the actuating mechanism (21) is a pneumatic mechanism adapted to deform the membrane (15) by exerting pressure via actuating holes (27) formed in the second substrate ( 13) and opening at the levels of microfluidic chambers (5), the change of state of any microfluidic chamber being achieved by a change in the pressure value exerted on the membrane via the actuating hole corresponding to said microfluidic chamber. Microfluidic device according to any one of the preceding claims, characterized in that said at least one series of cavities and said at least one series of microfluidic communication channels are formed on the first microfluidic surface (111), a microfluidic chamber (5) being in an open state when the deformable membrane (15) is pressed onto the second microfluidic surface (131) so that the membrane and the corresponding cavity delimit a reservoir of predetermined volume equal to that of the cavity thus allowing the flow of the fluid of interest in said reservoir, and a microfluidic chamber being in a closed state when the membrane is plated on the first microfluidic surface (111) while conforming to the shape of the corresponding cavity so that the volume between the membrane and the cavity is virtually zero blocking the flow of the fluid of interest through said chamber. A microfluidic device according to any one of claims 1 to 8, characterized in that said at least one series of cavities is formed on the second microfluidic surface and said at least one series of microfluidic communication channels is formed on the first microfluidic surface, a microfluidic chamber (5) being in an open state when the membrane (15) is pressed onto the second microfluidic surface (131) while conforming to the shape of the corresponding cavity so that the membrane and the first microfluidic surface (111) delimit a reservoir of predetermined volume equal to that of the cavity thus allowing the flow of the fluid of interest into said reservoir, and a microfluidic chamber being in a closed state when the membrane (15) is plated on the first microfluidic surface (111) so that the volume between the membrane and the first surface is virtually zero thus preventing the flow of the fluid of interest through said chamber. Microfluidic device according to one of the preceding claims, characterized in that : the cavities (51) have a spherical cap shape whose base has a diameter of between approximately 1 mm and 1 cm and whose height is between approximately 100 μm and 1 mm, each channel of said at least one series of microfluidic communication channels (25) has a length of between approximately 1 mm and 5 mm and a cross section between approximately 50 μm and 500 μm on one side, and each of said first and second substrates (11, 13) has a thickness of between about 200 μm and 4 mm and a surface of the order of several square centimeters. Microfluidic device according to any one of the preceding claims, characterized in that the microfluidic network comprises a set of microfluidic branches (37) connected to the same inlet orifice (7) and each comprising a series of microfluidic communication channels (25). ) sequentially connecting a corresponding sequence of microfluidic chambers (5), the branches being arranged in comb or star form from said inlet port. An analysis method implemented by means of a microfluidic device according to any one of the preceding claims, all the microfluidic chambers being initially in the closed state and at least one of the microfluidic chambers containing an on-board reagent, said method comprising the following steps : injecting a liquid corresponding to the fluid of interest into the microfluidic device, and - Switching the state of the microfluidic chambers according to a predetermined sequence of operations adapted to control the passage of the liquid between the different chambers. An analysis method according to claim 13, characterized in that it comprises stirring steps by flipping the liquid between two microfluidic chambers a specified number of times.

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