WO1998040466A1

WO1998040466A1 - Integrated fluid circuit for the execution of a chemical or biological process

Info

Publication number: WO1998040466A1
Application number: PCT/US1998/004748
Authority: WO
Inventors: Thierry L. A. Dannoux; Isabelle M. J. Geahel
Original assignee: Corning Incorporated
Priority date: 1997-03-13
Filing date: 1998-03-11
Publication date: 1998-09-17
Also published as: FR2760838B1; EP1007622A1; FR2760838A1

Abstract

The circuit comprises an inlet (3) for the injection of a sample of material to be analyzed into the circuit, cuvettes (6, 7, 9, 11, 12) filled with fluid compositions intervening in the analysis of the sample and a network of ducts (4, 6', 8, 13, 14) for the flow of the fluids present in the circuit. The inlet (3), the cuvettes (6, 7, 9, 11, 12) and the ducts (4, 6', 8, 13, 14) each comprise a wall element that is deformable between a first position where it allows the presence of a fluid under its surface and a second position where it is applied against a nondeformable surface placed opposite it, after expulsion of the fluid contained between said surfaces.

Description

INTEGRATED FLUID CIRCUIT FOR THE EXECUΗON OF A CHEMICAL OR BIOLOGICAL PROCESS

BACKGROUND OF THE INVENTION

The present invention relates to an integrated fluid circuit for the execution of a chemical or biological analysis process; more particularly it relates to such a circuit comprising at least one inlet for the injection of a sample of a fluid material to be analyzed m the circuit, at least one cuvette filled with a fluid composition used in the analysis of the sample, and a network of ducts for the flow of the fluids present m the circuit.

For the analysis of biological molecules, such as DNA molecules for the identification of infectious agents (viruses, bacteria, parasites, etc.) several biotechnological methods are known today, which work either by the recognition of genes or by the identification of products expressed by the genes. The identification of the genes is classically carried out by the analysis of the DNA molecules, with this analysis involving a series of operations involving purification, neutralization, denaturing, mixing with nitrogen bases, amplification m thermal cycles using the PCR technique, hybridization, and detection. The identification of proteins expressed by the genes takes place by lmmuno- 2 enzymatic assays, for example, according to the method known by the name of ELISA.

In all cases, the analysis requires a certain number of successive steps that are delicate and thus expensive to implement, when they are performed with the standard means and by specialized personnel.

To lower the cost of these analyses, m the article entitled "Miniaturized Genetic-Analysis System" by Rolfe

C. Anderson, Gregory J. Bogdan, and Robert J. Lipshutz presented at the conference entitled "Microfabrication technology for biomedical applications" held m San Jose, California, USA, October 24 and 25, 1996, a "charger" or miniature circuit has been proposed, for genetic analysis, by the hybridization of a sample of nucleic acid; this cartridge has the shape of a polycarbonate plate into which cavities or "reactors" are molded and m which various successive steps of the analytical method used are carried out, with ducts also being molded m the plate, forming connections between these cavities . It has also been proposed to manufacture circuits of this type made of quartz by microengravmg cavities and ducts by means of NH₄F/HF, followed by the welding of a cover onto the circuit, as described m the publication of the Oak Ridge National Laboratory Institute entitled "Integrated Microdevice for Chemical and Biochemical Analysis" by Michel Ramsay, published at the above- mentioned conference, or by micromolding silicone or PDMS from a mold made by engraving with reactive ions (RIE) as described m the publication entitled "New Materials for Chip-Based Bioanalytical Devices: Molded Silicone

Elastomer Microstructures for Capillary Electrophoresis" by Carlos Essenhauser et al., Ciba, Ltd., published at the above-mentioned conference.

In all the devices mentioned above, the reagents and other liquid media required are introduced into the circuits by means of pipettes or reservoirs, from the 3 outside of the circuits. Thus, they require manipulations that are incompatible with a complete automation of the various steps of the analyses to be used, an automation that is desirable notably for economic and practical reasons .

SUMMARY OF THE INVENTION

The purpose of the present invention is precisely to construct an integrated fluid circuit for the execution of a chemical or biological analysis process, designed to ensure an automated sequence of the different steps of said process, with this circuit integrating all the means, media, or reagents required for the execution of this process . The present invention also relates to the preparation of such a circuit that is inexpensive to manufacture, ready for use, and disposable after use.

The present invention is also intended to provide an apparatus for the use of such a circuit and to provide a method for the manufacture of this circuit.

These purposes of the invention have been obtained, as well as others that will become apparent upon reading the following description, with a fluid circuit for the execution of a process of analysis of a sample of fluid matter, the type that comprises at least one inlet for the injection of a sample into the circuit, at least one cuvette filled with a fluid composition used m the analysis of the sample, and a network of ducts for the flow of the fluid present m the circuit. According to the invention, at least one of said inlets, of said cuvettes and of said ducts comprises a wall element that is deformable, between a first position where it holds a fluid under its surface and a second position where it is applied against a nondeformable surface placed opposite it, after expulsion of the fluid contained between said surfaces . 4

As will be seen below, the crushing of the deformable wall elements of the circuit allows a very simple triggering of the different steps of the analysis process, according to a rigorously predetermined sequence, and this with a means that is simple and inexpensive, and therefore particularly economical.

According to another characteristic of the present invention, the circuit comprises a rigid plate and a sheet made of a plastically deformable material attached to said rigid plate, with surface elements opposite said plate and said sheet delimiting the internal volume of each of said ducts, cuvettes, and inlet.

According to a preferred embodiment of the circuit according to the invention, at least one of said volumes is formed m said sheet. According to a variant, at least one of said volumes is hollowed out m the rigid plate.

According to another characteristic of the circuit of the invention, the circuit comprises a rectilinear central duct that is connected, at one end, to the inlet of the circuit and to at least one cuvette that is arranged laterally with respect to said central ducu and is connected to it by a lateral duct that is slanted on the axis of the central duct of an acute angle (elbow piece) that opens towards the inlet of the circuit. The invention also provides an apparatus for the operation of this circuit comprising a circuit support means, a roller made of a plastic material, and a means to cause said roller to move on said circuit so as to sequentially crush the ducts, cuvettes, and inlet of the circuit, allowing the execution of a process of analysis of a sample of fluid material injected m the inlet of the circuit .

The invention also supplies a method for the manufacture of the circuit according to the invention, according to which ducts, cuvettes, and an inlet of the circuit are formed m a first element, generally flat, 5 forming a part of this circuit, with breakable capsules containing the analysis liquid, as well as other devices, being inserted m said cuvettes of the circuit, and with a second, generally flat, element being attached against said first element, to close said cuvettes. According to an embodiment, said breakable capsules are formed by the deformation under a vacuum of a sheet of flexible material, the filling of recesses formed by local aspiration of said sheet with a predetermined fluid, the sealing of said recesses by welding a second sheet onto the first one, and mdividualization of the capsule so formed.

Other characteristics and advantages of the present invention will become apparent m a reading of the following description.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a diagrammatic planar representation of a preferred embodiment of the integrated fluid circuit for the execution of an analysis process according to the invention,

Figure 2 is a diagrammatic representation of the essential devices of an apparatus for the operation of the circuit of Figure 1,

Figure 3 is a diagrammatic illustration of the operation of another embodiment of the circuit of the invention,

Figure 4 illustrates a method for the manufacture of capsules forming a part of the circuit according to the invention, and

Figure 5 is a diagrammatic illustration of a method for the manufacture of the integrated fluid circuit according to the invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS 6

Reference is made to Figures 1 and 2 where the circuit represented consists of a generally flat element formed in a rectangular sheet 1 made of a plastically deformable material, embossed in such a manner so as to form in this sheet a group of cuvettes or chambers and ducts, with these cuvettes and ducts being closed on one side by a second, generally flat, element formed by a rigid plate 2 (see Figure 2) attached against the sheet 1. The sheet 1 can be formed of a complex, such as the aluminum/polyethylene complex that is routinely used in industries involving the conditioning and packaging of food and pharmaceutical products. The aluminum layer of the complex imparts to it the necessary deformability for the permanent formation of ducts and cuvettes, by the embossing of a matrix having a form that is complementary to that of said ducts and cuvettes. The polyethylene layer ensures a good insulation by its chemical inertia; it has a hydrophobic character and it can be heat welded. The plate 2 can consist of glass coated with a silane to allow its attachment to the polyethylene of the complex 1 by welding or gluing. It can also consist of a plastic material having the chemical inertia necessary in the desired application. The sheet 1 and the plate 2 are attached to each other by means of an adhesive for polymerization under ultraviolet radiation or by heat welding, for example.

The circuit represented in Figures 1 and 2 will now be described in detail, with it being understood that the structure of this circuit, which is adapted to a specific application, can have numerous variations intended to adapt said circuit to a given biological or chemical analysis process.

One application of the present invention involves the identification of infectious agents such as viruses, bacteria, or parasites, which are present in body fluids or m food products, for example. This identification can be carried out, as seen above, by the recognition of the genes of said agents, m processes that require, as is known, a segment of a DNA molecule, an amplification by the so-called PCR (Polymerase Chain Reaction) , followed by hybridization. The circuit of Figures 1 and 2 is designed, more particularly, for performing such an identification process .

For this purpose, the circuit comprises (see Figure 1) an inlet 3, which is open on the edge of the circuit to receive a sample of DNA molecules to be identified, with these molecules being obtained by any of ^+~ e known extraction methods for this purpose (centπfugation, filtration, etc.) Moreover, the circuit comprises, from the inlet 3, a rectilinear central duct 4 that opens into a filter 5 that itself is connected to a reservoir or cuvette 6 by a lateral duct 6' opening at an upstream location. The filter 5 is intended to stop the analysis of any debris or precipitates contained m the sample. The reservoir 6 contains a liquid medium, called a "PCR buffer, " which is capable of allowing the amplification by the PCR technique m a chamber 7 located downstream from the filter 5 m the central duct 4. This chamber 7 is also connected by a lateral duct 8 to a reservoir or cuvette 9 that is filled with a replication-error correction medium, known as a stringent buffer, which acts m chamber 7 during the PCR amplification.

The PCR chamber comprises different materials that are involved m the amplification of a DNA strand, conditioned m the form of points of dry matter, that is: the nitrogen bases adenme, cytosme, thymine and guanme, the polymerization enzyme (a polymerase), and primers.

Also, downstream from the PCR chamber 7, a chamber 10 is located; it comprises a network of miniature wells 8 such as the network described in French Patent Application

No. 9,513,878, filed November 22, 1994 in the name of the applicant. This network can contain, for example, 200 miniature wells. Each well is equipped with probes having a purpose that will be described below.

The network can thus assume the shape of a glass plate placed in the chamber 10 formed by embossing of the complex 1, so that the wells of the network are filled with liquid originating from the central duct 4. In a variant, the network of wells can be formed by embossing of the sheet 1, as in the case of the different cuvettes and ducts of the circuit according to the invention.

One of several reservoirs or cuvettes 11,12 of rinsing liquid are arranged between the chamber 7 and the network contained in the chamber 10, with ^~.hese reservoirs being connected to the central duct 4 by lateral ducts 13,14, respectively. The liquid put out by the reservoirs 11,12 is used for rinsing the miniature wells after hybridization of the segments of the DNA molecule, as will be seen below, so as to evacuate from these wells any DNA segment that has not grafted onto the probes placed in the wells. The rinsing liquid and said segments that have not been grafted are then gathered in a collecting cuvette or chamber 15. Downstream from the latter, there is a duct 16, which is used for ventilation; this duct passes through a hydrophobic filter 17 that is air permeable but not permeable to liquids. This filter thus leaks towards the exterior of liquid contained in the circuit, and thus causes environmental pollution by this liq lid.

The integrated fluid circuit for biological analysis represented in Figure 1 has, as an illustrative and nonlimiting example only, a width of 2.5 cm and a length of 6 cm. The dimensions of the reservoirs and chambers formed in the complex are measured in millimeters. The connection ducts typically have a section of 150 mm x 150 9 mm. The complex 1 m which these ducts, reservoirs, and chambers are formed can be made of an aluminum sheet having a thickness of 40 mm, which is covered with a 15-mm layer of polyethylene. Diagrammatically, m Figure 2, m a perspective view and m cross section through a plane passing through the central duct 4 of a fluid circuit installed m the apparatus, the essential devices of an apparatus 20 are represented for the operation of the circuit of Figure 1. In the case of a circuit designed to carry out the analysis of a sample of biological material using PCR amplification, the apparatus 20 essentially comprises a support means (not shown) of the integrated fluid circuit, a roller 21 made of a plastic material, m rared-radiation heating means 22 and reading means 23. The roller 21 is arranged so as to sequentially crush the inlet 3, the duct 4, the filter 5, the reservoirs 6,9,11,12, and the cuvette or chamber 7, as will be discussed below. At 21A of Figure 1, m broken lines, the projection of the roller 21 onto the plane of the circuit is represented, with the latter being m the starting position. The shaft of the roller 21 is motorized so that this roller can move on the circuit, m the direction of arrow F, along a rectilinear trajectory parallel to the axis of the central duct 4. Heating means 22, diagrammatically represented by a bundle of infrared radiation focused by a lens 24 onto chamber 7, ensures the PCR amplification of a DNA sample to be analyzed. It is known that this technique allows the amplification of a DNA sequence several thousand times within a few tens of minutes. It is achieved by thermal cycles (which are controlled by the heating means 22) , for example, heating to 90° followed by cooling to 60°.

The reading means 23 are arranged perpendicularly to the network of miniature wells contained m the chamber 10; they consist of a matrix of photosensitive cells arranged at the pitch of the wells to deliver signals that 10 are representative of the content of these wells. The miniature wells constitute chambers with hybridization of

DNA strands or segments formed m the PCR chamber 7, with

"probes" attached m these cnambers, and with the strands becoming grafted to these probes. These probes are labeled such that they are fluorescent, which allows their detection by means of the network of photosensitive cells, and thus the identification of the DNA strands or segments constituting the sample of analyzed DNA. The circuit of Figure 1 and the apparatus of Figure 2 then operate as follows. The circuit is loaded with a sample of biological material to be analyzed, such as a sample containing DNA. The sample is introduced into the inlet 3 of the circuit according to the invention, for example, using a pipette. This inlet is then closed by any appropriate means. The circuit is then placed into the apparatus of Figure 2, between the support means constituting a plate (not shown) and the roller 21. Initially, the roller is m the position identified as 21A m Figure 1. The roller is borne by a shaft X that is movable m parallel to the plate so that the roller moves on the circuit successively and sequentially crushing the relief forms of the complex 1 of the circuit on which it moves, with said forms delimiting the ducts, reservoirs, and chambers described above. In a vaπan^4", the roller 21 could occupy a fixed position and the circuit could be displaced m a translational movement against this roller by appropriate means.

The roller first crushes the inlet 3 so as to push back its content into the duct 4 and the filter 5. The contact-generating line of the roller 21 with the circuit then reaches position 21B, which is tangent to the reservoir 6 containing the PCR buffer liquid medium. The continuation of the displacement of the roller 21 then causes the crushing of this reservoir and thus the return of its content into the filter 5, through the duct 6'. 11

In this regard, it is noted that the inclined position of the duct 6' on the duct 4, where the ducts define an acute angle a opening towards trie inlet 3, ensures that the roller 21 does not close the duct 6' by pressure before the complete emptying of the reservoir 6. It should also be noted that the roller 21, by gradually crushing the duct 4 at the same time as the reservoir 6, ensures that the content of the reservoir 6 does not flow towards the inlet 3. This content then can only pass through the filter 5 before being collected in the PCR chamber 7, where it is necessary for the PCR amplification operation to be carried out therein. As indicated above, the filter 5 has the function of retaining the debris of materials (for example, pieces of cell membranes) that must not penetrate into the PCR chamber 7.

The heating means 22 are then activated to ensure the thermal-cycle processing of the materials contained in the PCR chamber, between 60° and 90°, in 20-30 cycles of 2 in, for example. This thermal cycle allows the multiplication of the DNA fragments and strongly increases the sensitivity of the subsequent detection by the reading means 23 of the content of the miniature well of the network contained in the chamber 10. During this amplification, the roller is naturally started in the position 21C.

After the amplification, when the roller extends beyond position 21C, it is the reservoir 9 of replication- error correcting medium that is crushed by the roller and that empties into the PCR chamber 7. The roller then stops in position 21D.

Once the thermal cycle is completed, the roller 21 resumes its course successively crushing the chamber 7 and the reservoirs 11 and 12, with the DNA fragments present in the chamber 7 being then expelled into the wells of the hybridization chamber 10, where they become attached to 12 the probes that have been selectively implanted in these wells. Any unattached DNA fragments are expelled from these wells by the rinsing carried out by the emptying of the reservoirs 11 and 12 successively crushed by the roller 21, which stops in position 21E.

As mentioned above, the wells are equipped with probes, which are synthesized DNA fragments capable of recognizing and fixing, in the wells, the DNA strands resulting from the PCR amplification. After the grafting of the strands that recognized a compatible probe, the nongrafted strands are expelled by the rinsing fluid contained in the reservoirs 11 and 12, in the collecting cuvette or chamber 15. The latter communicates with the ventilation duct 7 and the filter 17; the opening to the air achieved by the duct 16 allows the fluids propelled by the roller 21 to progress in the circuit according to the invention.

With the preinstalled probes in the wells of chamber 10 being labeled with fluorescent labels, the strands grafted in the wells are identified by the fluorescences detected in these wells by the photosensitive cells of the reading means 23. The chamber 10 thus operates as a "detection chamber" to allow the decoding of the genes contained in the DNA fragments analyzed. In an application in medical diagnosis, for example, this decoding allows the identification of a given pathogenic virus.

The diagram of Figure 3 explains the operation of a variant of the integrated fluid circuit of Figure 1. The circuit of Figure 3 consists, like that of Figure 1, of a flexible and deformable sheet 1', consisting of an aluminum/polyethylene complex, for example, and a nonndeformable plate 2', made of glass or a plastic material, for example.

In the embodiment of Figure 1, the p-Nte 2 is flat and the ducts, cuvettes, chambers and reservoirs are formed in the complex 1, being glued or welded to the plate. Thus, these ducts, cuvettes, chambers, and reservoirs each comprise a deformable wall element formed from the complex. The passage of the roller 21 over these wall elements, which extend above the planes of welding or gluing of the complex to the plate, crushes the latter elements, expelling their contents. In the variant of Figure 3, the ducts, cuvettes, chambers, and reservoirs are hollowed out in the surface of the plate 2'; the complex 1' is flat and welded to this surface. The ducts, cuvettes, chambers, and reservoirs always comprise a deformable wall element; the latter is initially flat and extends above a cavity such as the cavity 9' represented in Figure 3. When the roller 21 of the apparatus represented in Figure 2 passes over such a wall element, the latter collapses under the pressure exerted by the roller, as shown in Figure 3, to become applied against the surface of the bottom of the cavity 9', expelling the contents, for example, into an adjacent duct (not shown) , which plays the role of the duct 8 of the circuit of Figure 1.

The formation of spaces corresponding to the ducts, cuvettes, chambers, and reservoirs mentioned above, in the plate 21, can be obtained by the molding of the plate 2' against a matrix having solid parts corresponding to the hollowed out parts to be formed in the plate. Other methods for the formation of these hollowed out parts could be used, for example, mechanical or chemical engraving methods .

According to another embodiment of the circuit according to the invention, which is clearly derived from those described above, the volumes of certain ducts, cuvettes, etc., could be delimited in a plastically deformable sheet, whereas the volumes of other ducts or cuvettes could be delimited in the nondeformable plate. Reference is now made to Figures 4 and 5 to describe a method for the manufacture of the circuit according to 14 the invention, for example, the one shown in Figure 1. It is apparent to a person skilled in the art that this method is immediately transposable to the circuit represented in Figure 3. First, in Figures 4A-4D a method for the manufacture of breakable capsules is described; such a method is designed to constitute the different reservoirs of fluid provided in the fluid circuit according to the invention. These capsules are designed to be installed in the cuvettes 6,9,11,12 of the circuit of Figure 1 and are designed to tear due to the crushing effect developed on them by the roller 21, and thus allow the escape of their contents (see Figure 4D) , which then flow into the circuit according to the program resulting from the topology of the circuit and the sequence of passage of the roller 21 on the latter.

In Figure 4, a sheet made of flexible plastic material 26 is represented, placed above a matrix 27 of recesses 28ι, 28₂, 28₃, etc., which communicates with the base of the matrix 27 that is opposite to that into which these recesses are hollowed out, by ducts 29ι, 29₂, 29,, etc., respectively.

The sheet 26 is applied to the recesses 28_α, as represented in Figure 4A, and the other face of the plate 27 is placed in communication with the vacuum source (not shown) which forcibly applies the sheet 26 against the bottom of the recesses.

The alveoli are then filled with the liquid to be held therein (Figure 4D) ; the filled recesses are covered with a second sheet made of plastic material 30 welded to the sheet 26 around the opening of each recess (Figure 4C) and, at the part support 31, each of the capsules 32₂, 32₂, etc., so formed is detached. The latter capsules are then put in place in the circuit according to the invention as will now be explained with reference to Figure 5. 15

In Figure 5, a band 33 of the aluminum/polyethylene complex mentioned above is represented, which enters into a press 34 compressing a tool designed to emboss, on successive areas of this band, the fluid circuit represented m Figure 1. The embossed areas then pass into one or more loading stations, such as sta Lon 35, to load these areas with breakable capsules 32_x arranged on a tray

36, or with other circuit devices (filters, plate of miniature wells, etc.) . The loading station can assume the shape of those routinely used m the electronics industry to deposit components on circuit maps. The same station can be programmed to deposit capsules of various types and other devices m the fluid circuits according to the invention. In a variant, several successive stations can be provided, with each specializing m the deposition of a single type of capsule or other device. In Figure 5, one loading station has been represented, for the clarity of the drawing, but it should be understood that other stations could be staggered on the trajectory of t\- circuits so as to gradually equip them with the different capsules and other devices that they must receive. Thus, the manufacturing line represented m Figure 5 comprises a station 37 for loading networks of miniature wells; such networks assume their place m the hybridization chamber 10 (see Figure 1) .

When all the recesses and chambers of the embossed complex are equipped with breakable capsules, filters, networks of wells, etc., the complex so equipped receives the nondeformable plate 2 that holds these different elements. The plate 2 is welded to the complex 1 by any known means, by ultrasound welding, gluing with an adhesive activated under UV radiation, etc.

Naturally, the invention is not lιmι^J -d to the described and represented embodiments, which were given only as examples. Thus, the fluid circuits represented 16 could have a shape other than the one applied for a biological analysis in which PCR amplification is used, for a hybridization in a network of miniature wells, and for a reading by the detection of fluorescent labels. Indeed, it is known that another biotechnology method for the recognition of infectious agents (viruses, bacteria, and parasites), known by the name of ELISA, proceeds to this elucidation by the identification of the expression of the genes of these infectious agents. A fluid circuit according to the invention, adapted to the implementation of this technique, then comprises means for preparing a specimen to be analyzed (filter, diluant) , a scanning matrix comprising capturing antibodies, means for rinsing, and labeled antibodies. The apparatus for using such a circuit comprises reading means adapted to these antibodies .

Similarly, the present invention is not limited to a fluid circuit designed to perform processes of biological analysis. It should be understood that a fluid circuit according to the invention could be adapted to the execution of a purely chemical analysis process, characterized by a program sequence of operations, such as operations of dilution, mixing with a reagent (s), and observation or measurement of phenomena ch racterizing intermediate or final reactions.

Thus, it appears that the invention can be applied to any analytical process that uses fluids whose actions are organized around a predetermined sequence. The circuit according to the invention, which is inexpensive to manufacture, ready for use, and disposable, can thus be used in a very great number of applications. In the field of biotechnology, it automates the complex analytic processes that can then be executed at a reduced cost by personnel with fewer qualifications than in the prior art and at the sites themselves where the results of these analyses must become available (workshops for 17 manufacturing and controlling agro-food products, for example) .

Claims

18 What is claimed is:

1. An integrated fluid circuit for the execution of a process of analysis of a sample of fluid material, comprising an inlet (3) for the injection of said sample in the circuit, at least one cuvette (6,7 .',11,12) filled with a composition of fluid involved in the analysis of the sample and a network of ducts (4, 6 ', 8, 13, 14, 16) for the flow of the fluids present in the circuit, characterized in that at least one of said inlets (3), said cuvettes (6,9,10,11,12) and said ducts

(4, 6 ', 8, 13, 14 , 16) comprises a wall element that can be deformed between a first position where it allows the presence of a fluid under its surface and a second position where it is applied against a nondeformable surface placed opposite it, after expulsion of the fluid contained between the surfaces.

2. Circuit according to Claim 1, characterized in that said circuit further comprises a rigi . plate (2;2') and a sheet (1;1') made of a plastically deformable material attached to said rigid plate, the surfaces opposite said plate and said sheet delimiting the interval volume of each of said ducts, cuvettes, and inlet.

3. Circuit according to Claim 2, characterized in that at least one of said volumes is formed in said sheet (1) .

4. Circuit according to Claim 2, characterized in that at least one of said volumes is hollowed out in the rigid plate (2 ' ) .

5. Circuit according to any one of '.laims 1-4, characterized in that said ducts, cuvettes, and inlet are arranged so as to be able to be sequentially crushed by a 19 roller (21) made of a plastic material rolling over said circuit in the form of a rectilinear trajectory.

6. Circuit according to Claim 5, characterized in that it comprises a central duct (4) that is rectilinear and connected, at one end, to the inlet (3) and at least one cuvette (6,9,11,12) arranged laterally with respect to said central duct (4) and connected to it by a lateral duct (6', 8, 13, 14) inclined on the axis of the central duct (4) by an acute angle (a), which opens towards the inlet (3) of the circuit.

7. Circuit according to any one of Claims 1-6, applied to the analysis of a DNA molecule and characterized in that it comprises a chamber (7) for PCR amplification and a chamber (10) for hybridization.

8. Circuit according to Claim 7, characterized m that said hybridization chamber is in the lorm of a network of miniature wells, each equipped with probes.

9. Circuit according to any one of Claims 1-8, characterized in that it comprises, at the end of the central duct (4) opposite the inlet (3), a collection chamber (15) for displaced fluids.

10. Circuit according to any one of Claims 1-8, characterized in that it comprises a duct (10) for ventilation of the cuvettes and ducts.

11. Circuit according to any one of Claims 1-6, applied to the execution of an immunoenzymatic assay using the ELISA technique.

12. Apparatus for the use of the circuit according to any one of Claims 1-11, characterized in that it 20 comprises a means for the support of said circuit (1,2; l',2')_/ a roller (21) made of a plastic material and a means (X) to cause said roller to move on said circuit (1,2; l',2') so as to sequentially crush the ducts, cuvettes, and inlet of the circuit (1,2; l',2') for the execution of a process of analysis of a sample of fluid material injected in the inlet (3) of the circuit (1,2; l',2') ΓÇó

13. Apparatus according to Claim 12, adapted for an integrated fluid circuit for the analysis of DNA molecules, characterized in that it comprises a means (22,24) for the thermal cycling of the content of a PCR amplification chamber present in the circuit and a reading means (23) for the content of a hybridization chamber (10), also present in said circuit.

14. Method for the manufacture of a circuit according to any one of Claims 1-11, characterized in that ducts, cuvettes, and an inlet of the circuit are formed in a first element, generally flat, forming a part of the circuit, with breakable capsules of analytical fluid being inserted, as well as other devices, into said cuvettes of the circuit, and with a second element, generally flat, being attached against said first element to close said cuvettes.

15. Method according to Claim 14, characterized in that said ducts, cuvettes, and inlet are formed by the embossing of a sheet (1,1') made of a plaj ically deformable material, constituting said first element.

16. Method according to any one of Claims 14 and 15, characterized in that said breakable capsules are made by deformation under a vacuum of a sheet made of flexible material (Figure 4A) , filling of recesses formed by a 21 local aspiration of said sheet with a predetermined fluid

(Figure 4B) , sealing of said recesses by the welding of a second sheet to the first one (Figure 4C) , and individualization of the capsules so formed (Figure 4D) .