FR2941531B1 - MICROFLUIDIC DEVICE, SYSTEM AND METHOD FOR IMPLEMENTING THE SAME - Google Patents

Abstract

The invention provides a microfluidic reaction device with particles of interest (PI) present in a carrier liquid circulated by a pump from a reservoir, allowing routine use, at a rate of economic analysis, with reliability improved, and allowing reliable logistics within an analysis laboratory. The device according to the invention comprises a cover intended to be hermetically disposed on a blade, one face of which is at least partially functionalized so as to react the particles of interest, in which the cap comprises: a liquid supply channel; intended to be connected to the reservoir, and provided with N channel segments, N being an integer greater than or equal to 2, each segment being in fluid communication with at least one liquid inlet pipe for circulating the liquid in N chambers the feed channel being provided with means for accelerating the liquid along the channel; and o N liquid outlet lines out of the N chambers.

Description

MICROFLUIDIC DEVICE, SYSTEM AND METHOD FOR IMPLEMENTATION The invention relates to a microfluidic device, a system and a method of implementation.

In many technical fields, it is necessary to react certain particles of interest present in a complex liquid, that is to say having many types of particles.

For example, one can seek to detect dangerous particles present in a carrier liquid. To this end, it is sought to react these particles with suitable molecules, so that their interaction causes a signal, such as a color change, for example. It is also possible to neutralize certain particles of a liquid by reacting them with suitable neutralization molecules. We can finally try to capture molecules of interest, using capture means, to analyze them later.

In the remainder of the description, reaction is understood to mean any interaction between a molecule of interest and a suitable molecule. The interaction can be the formation of a covalent bond or not. This reaction may therefore allow capture, chemical modification or steric modification of the particles of interest.

By "liquid" will be understood in the rest of the description, the carrier liquid and the particles it contains, including the particles of interest.

A particular example is the capture of circulating tumor cells (CTCs) present in blood samples of people suffering from cancer.

Thus, breast cancer is the most common cancer in women whose main cause of death is due to the invasion of metastases. The dissemination of tumor cells in the peripheral blood is an early event in the process of tumorigenesis. The detection and the characterization of CTC represent an essential stake for the diagnosis and the prognosis of this pathology.

Three techniques are currently used for the detection of CTCs: two cytometric methods: immunomagnetic separation and cellular ultrafiltration, and a multiple marker assay using RT-PCR methodology in real time.

Immunomagnetic separation uses magnetic beads (CellServe System, Immunicon-Veridex), while cell filtration uses a Poretic Polycarbonate Track-Etch-type (PCTE) membrane calibrated with 8 μm cylindrical pores.

The complementarity of the RT-PCR assay and cytometric methods has been demonstrated in a comparative study by Ring AE et al (Detection of circulating epithelial cells in the blood of patients with breast cancer: comparison of three techniques.) AE Ring, L Zabaglo, MG Ormerod, IE Smith and M Dowsett, Brit J Cancer (2005) 92,906-912). Nevertheless, the cytometric approach offers an advantage over the RT-PCR assay: the apprehension of a circulating individual tumor cell. Its phenotypic and molecular characterization makes it possible, in addition to a better understanding of its characteristics during a recurrence of the disease and mechanisms of resistance during the treatment, a more appropriate therapeutic targeting. However, there is an imperative need to improve CTC capture efficiency.

Detection technology has already been developed by the Applicant and described in patent FR2872912. The device, a microfluidic immunosensor of cells, comprises a laminar flow chamber of liquid circulation, a wall of which has been functionalized by an organized monolayer of self-assembled silane chains, so as to fix specific antibodies capable of capturing a subunit. -population of cells within a biological sample, put into circulation in this room.

This immunosensor is powerful but it requires the manufacture of a specially adapted microscope stage.

In addition, the handling of this immunosensor is satisfactory for point experiments, but it is necessary to disassemble the glass or silicon plate at each new analysis. However, to obtain significant results, it is necessary to perform several repetitions of the analysis under the same physicochemical and biological conditions.

By dismounting each time a used plate and going up a new plate, the maintenance of the same physicochemical and biological conditions is delicate and requires a lot of precautions.

This immunosensor is therefore not very compatible with carrying out analyzes at the rates practiced in the analysis laboratories.

However, performing tests in parallel from a single source of liquid and with several previous immunosensors poses fluid circulation problems and, in particular, homogeneity of CTC distribution between the devices.

Indeed, tests have been performed with "Y" tubes used to distribute liquid comprising CTC between several immunosensors from the liquid source. Now, it can be seen that the quantity of liquid on the one hand, and the quantity of CTC on the other hand, are unequally distributed between the immunosensors (FIG. 1), and even within the same immunosensor. Moreover, when one renews the experiment, one realizes that the distribution of the liquid and the CTC is not only unequal, but also random. In other words, if an immunosensor has received a lot of fluid and / or a lot of CTC in a first experiment, it can get a lot less in a second experiment. The paralleling of several immunosensors with Y-shaped tubes thus implies capillary resistance problems that are incompatible with good reaction control of the particles of interest and therefore a satisfactory reliability of the device. In addition, such a system requires a lot of equipment (pumps and pipes) and a large amount of liquid, a significant part of which is unused since remaining in the pipes and pumps. This not insignificant amount of liquids defines the dead volume of the system.

In an attempt to solve this problem, and in order to optimize the position of the source with respect to the chamber entrances by reducing the dead volumes of liquid, the Applicant has realized a single immunosensor integrating several chambers served by a single feed channel to constant section. However, such a system also generates capillary resistance phenomena, favoring, randomly, the circulation of liquid in certain chambers.

To avoid this problem of poor distribution of the particles of interest, one solution is to provide as many identical pumping means as chambers to reproduce the experiments simultaneously, and under the same physicochemical and biological conditions. However, it can be seen that such a device allows a better distribution of the liquid in the chambers, but does not allow a homogeneous distribution of the particles of interest.

It therefore does not currently seem possible to carry out in parallel several experiments, identical or otherwise, satisfactorily.

One could consider explaining this problem of distribution of the liquid and the particles it contains by a phenomenon of sedimentation of the particles in the feed channel. Indeed, the distribution defects in the different chambers arise for liquids, aqueous or otherwise, containing particles of interest in dispersion or suspension: for example, cells, microorganisms, macromolecules, etc. Among the liquids concerned, mention may in particular be made of blood.

However, given the dimensions of the device and the flow rates used in the envisaged applications of the device according to the invention, it is not possible to explain the differences in distribution by a phenomenon of sedimentation of the particles contained. in the liquid, because these particles do not physically have the time to sediment in the pipes. Sedimentation is therefore not significant.

However, the Applicant has found that, surprisingly, the poor distribution of particles in the different chambers arises for certain liquids only, in which the carrier liquid has a dynamic viscosity of between about 5.10 -4 Pa.s and about 3.10-2 Pa.s, the particles of interest have a diameter of between approximately 1 μm (as for example bacteria) and 25 μm (for example cells, such as tumor cells), the particles of interest and the carrier liquid have a difference in density, in absolute value, between about 30 mg / ml and about 3.5 g / ml.

The viscosity values are given for a temperature of 37 ° C and at normal pressure. Thus, at this temperature, at this pressure and under the same conditions for measuring the dynamic viscosity of the aforementioned liquid, the blood has a dynamic viscosity of between approximately 4.10'3 Pa.s and 2.5 χ 10'2 Pa.s. according to its composition. Under these same conditions, the dynamic viscosity of the water was measured at about 0.7 × 10 -3 Pa.s. and the dynamic viscosity of a mixture of water and 65% of glycerol was measured at about 3.10'2 Pa.s. This mixture simulates an aqueous liquid comprising a high concentration of cells. This latter measurement was carried out with a British-Standard U-tube capillary viscometer from Cannon Instrument Company.

This problem arises, more particularly for liquids in which the carrier liquid has a viscosity of about 1.10 3 Pa.s. Such a liquid may be a diluted blood sample.

The particle distribution problem arises, especially for liquids in which the particles of interest have a diameter of from about 15 microns to about 25 microns.

It arises, more particularly for liquids in which the particles of interest and the carrier liquid have a difference in density, in absolute value, between about 2 g / ml and 3 g / ml and, in particular about 2, 5 g / mL.

The present invention therefore aims at providing a device allowing a homogeneous and reproducible diffusion of a aforementioned liquid in several rooms in parallel, usable routinely, at a fast rate and under satisfactory economic conditions, with improved reliability by performing in parallel several identical or different experiments on a large capture surface, limiting the amount of liquid needed, and allowing a reliable logistics within an analysis laboratory. To this end, the subject of the invention is a microfluidic reaction device with particles of interest present in a liquid circulated by a pump from a reservoir, comprising a cap intended to be placed hermetically on a slide, one face of which is at least partially functionalized so as to react the particles of interest present in the liquid, the cap comprising: o a liquid supply channel, intended to be connected to the reservoir, and provided with N channel segments, N being a integer greater than or equal to 2, each segment being in fluid communication with at least one liquid inlet pipe for circulating the liquid in N chambers, the feed channel being provided with liquid acceleration means along the channel ; and ο N liquid outlet pipes out of the N chambers.

According to other embodiments: the liquid acceleration means may consist of at least two segments of the supply channel of different sections, the sections being smaller and smaller in the direction of circulation of the liquid; • the intake ducts may show a sudden enlargement of the section in the direction of circulation of the liquid; • the cover can be transparent; and / or • the cover may be of polymethyl methacrylate (PMMA). The invention also relates to a reaction system with particles of interest present in a liquid coming from a reservoir, comprising: a blade, one face of which is at least partially functionalized so as to react the particles of interest present in the liquid; a seal having a planar portion, to be arranged hermetically on the blade, and provided with N openings each defining a laminar flow chamber, N being an integer greater than or equal to 2; a previous device, arranged such that the hood is hermetically disposed on the blade; a liquid reservoir in fluid communication with the supply channel of the device; at least one evacuation tube connected to the outlet pipes of the device; at least one pump controlling the circulation of the liquid from the reservoir to the outlet pipes, via the N chambers of the device; a sensor arranged to allow analysis of the glass slide when the liquid has circulated in the chambers.

According to other embodiments: the system may further comprise at least one liquid circulation tube making it possible to recycle the liquid from the outlet pipes to the tank; The system may further comprise a casing arranged in a hermetic manner between the cover and the seal; The casing may be made of polymethylmethacrylate (PMMA), aluminum or aluminum alloy; The housing may comprise the liquid reservoir; The seal may be polydimethylsiloxane (PDMS); and / or • the seal may further comprise a rod provided with a distal tubular portion, providing the sealed connection between the reservoir and the hood supply channel. The invention also relates to a reaction method with particles of interest present in a liquid from a reservoir, comprising the following steps: - providing a previous reaction system; generating a liquid flow in the supply channel in fluid communication with at least one liquid inlet pipe per chamber; accelerate the liquid along the feed channel; drain the liquid out of each chamber.

According to other embodiments: the method may further comprise a step of homogenizing the liquid in each intake pipe; The method may comprise the following steps: functionalizing a slide, so as to react the particles of interest (PI) present in the liquid; - providing a seal having a flat portion provided with N openings each defining a laminar flow chamber, N being an integer greater than or equal to 2; sealing the seal on the blade, - providing a cover comprising a liquid supply channel provided with N channel segments each being in fluid communication with at least one liquid inlet pipe in a chamber; - Hermetically placing the cover on the seal, - generate a liquid flow in the supply channel in fluid communication with at least one liquid inlet pipe per chamber; - accelerate the liquid along the supply channel; recover the liquid, at an exit from each room; • the acceleration of the liquid can be achieved by reducing the section of the feed channel before each intake pipe, the sections being smaller and smaller in the direction of liquid flow; The carrier liquid may have a viscosity at 37.degree. C. of between approximately 10.sup.-10 Pa.s and approximately 3.times.10.sup.-2 Pa.s, the particles of interest may have a diameter of between approximately 1 μm and 25 μm, the particles of interest and the carrier liquid may have a difference in density, in absolute value, of between about 30 mg / ml and about 3.5 g / ml; The carrier liquid may have a viscosity of approximately 10 3 Pa.s; The particles of interest may have a diameter of between about 15 μm and about 25 μm; and / or the particles of interest and the carrier liquid may have a difference in density, in absolute value, of between approximately 2 g / ml and 3 g / ml and, in particular, approximately 2.5 g / ml. . Other characteristics of the invention will be set forth in the following detailed description, with reference to the appended figures which represent, respectively: FIG. 1, a diagrammatic view from above of two single-chamber immunosensors, connected in parallel, powered by "Y" branch tubes, after circulation of a liquid comprising MCF7 breast cancer cells; Figure 2 is a schematic longitudinal sectional view of a system according to the invention for the detection of particles of interest using a device according to the invention; Figure 3 is a schematic cross-sectional view of an immunosensor of the device of Figure 2 along the line II-II; Figure 4, a photograph of an alternative embodiment of the immunosensor of Figure 2 disassembled; FIG. 5 is a diagrammatic cross-sectional view seen from the side of a first embodiment of the hood supply duct of the immunosensor according to the invention; Figure 6, a top view of a blade obtained after circulation of a liquid comprising MCF7 breast cancer cells in an immunosensor according to the invention provided with the supply channel of Figure 5; Figure 7 is a schematic sectional view of a second embodiment of the hood supply channel of the immunosensor according to the invention; FIG. 8 is a view from above of a blade obtained after circulation of a liquid comprising CTCs in an immunosensor according to the invention provided with the supply channel of FIG. 7; Figure 9 is a schematic perspective view of a variant of an immunosensor according to the invention, provided with a housing; Figure 10 is a bottom view of the immunosensor of Figure 9; Figure 11 is a schematic perspective view of an embodiment of a seal for an immunosensor according to the invention; Figure 12 is a partial schematic side view of the seal of Figure 11; Figure 13 is a schematic perspective view of mounting the blade under the housing of the immunosensor of Figure 9; Figure 14 is a schematic perspective view of mounting the cover on the housing of the immunosensor of Figure 9; and Figures 15 and 16, two partial enlargements of Figure 14 at two different angles, showing a means for positioning the cover on the housing. The exemplary system according to the invention described with reference to FIGS. 2 to 16 is an immunosensor for capturing CTC in a blood sample. Specifically, CTCs are circulating tumor cells in the peripheral blood of a patient. The immunosensor 1 illustrated in FIG. 2 comprises a plate 2, preferably made of glass or silicon, one face of which 2a is functionalized by means of recognition molecules 3, adapted to the particles of interest which it is desired to capture in the liquid. In the application described, the face 2a is chemically modified by silane chains. These chains are described in patent FR2872912.

A seal 4 is disposed on the blade 2. It is provided with four openings 5 each defining the contour of a laminar flow chamber.

Of course, the seal has a broad meaning. It may consist of several independent loops, that is to say not connected to each other, but each defining the contour of a laminar flow chamber. However, it is more practical that the seal consists of a single piece comprising several openings, that is to say forming several loops connected together.

A cap 6 is disposed above the seal and comprises a supply channel 7 for supplying each chamber with liquid, and liquid outlet lines 8 out of each of the chambers.

More particularly, the feed channel 7 is provided with as many channel segments as the seal comprises apertures. In the illustrated example, the supply channel 7 comprises four channel segments 7a, 7b, 7c and 7d. Each segment is in fluid communication with a liquid inlet line 9a, 9b, 9c and 9d in a chamber.

According to the invention, the feed channel 7 is provided with liquid acceleration means along the channel. In the exemplary embodiment illustrated in FIG. 2, the liquid acceleration means are constituted by four segments of the feed channel each having different sections, the sections being smaller and smaller in the direction of circulation of the liquid, according to the arrow F1. This means of acceleration makes it possible to keep the speed of the liquid substantially constant along the supply channel 7, and ensures a homogeneous distribution and circulation of the particles of interest in each chamber (see FIG. 8). The immunosensor 1 is in fluid communication with a reservoir 10 of liquid 11. On the other hand, in the embodiment illustrated in FIG. 2, each output line 8 of the immunosensor 1 is connected to a pump 13 by the According to an alternative illustrated in FIG. 4, each outlet pipe 8 can be connected directly to the pump 13.

Pump means any pumping means for circulating the liquid in the system and in the immunosensor. Thus, for example, a syringe used to circulate the liquid is considered a pump within the meaning of the present invention.

The pump 13 controls the circulation of the liquid 11 from the reservoir 10 to the outlet lines 8 via the chambers of the immunosensor. The pump 13 can be mounted in pressure, that is to say it pushes the liquid 11 from the reservoir 10 to the immunosensor 1, or in suction, that is to say, it sucks the liquid through the outlet lines, which sucks the liquid from the tank 10 to the immunosensor 1.

In other words, a flow rate is imposed in each chamber by a pump implemented upstream or downstream of the immunosensor. According to a preferred mode of use, the pump 13 is a multi-channel peristaltic pump placed downstream of the immunosensor. In this case, each outlet pipe of each chamber is connected to a channel of the pump. When the pump is downstream of the immunosensor, it operates on aspiration. This allows a better evacuation of the gas bubbles present in the liquid.

The pump 13 can simply evacuate the liquid according to the arrow F2 or, thanks to a liquid recirculation tube 14, recycle the liquid along the arrow F3 from the outlet pipes 8 to the tank 10.

A sensor 15 is disposed with respect to the immunosensor so as to allow analysis of the blade 2 when the liquid has circulated in the chambers. Preferably, the sensor 15 is an optical sensor which can be arranged above the cover 6, as illustrated in FIG. 2, or below the blade 2. In order to detect the particles, the position of the sensor may require optical transparency of the cover .

Thus, thanks to the device for detecting particles of previous interest, and in particular thanks to the immunosensor 1 according to the invention, a flow of liquid is generated in the supply channel of the immunosensor and the liquid is accelerated. along this channel, preferably before it enters each chamber, to maintain its substantially constant velocity along the channel.

With this method, the distribution of the liquid and the particles of interest it contains is homogeneous in each chamber. In other words, the invention makes it possible to simultaneously perform several capture experiments, identical or not, of particles of interest with the same physicochemical and biological conditions for each repetition.

The number N of chambers, and therefore the number of repetitions, is an integer greater than or equal to 2.

According to a preferred embodiment, the blade 2 has a standard size microscope slide, that is to say a size of 76x25mm (3 "x1"). With a blade of this size, it is preferable to produce four individual laminar flow chambers having an oblong shape circumscribed by a rectangle of length 16 mm and width 6 mm.

According to the embodiment, illustrated in FIG. 3, the gasket 4 has a flat part 4a provided with openings 5 (only one is visible in FIG. 3) each defining a laminar flow chamber and means 4c, 4d of hermetic retention. of the cover 6 and the blade 2. The thickness "e" of the flat portion 4a of the seal 4 determines the height of each chamber. Preferably, this thickness e is chosen equal to about 0.5 mm. With a microscope slide 2 of standard size and having four chambers 16 mm long and 6 mm wide, an immunosensor is obtained with laminar flow chambers of 48 mm 3 each.

The small volume of each of these chambers allows, nevertheless, a laminar flow and a reaction of the particles of interest with little liquid.

The device according to the invention thus achieves a miniaturization making it possible to carry out several analyzes simultaneously, with a single sample of liquid in a small quantity.

Figure 4 illustrates an alternative embodiment of the immunosensor of Figures 2 and 3 disassembled.

The seal 4 has two C 4b parts between which the flat portion 4a of the seal extends.

This is provided with five openings 5 each defining a laminar flow chamber.

The seal thus has two longitudinal grooves 4c in which the cover 6 must be slid, and two groove 4d in which the blade 2 must be slid.

The seal is of a flexible material, such as polydimethylsiloxane (PDMS). The dimensions of the grooves of the seal are calculated so that the seal is itself clamped between the cover and the blade once the system is mounted, so as to seal each laminar flow chamber.

In other words, the sealing of the laminar flow chambers is provided by the seal whose crushing by the cover 6 and the blade 2 makes each chamber hermetic.

The blade 2 illustrated in FIG. 4 has five functionalized surfaces 2b so as to fix the particles of interest 3 present in the liquid. For example, if it is desired to capture cultured breast cancer tumor cells, MCF7, these 2b surfaces, chemically modified by silane chains, are coated with anti-epithelial cell adhesion molecule 3 antibodies recognizing the EpCAM human antigen expressed on the surface of MCF7 cells.

Each surface 2b functionalized by the antibodies is intended to be opposite each opening 5 of the seal 4.

In a non-illustrated embodiment, it is possible that the entire face 2a of the plate 2 is chemically modified by silane chains and functionalized with antibodies. However, the parts in contact with the seal 4, that is to say all parts of the seal other than the openings 5, will not be used to capture the particles of interest.

The cover 6 has five lines 8 out of the liquid out of the chambers. Each pipe 8 is intended to be connected directly to a pump 13. According to an alternative illustrated in FIG. 2, each outlet pipe 8 can be connected to the pump 13 via a discharge pipe 12.

The cover 6 may have stops 6a to precisely position the cover in the grooves 4c of the seal 4.

The acceleration means (not visible) of the liquid along the channel 7 make it possible to obtain a homogeneous distribution of the particles of interest in each chamber and substantially identical between the chambers.

An example of a first embodiment of a supply channel having such acceleration means is illustrated in FIG. 5. The channel 7 has four channel segments 7a, 7b, 7c and 7d. The segments 7a to 7c have identical sections. The segment 7d, the most downstream with respect to the direction of flow of the liquid according to the arrow F4, has a lower section than the segments 7a to 7c. In the example shown, the latter have a section of 2 mm in diameter, while the segment 7d has a section of 1 mm in diameter. These dimensions are chosen to be able to apply a flow rate of 90 μl per minute per chamber with an average speed in the channel of 19 mm per second. Each segment 7a to 7d is in fluid communication with a pipe, respectively 9a, 9b, 9c and 9d, for admitting the liquid into a chamber.

Preferably, as illustrated in FIG. 5, the inlet ducts 9a, 9b, 9c and 9d have a sharp enlargement 15 of section in the direction of circulation of the liquid according to the arrow F5. In the illustrated example, the inlet ducts have an upstream section of 0.4 mm in diameter, and a downstream section of 0.8 mm in diameter.

This sudden enlargement disrupts the flow in the inlet ducts of the chambers and homogenizes the concentration of particles (PI or CTC) in these ducts. This is illustrated in FIG. 6, which represents a view from above of a plate obtained after circulation of a liquid comprising particles of interest in an immunosensor equipped with the supply channel of FIG. 5.

It is also advantageous for the supply channel 7 to terminate above the last intake duct, here duct 9d. The end of the feed channel 7 advantageously has an angle α of about 60 ° with respect to the axis of the channel.

An example of the embodiment of the feed channel 7 of Figures 2 and 3 is illustrated in Figure 7.

The channel 7 has four channel segments 7a to 7d, each of different sections, the sections being smaller and smaller in the direction of flow of the liquid according to the arrow F4. In the illustrated example, the segment 7a has a section 2 mm in diameter, the segment 7b has a section 1.7 mm in diameter, the segment 7c has a section 1.4 mm in diameter and the segment 7d has a section of 1 mm in diameter, the flow rate, in use, being about 90 pL / min.

The reduction in section along the channel makes it possible, in response to the significant reduction in flow rate in the channel, to maintain a quasi-constant liquid velocity. It is in this sense that one speaks of "means of acceleration of the liquid along the channel". This is illustrated in FIG. 8 which represents a view from above of a plate obtained after circulation of a liquid comprising particles of interest in an immunosensor equipped with the supply channel of FIG. 7.

White zones W, without cell capture, illustrated by way of example, may be the consequence of non-homogeneous functionalization of the surface of the blade.

It is preferable that the seal 4 is disposable to avoid leaks and obtain accurate results.

According to a variant of the invention illustrated in FIG. 9, an immunosensor 20 according to the invention may be provided with a housing 21.

In this embodiment, the housing 21 comprises a body 210 provided with a reservoir 211 and holding means 212 of a cover 6. The housing also comprises openings 213 in number equal to the number of chambers, here four.

FIG. 10 shows a view from below of the variant of FIG. 9. The housing 21 comprises means 214 for holding a functionalized blade 2.

Preferably, on the lower face, the casing has a shoulder 215 at the edge of the openings 213 and between the openings.

This shoulder 215 is intended to receive a seal 300 illustrated in FIG. 11. This seal may be produced by injection molding the casing, making it integral with the latter.

The latter has a planar portion 301 comprising four loops interconnected and defining four openings 302. The seal optionally comprises a rod 303 provided with a distal tubular portion 304 intended to ensure the sealed connection between the reservoir 211. and the feed channel 7 of the hood 6.

The seal 300 has a crushable lower lip 305 (FIG. 12) intended to come into contact with the glass slide for sealing between the blade and the seal 300.

The upper face 306 of the gasket 300 is intended to come into contact with the casing 20 via the shoulder 215.

In a first variant, not shown, the cover 6 has a flat bottom surface, intended to be applied to the housing 20. However, in this variant, it is necessary to ensure that the application between the cover and the housing is waterproof. For this purpose, it is possible to provide a second adapted seal.

In a second variant, more advantageous, the cover has counterforms 6b (see Figure 14) adapted to the openings 213 of the housing 20. In addition, the counterforms have a height slightly greater than the thickness of the housing so that when the cover 6 is positioned on the casing 20, the counter-forms pass through the openings 213 and crush the edges of the seal 300.

Thus, the floor of the laminar flow chambers is constituted by the upper surface of the blade 2 chemically modified by silane chains, and functionalized by antibodies, the wall of the chambers is constituted by the edge of the openings 302 of the seal, and the Ceiling of the chambers is constituted by the surface of the counterforms of the cover 6. The assembly is sealed thanks to the crushing of the seal between the counterforms of the cover 6, the casing 20 and the glass plate 2. Other on the other hand, the same seal 300 can also ensure the seal between the reservoir 211 and the feed channel 7 of the cover 6 thanks to the distal tubular portion 304.

The mounting of the immunosensor of Figure 9 is illustrated in Figures 13 to 16.

Thus, the seal 300 is positioned so that its planar portion 301 is under the casing 20. When the seal 300 comprises a rod 303, the rod is passed through the passage opening (not shown) so as to position the seal. distal tubular portion 304 against the opening 211a (FIG. 15) of the reservoir 211.

Then, according to the arrow F6, the blade 2 is inserted so that the at least partially functionalized face is in contact with the seal 300. The blade 2 is held in position by the elastic means 214.

Then, the cover 6 is positioned on the casing 20.

Preferably, the cover 6 and the casing 20 have positioning means of keying type to avoid assembly errors. In the example illustrated in Figures 14 to 16, the cover 6 has a stud 6c and the housing 20 has a notch 216 of complementary shape to the stud 6c.

Thus, the cover 6 is engaged on the casing 20 along the arrow F7 so as to connect the stud 6c and the notch 216. Then the cover 6 is plate on the casing 20 along the arrow F8 so as to block the cover 6 with the blocking means 212.

The capture of particles of interest (PI) present in the liquid 11 coming from the reservoir 211, then necessitates generating a flow of the liquid in the feed channel and then in the intake ducts to each chamber and finally to recover the liquid, at an exit from each room.

According to the invention, the liquid is accelerated along the feed channel, preferably before it enters each intake line. The acceleration of the liquid is preferably achieved by reducing the section of the feed channel before each intake pipe, the sections being smaller and smaller in the liquid flow direction.

The liquid may also undergo a homogenization step in each intake pipe.

Thus, the immunosensor according to the invention has a structure that makes it easy to handle because it is quick to assemble and disassemble. In addition, it is possible to achieve it in standard dimensions for routine use, at a fast rate of analysis and under satisfactory economic conditions. On the other hand, thanks to the multiple chambers and to the good homogeneity of distribution of the liquid between them, the immunosensor according to the invention makes it possible to carry out in parallel several experiments, identical or not, simultaneously and under physicochemical conditions. and identical biologicals. The reliability of the analyzes is thus improved.

Finally, the assembly can carry machine-readable identification means, such as a bar code or an RFID chip. This allows reliable logistics within an analysis laboratory. The immunosensor according to the invention is particularly well suited to the detection of circulating tumor cells (CTC) present in a blood sample.

Tests have shown that the blood sample can include up to one hundred million nucleated cells without affecting the immunosensor function.

Of course, it will be necessary to adapt, depending on the applications, the dimensions of the segments of the supply channel, the intake ducts and all the liquid lines depending on the liquid used, particles of interest to detect and their concentration.

The devices of the invention can be used in all kinds of applications, especially for high throughput screening and high reagent content;

In chemistry, they can be used to make reactions, in particular to implement combinatorial chemistry processes, separations, liquid-liquid extractions, crystallization and solubilization tests of mineral or organic molecules / particles.

They offer the possibility of synthesizing and transporting organic molecules but also micro or nano particles organic, inorganic or metallic in situ (such as latex or colored pigments for cosmetics or painting to fluorescent nano particles).

Another important application is the implementation of formulation assistance protocols (optimization of chemical mixtures to access a given physical and / or chemical property).

They can be used for the construction of DNA chips and protein chips.

In biology, they allow testing on cells inside a device of the invention. They can be used for the detection of rare cells in a biological fluid: - CTC detection for diagnosis and therapeutic monitoring; - detection and purge of contaminants by CTCs in stem cells; - detection of fetal cells in the maternal blood for prenatal diagnosis (Rhesus factor, trisomy, genetic abnormalities ...).

They can also be used to detect the evolution of pathological cells for the diagnosis of autoimmune diseases.

They can also be used to detect the evolution of specific post-vaccine cells.

They can also have applications in clinical, food or environmental microbiology such as the detection of pathogens from various samples (blood, food, water ...).

Cellular selection by a functionalized surface in a fluidic microsystem is particularly aimed at the pharmaceutical industries involved in the clinical diagnosis fields from a blood sample or a biological fluid.

The devices of the invention can also be used for the study of products extracted / used in the exploitation of petroleum deposits (crude oil, drilling fluids, sludge), paints, cosmetic creams or food formulations (quality control ).

The foregoing device is generally used in an arrangement such that the feed channel and the intake ducts are disposed at a greater height than the chambers. This arrangement is preferably used when the particles of interest have a density greater than that of the carrier liquid, in the previously described ranges of values.

The system can also operate "flipped": the arrangement is then such that the feed channel and the intake ducts are arranged at a lower height relative to the chambers. In this case, the liquid is sucked upwards, that is to say towards the chambers. This arrangement is preferably used when the carrier liquid has a density greater than that of the particles of interest (the particles tend to float on the liquid), in the ranges of values previously described.

Numerous variants and alternatives can be made without departing from the invention and in particular: • The materials for producing the cover and the housing can be of all types provided that they are non-cytotoxic, and biocompatible. If optical transparency is required to allow optical particle detection or handling comfort, a smaller choice of materials will be possible. According to the embodiment chosen, the cover is made of polymethylmethacrylate (PMMA) and the housing made of polymethylmethacrylate (PMMA), aluminum or aluminum alloy. PMMA has the advantage of reducing the capillary resistance of the feed channel relative to a material such as PDMS. The device according to the invention is also adapted to react particles of interest different from the CTCs. In this case, the partial or total functionalization of one of the faces of the slide must be adapted according to the particles of interest and the reaction that one wishes to obtain: capture, chemical or steric reaction.

Claims (19)

1. Microfluidic reaction device with particles of interest (PI) present in a carrier liquid circulated by a pump from a reservoir, comprising a cap intended to be hermetically disposed on a blade, one face of which is at least partially functionalized with in order to react the particles of interest present in the liquid, characterized in that the cap comprises: a liquid supply channel, intended to be connected to the reservoir, and provided with N channel segments, N being an integer greater than or equal to 2, each segment being in fluid communication with at least one liquid inlet pipe for circulating the liquid in N chambers, the feed channel being provided with liquid acceleration means such as the speed of the liquid; liquid along the feed channel is maintained substantially constant along the channel; and ο N liquid outlet pipes out of the N chambers. 2. microfluidic device according to claim 1, wherein the liquid acceleration means are constituted by at least two segments of the supply channel of different sections, the sections being smaller and smaller in the direction of flow of the liquid. 3. Microfluidic device according to any one of claims 1 or 2, wherein the inlet ducts have a sharp sectional enlargement in the direction of flow of the liquid. 4. microfluidic device according to any one of claims 1 to 3, wherein the cover is transparent. The microfluidic device according to claim 4, wherein the cap is made of polymethylmethacrylate (PMMA). 6. Reaction system with particles of interest (PI) present in a liquid from a reservoir, comprising: a blade, one face of which is at least partially functionalized so as to react the particles of interest present in the liquid; - A seal having a flat portion, intended to be arranged hermetically on the blade, and provided with N openings each defining a laminar flow chamber, N being an integer greater than or equal to 2; a device according to any one of claims 1 to 5, arranged such that the hood is hermetically disposed on the blade; a liquid reservoir in fluid communication with the supply channel of the device; at least one evacuation tube connected to the outlet pipes of the device; at least one pump controlling the circulation of the liquid from the reservoir to the outlet pipes, via the N chambers of the device; a sensor arranged to allow analysis of the glass slide when the liquid has circulated in the chambers. 7. System according to claim 6, further comprising at least one liquid circulation tube for recycling the liquid from the outlet pipes to the tank. 8. System according to any one of claims 6 or 7, further comprising a housing disposed hermetically between the cover and the seal. 9. System according to claim 8, wherein the housing is polymethyl methacrylate (PMMA), aluminum or aluminum alloy. 10. System according to any one of claims 8 or 9, wherein the housing comprises the liquid reservoir. 12. System according to any one of claims 6 to 11, wherein the seal further comprises a rod provided with a distal tubular portion, providing the sealed connection between the reservoir and the hood supply channel. 13. A reaction method with particles of interest (PI) present in a liquid from a reservoir, comprising the following steps: - providing a reaction system according to any one of claims 6 to 12; - Generating a liquid flow in the supply channel in fluid communication with at least one liquid inlet pipe per chamber; accelerating the liquid along the feed channel so that the velocity of the liquid along the feed channel is kept substantially constant along the channel; - evacuate the liquid from each chamber. 14. The method of claim 13, further comprising a step of homogenizing the liquid in each intake pipe. 15. A method according to any one of claims 13 or 14, comprising the steps of: - functionalizing a blade, so as to react particles of interest (PI) present in the liquid; - providing a seal having a flat portion provided with N openings each defining a laminar flow chamber, N being an integer greater than or equal to 2; - Sealing the seal on the blade, - providing a cover comprising a liquid supply channel provided with N channel segments each being in fluid communication with at least one liquid inlet pipe in a chamber; - Hermetically placing the cover on the seal, - generate a liquid flow in the supply channel in fluid communication with at least one liquid inlet pipe per chamber; accelerate the liquid along the feed channel; recover the liquid, at an exit from each room. 16. The method of claim 15, wherein the acceleration of the liquid is achieved by decreasing the section of the feed channel before each intake pipe, the sections being smaller and smaller in the liquid flow direction. 17. A process according to any one of claims 13 to 16, wherein the carrier liquid has a viscosity, at 37 ° C, between about 5.10'4Pa.s and about 3.10'2Pa.s, the particles of interest have a diameter of between about 1pm and 25pm, the particles of interest and the carrier liquid have a difference in density, in absolute value, between about 30 mg / ml and about 3.5 g / ml. 18. The process according to claim 17, wherein the carrier liquid has a viscosity of about 10-3 Pa.s. The method of any one of claims 17 or 18, wherein the particles of interest have a diameter of between about 15 μm and about 25 μm. 20. Process according to any one of claims 17 to 19, in which the particles of interest and the carrier liquid have a difference in density, in absolute value, of between approximately 2 g / ml and 3 g / ml and, in particular about 2.5 g / mL.