WO2003075017A1

WO2003075017A1 - Method for analyzing a molecule comprising a support for molecules and a receiving chamber

Info

Publication number: WO2003075017A1
Application number: PCT/FR2003/000621
Authority: WO
Inventors: Antonios Vekris
Original assignee: Antonios Vekris
Priority date: 2002-03-01
Filing date: 2003-02-26
Publication date: 2003-09-12
Also published as: FR2836720A1; AU2003229841A1

Abstract

The invention concerns a method for analyzing a molecule, comprising: a support (8) for bearing at least one molecule fixed to the support and a chamber (26) for holding a liquid (52) while contacting the liquid with the support. The chamber is designed to be rigidly fixed to the support.

Description

MOLECULE ANALYSIS DEVICE COMPRISING A SUPPORT AND A RECEPTION CHAMBER

The present invention relates to devices for analyzing biological compounds.

DNA chips make it possible to measure and visualize very quickly the differences in expression between genes, and this on the scale of a complete genome. If the implementation of the technique is quite complicated, its principle is very simple. This technique consists of placing on a surface of a few millimeters or square centimeters several hundred DNA sequences specific to a given organism generally obtained by RT-PCR (Real Time Polymerase Chain Reaction, namely real-time PCR) and called probes. These fragments are brought into contact with a population of DNA strands whose composition we wish to know. The possible hybridization between a probe and the studied DNA is then observed, and indicates the presence or absence of expression of a gene, in the case of use of complementary DNA resulting from the retrotranscription of RNA m, or of the organism sought in the case of use of DNA extracted from a biological sample within which a pathogen is sought.

It is also possible to develop protein chips on which antibodies or antigens are fixed, for example, and the presence of, respectively, the antigen or the complementary antibody is detected in the biological sample.

Other macromolecules can also be attached and such supports used to determine the presence of ligands of said macromolecules in studied samples (in particular blood, urine, or any other biological fluid).

DNA chips have a major advantage over conventional methods of hybridization: thanks to miniaturization techniques, they allow the simultaneous hybridization of a considerable number of probes on a total useful surface area of less than 1 cm ² . The supports on which the probes are fixed are in the form of flat or porous surfaces (naturally or pierced with wells) composed of materials such as:

- glass, an inexpensive, inert and mechanically stable material. The small glass plate, substrate of the chip, can in particular be covered with a grid of PTFE (which determines hydrophilic and hydrophobic zones),

- polymers which are the basis of other techniques, and more specifically polypyrrole, polypropylene, polycarbonate ...

- silicon, commonly used for the manufacture of electronic chips because of its semiconductor properties; and

- metals, in particular gold and platinum.

Whatever the support chosen, it can be treated to form a dense and regular network of microsurfaces and on each of these is grafted a synthetic DNA probe. To this end, there are two methods: the transfer of DNA strands obtained by biosynthesis or chemical synthesis or its chemical synthesis in situ. The synthesis prior to the attachment of the probes makes it possible to attach relatively long probes, exceeding 40 nucleotides. The transfer of these probes on the chip can be done by means of micropipettes, microdots, by devices of inkjet type or by electrochemical addressing. In the latter case, the chip is composed of a silicon support covered, at each pad, with a gold mini-electrode. Each probe joins a specific pad when the mini-electrodes are energized selectively.

In situ synthesis: in this case, the construction of the probes is done by depositing successive layers of the four bases of DNA on a glass support. It is a mask, the configuration of which varies with each deposition of a layer, which allows the bases to stack correctly.

The role of each probe is to recognize, in a mixture applied to the surface of the biochip, a DNA sequence. Generally this sequence is amplified by PCR then labeled with fluorescence, prepared in solution and brought into contact with the biochip.

Other marking methods can be used. There are generally three families: a) optical, namely chemiluminescent, fluorophores, chromophores, b) radioactive and c) electromagnetic.

The hybridization phase is carried out in an incubator (fluidic station) and is followed by washing intended to rid the chip of the non-hybridized nucleic targets. It makes it possible to determine which probe has paired the DNA sequence analyzed.

Most of the methods are based on the use of a scanner which makes it possible to identify DNA probes which have become fluorescent, that is to say which have given rise to hybridization. Other methods are based on the modifications of the electric charges of the silicon support where the probes are fixed, or on the difference in conductivity observed after pairing. The measurement of these modifications makes it possible to identify the specific associations between the probes and the target DNA.

The possibilities of using DNA chips, proteins and other microsystems (also called micro-arrays) are multiple, and cover several fields:

- diagnosis, in particular immunoassays (hormone, cancer), field of microbiology (detection of the DNA of pathogens or of antigen / antibody complex), aggravating cardiovascular factors ...

- pharmacy, in particular the screening of products (study of the binding of compounds with their targets), the development of drugs (study of the effects on the expression of certain genes), pharmacogenomics (study of genomic, specific or no, between individuals) ...

- research, with sequencing and sequence analysis possibilities, molecular sorting, simplified study of the different metabolic pathways ...

- the food industry, in particular the classification of products, the study of the origin of certain ingredients, the study and characterization of contamination ...

- the environment, in particular - the study of microbiological contamination, pollutants, plants ...

Sequencing by hybridization (SPH) is different from the enzymatic method (Sanger method), which can to be considered as a spelling of the sequence, that is to say a reading base by base. The SPH proceeds by reading small blocks. The analysis relates to overlapping sub-sequences which are read and reassembled using a computer program which reconstructs the studied sequence.

Biochip sequencing is an interesting and more precise alternative to the classic sequencing method in terms of automation and reduction of costs and execution times.

The identification of targets for therapeutic research is carried out by a better understanding of the genome and its regulation provided by biochips. Thus, thanks to DNA chips, new genes have been identified that express themselves specifically in the brain tissue of children, or appear in association with inflammatory rheumatic or intestinal pathologies. Biochips can therefore contribute to the identification of therapeutic targets for pharmaceutical research. They can also be used to determine the antibiotic resistance of certain microbial strains to help better fight against them.

Pharmacogenetics consists of identifying the alleles involved in the efficacy (or ineffectiveness) of a product, or its undesirable effects. It leads to a better understanding of the mechanisms of action of drugs. By showing that a molecule has a variable action on a target, the biochip opens up the field of therapeutic potentials. It also makes it possible to identify the side effects of a product and, during clinical trials, to make measurements of toxicity. This is based on the slight differences that may exist in the genomic sequence between different individuals.

The diagnosis of infectious and genetic diseases is carried out by analysis of the presence of a specific sequence or antigen of a given infectious agent. Biochips can carry the specific determinants of several agents and thus allow rapid and precise identification. This also makes it possible to define the subtypes of the infectious agents, which is an advantage for the quality of the treatment to be provided.

Apart from the medical sector, three significant industrial sectors offer outlets for biochips:

- Agri-food: for example the monitoring of bacteria producing lactic ferments, or the detection of sequences from genetically modified organisms in seeds.

- The environment: bacterial analysis of drinking water, detection of infectious agents in food, air or water (Salmonella, Listeria, Legionnella).

- The bacteriological or chemical threat: by determining in advance the modifications of the genetic functioning of the immune cells caused by toxic agents, one can quickly identify the chemical products (mercury, dioxin ...) or bacteriological (anthrax or diphtheria bacillus ...) disseminated by a possible attacker.

The appearance of new technologies has upset the search for new drugs in its phase initial. This includes first the synthesis and isolation of new molecules, then their test on biological systems allowing to presuppose a possible therapeutic interest.

Three approaches have profoundly transformed this research.

1. Conformational techniques

The receptor theory postulates that it is the union of the drug molecule with a macromolecule (a protein in general) which is at the origin of the pharmacodynamic effect and more generally of the therapeutic response. This union is highly specific, only a few privileged molecules are capable of it. The drug is said to be like "the key in the lock". We now know how to determine the conformation in space of proteins, in particular thanks to X-ray radiocrystallography, and therefore that of receptors. In certain cases, it is therefore possible to predict which structures will have to present the molecules in order to be able to unite with them, in particular using computer modeling (computer-aided design).

It is essential to know at the outset the relevant receptor, that is to say to have a physiopathological hypothesis and to have been able to identify and isolate the protein which carries it.

2. Combinatorial chemistry

It is now possible to synthesize several hundreds of molecules in a single operation. This is called combinatorial chemistry. We start from the basic structure determined a priori as just said and we systematically generate all the possible variations by grafting chemical radicals, side chains, modifying the skeleton, etc. This is no longer done step by step, but by bringing together the necessary reagents. Several hundred molecules are thus obtained in one go. All operations, synthesis, isolation and identification, are miniaturized and automated. This saves time and lowers costs. We can thus build a library of several thousand derivatives in a few months. 3. High throughput screening

The problem is then to identify among all these molecules those which have the most interesting biological properties. The time savings and the increase in productivity brought about by combinatorial chemistry would have been in vain if the productivity of this phase of tracking, called "screening", had not been so improved. Long and limited trials of classical experimental pharmacology have been followed by a technique which allows thousands of molecules to be tested in the shortest possible time.

The test consists in bringing together the substance to be tested and a biochemical system (an enzyme for example) and measuring the importance of the possible reaction. The test can be done simultaneously with a large number of very different systems of meanings. In fact, everything is miniaturized and automated and all operations take place without human intervention.

Everything depends on the model used, which emphasizes the importance of pathophysiological hypotheses. The biological systems tested do not are not indifferent: they are those who are believed to play a crucial role in the determinism of the disease. Thus, this applied research, whose progress is so remarkable, it is totally dependent on basic research upstream.

Finally, the most promising macromolecules are selected, taking into account their test results, but also other considerations such as their ease of synthesis or their pharmacokinetics.

It is therefore essential to be able to screen the new molecules produced by combinatorial chemistry and to obtain the best data, in particular of binding to the receptor, as quickly as possible. A high-throughput screening is then carried out, consisting in identifying the active molecules by contacting the biological target. These targets can for example be proteins whose implication in a particular pathological process has been identified experimentally.

Until the early 1990s, traditional test tubes allowed one person to test 2,000 compounds per year. The use of 96-well microplates then made it possible to carry out 6000 tests per day and by robot on the same target. Today the microplates have 384 wells, some of which can go up to 1,536 wells. These advances have made it possible to multiply tests and also to reduce costs since the tests are "miniaturized" and use very small sample volumes.

High-throughput screening is characterized by the use of robots capable of managing simultaneously large numbers of microplates, easy to handle and store, where thousands of tests are performed per day.

High-throughput screening is the result of advances in robotics. It is based on systems capable of performing independent sequential tasks such as dilution, pipetting and distribution of compounds in wells or wells, agitation, incubation and reading of the results. They are controlled by software specifically adapted to the type of analysis to be performed. The tests are carried out in standard microplates identified by a bar code and manipulated by the "hand" of a robot: this takes the empty plate, adds the necessary reagents and the compounds to be tested, manages the reaction time then pass the plate to a reader to know the result. To visualize the reactions resulting from the contact, in the wells, of the molecule and the target, methods based on radioactivity or, very often, fluorescence are used. These results are stored and analyzed using computers.

As seen above, it is extremely important to be able to have a system for studying the bonds between compounds and their targets, as well as the binding kinetics. Currently existing systems do not allow such a study to be carried out, since the analysis of the connections between the macromolecules fixed on a solid support and the macromolecules in solution is generally carried out after several rinsing steps to reduce non-binding and the risk of false positives. It is therefore impossible with current systems of be able to quickly and easily carry out an analysis of the kinetics of binding between molecules in solution and molecules on a solid support.

The present invention proposes to meet the needs thus demonstrated by providing a device making it possible to carry out studies of binding of compounds on a large scale, by increasing the speed and reducing the operating costs. This system can be easily automated and can be implemented for all the uses set out above.

It is important to note that in the examples defined preferentially below, and in particular the supports, it is easy to substitute supports made with materials as described above. The foregoing description is only intended to recall the context and the field of application of the invention, in particular the manufacture, the use and the methods of analysis of biological micro-supports.

With a view to achieving this goal, a device for analyzing a molecule is provided according to the invention, comprising:

- a support capable of carrying at least one molecule attached to the support; and a chamber capable of containing a liquid by bringing the liquid into contact with the support, the chamber being arranged to be rigidly fixed to the support.

Depending on the case, provision may be made for the molecule to be analyzed, in general an organic molecule, or even an organic macromolecule, to be fixed either to the support for bringing into contact with one or more molecules contained in the liquid, or on the contrary contained initially in the liquid for bringing into contact with one or more molecules initially attached to the support.

The device according to the invention may also have at least any one of the following characteristics:

the support has at least two zones capable of carrying molecules fixed to the zones, the device comprising at least two chambers capable of containing two respective liquids by bringing the liquids into contact with the respective zones, the chambers being arranged to be rigidly fixed to the support;

- the molecules of each zone are arranged annularly in the zone;

- the zones belong to a common room in one piece;

- The support comprises a base and support elements each forming one of the zones and able to be fixed to the base; the support elements have a disc shape; it comprises a frame, the support being mounted movable relative to the frame, for example movable in rotation relative to the frame, in particular around an axis of horizontal rotation and / or perpendicular to a thickness of the support;

- It comprises means for driving the support relative to the frame, for example arranged to cooperate with the contactless support, electromagnetically;

- the support has a disc shape; - The or each chamber has a shape with symmetry of revolution around a main axis of the chamber; the or each chamber is permanently fixed to the support; the or each chamber is mounted movable relative to the support;

- It comprises a compartmentalization member capable of being brought into contact with the support in a removable manner; it comprises a frame, the compartmentalization member and the support being arranged to be received in the frame while being movable relative to each other;

- It comprises a frame having an enclosure capable of receiving the support and means for introducing a liquid into the enclosure and / or extracting a liquid from the enclosure;

- the or each room includes an orifice; the orifice is obstructed by a compressible element capable of being crossed by a needle so that it opens into the chamber; it includes means for analyzing an interaction of the or each molecule attached to the support with a molecule present in the liquid; the interaction includes a bond between molecules; the analysis means are arranged to be sensitive to radiation resulting from the interaction;

- the molecules are nucleic acids, proteins, peptides or any other molecule capable of interacting with biological compounds; and it is arranged so that the molecule to be analyzed is in the liquid before bringing the liquid into contact with the support so that it interacts with the molecule attached to the support.

According to the invention, a method of analyzing a molecule is also provided, comprising the steps consisting in:

- attach at least one molecule to a support; and

- Put a liquid contained in a chamber in contact with the support, the chamber being rigidly fixed to the support.

In one embodiment, the molecule to be analyzed is found in the liquid before the liquid is brought into contact with the support.

Other characteristics and advantages of the invention will become apparent in the following description of a preferred embodiment of the invention and of the variants given by way of nonlimiting examples. With reference to the accompanying drawings:

- Figures 1 and 2 are two partial views in axial section of a device according to the invention in two different operating configurations;

- Figures 3 and 4 show two possibilities for the arrangement of. support elements on the base;

- Figure 5 is a partial view in axial section of a part of the device of Figure 1 showing the introduction of a sample into a chamber;

- Figure 6 is a sectional view of the device of Figure 1 along a plane perpendicular to the axis of rotation, showing the fluid circulation conduits in the enclosure; and

- Figure 7 is a view similar to Figure 2 showing an alternative embodiment of the device.

FIGS. 1 and 2 illustrate a preferred embodiment of the device for analyzing a macromolecule according to the invention. It could for example be, as explained above, a device for the analysis of a genome.

The device 2 comprises a frame 4 defining within it a closed enclosure 6. The device also comprises a support 8 as well as a compartmentalization member 10, both of which are here essentially planar. The support 8 and the member 10 each have a disc shape here and have substantially the same diameter. They are intended to be arranged concentrically with the same axis 12. Taking into account this disc shape which these two members have in the present example, we will call them in the following respectively "probe-carrying disc" and "compartmenting disc ".

The probe carrier disc 8 (DPS) comprises a base 14 in the form of a flat disc. The DPS also comprises several support elements 16 each having in this case a form of flat disc. The radius of these discs 16 is much smaller than the radius of the base 14 knowing that the support elements 16 are intended to be fixed next to each other on the same end face 18 of the base 14.

The radius of the support elements 16 will vary depending on the number of elements that one wishes to have simultaneously on the face 18. There is thus illustrated in FIG. 3 a possible embodiment in which the face 18 carries four support elements 16 while FIG. 4 presents an example in which this face carries eighteen support elements 16.

The number and the dimensions of the support elements 16 being determined, they can be arranged in different ways on the face 18. They can for example be arranged in a rectangular matrix as is the case in FIG. 3. Such a arrangement facilitates the handling and location of the support elements by robotic means. Alternatively, the support elements 16 may be arranged so that their centers form concentric circles around the axis 12 as is the case in FIG. 4. This arrangement generally makes it possible to occupy most of the face 18 of the base 14.

The support elements 16 are attached to the base 14 to be rigidly fixed thereto. In the present example, they each have, on their face 19 remaining free, test macromolecules or probes 20 intended to interact with macromolecules present in the liquid sample to be analyzed as will be seen below.

Preferably, the macromolecules 20, represented by a point on one of the discs of FIG. 3, are arranged annularly on the face 19 of the support elements with reference to the axis 22 of these, thus leaving unoccupied a central area of the disc. Alternatively, provision could be made for the entire surface 19 to be covered by the macromolecules 20. The macromolecules are arranged on the face 19 prior to the fixing of the support elements 16 on the base 14. This arrangement is carried out according to conventional techniques known in themselves and which will not be detailed here.

To facilitate the reading of the results of the analysis as will be seen below, the base 14 as well as each of the support elements 16 are produced according to a conventional technology such as that of compact discs (CDs), DVDs {digi tal versa tile disc), etc. allowing the coding of a signal and its reading by suitable means. CD type discs have the advantage of withstanding high temperatures due to their polycarbonate construction. In addition, they carry addressing signals allowing the location of the probes 20.

The compartmentalization disc (DC) 10 has a face 24 intended to extend opposite the face 18 of the DPS, concentrically with the latter. The face 24 of the DC has chambers 26 equal in number to the number of support elements 16 and arranged in a homologous manner at the disposal of the support elements so as to extend respectively opposite and in correspondence with them. Each chamber 26 has in this case a cylindrical shape coaxial with the axis 22 of the corresponding support element. In the present example, the section of the chamber 26 in a plane perpendicular to the axis 22 has a circular shape.

Figure 1 illustrates a configuration of the DC and DPS in which these are distant from each other while Figure 2 illustrates a configuration in which they are in contact with each other. So in FIG. 2, each chamber 26 is closed on the side of the face 24 by the corresponding support element, the macromolecules 20 thus extending into the chamber 26. Conversely, in FIG. 1, the chamber 26 is open from the side of the face 24 taking account of the distance from the support elements 16.

Sealing means are provided at each chamber 26, for example in the form of a seal 30, to ensure a sealed closure of the chamber 26 on the side of the face 24 by the corresponding support element 16.

At its end opposite to the support element 16, each chamber 26 is closed by the wall of the compartmenting disc 10. However, it has on the one hand a cylindrical opening 32 closed by a compressible element 34, for example made of elastomer as illustrated in Figure 5. On the other hand, it has a second opening 36 intended as will be seen later to avoid overpressure inside the chamber. This opening has sufficiently small dimensions to allow an outlet of gas out of the chamber while preventing an outlet of liquid through this same opening. This second opening 36 will preferably be arranged near the axis 22 of the chamber, or even on this axis.

The frame 4 is dimensioned to receive in the enclosure 6 the DC 10 as well as the DPS 8 both in the open position as in FIG. 1 and in the closed position as in FIG. 2. The device comprises mounting means making it possible to keep them facing each other and guide their movement relative to each other so that they can switch to inside the enclosure 6 from one to the other of the two positions.

The device comprises drive means 38 making it possible to set the assembly constituted by the two discs in rotation about their common axis 12. These drive means will preferably be arranged to cooperate electromagnetically with elements 40 arranged in the discs to drive them into the enclosure 6 without contact through the wall of the frame.

The device also includes means 42 for analyzing a possible interaction or the absence of interaction between one of the macromolecules 20 and a macromolecule present in the sample to be analyzed. The analysis means may be sensitive to a signal resulting from the interaction, this signal possibly being of an optical, electromagnetic or radioactive order for example. This reading can be done through a window in one of the walls of the frame. However, preferably, it will be ensured that the material or materials of the frame, of the DC 10 and of the DPS 8 are transparent to the analysis signal so as not to disturb it.

Given the rotation of the two discs in the enclosure 6 around the axis 12, it is sufficient that the analysis means 42 can read along a line 44 extending radially with reference to the axis 12 as illustrated in FIG. 6. It could for example be provided for the reading means 42 to have a movable reading head along this line 44.

As illustrated in FIG. 6, the frame 4 also includes means for introducing a fluid such as a liquid into the enclosure 6, extracting it, or still put it into circulation in the enclosure. These means include in particular conduits 46 opening about halfway up the enclosure or even in the lower part thereof to facilitate the evacuation of a liquid from the enclosure. The enclosure may also have an upper opening 48 making it possible to avoid overpressures in the enclosure or allowing the evacuation of an air bubble when the enclosure must be completely filled with a liquid. These means are also arranged in a known and non-detailed manner to allow, if necessary, to introduce or extract different types of liquids into the enclosure.

We will now describe an example of use of the device according to the invention.

The operator first installs the macromolecules 20 on the face 19 of the support elements 16. He then fixes the support elements on the base 14. Then the operator attaches the compartmentalization disc 10 to the probe-holder disc 8 thus formed so as to close the chambers 26.

The operator then introduces into the respective chambers 26 liquid samples 52 comprising compounds or macromolecules to be analyzed. Each sample may include, for example, a population of DNA fragments to be analyzed corresponding to one or more genomes. The present invention has the advantage that several analyzes which are different from each other can be carried out simultaneously. Thus, several samples can be analyzed simultaneously according to identical methods. In addition, analyzes obeying different execution methods can be carried out simultaneously. For example, in one of the rooms 26, one can carry out an HIV search while another of the rooms will be used at the same time for a typing of EBV (Epstein Bar Virus), other rooms being moreover devoted to a search for Chlamydiae or to a typing of the CF (Cystic Fibrosis) gene.

In the present embodiment, the operator introduces each sample 52 into the chamber 26 by means of a syringe by piercing the element 34 with the needle 50 of the syringe. The latter opening into the chamber 26, the sample 52 to be analyzed can be introduced into the latter as illustrated in FIG. 5. As can be seen, each chamber 26 forms a mini-reactor. This introduction can be done while the two adjoining discs extend horizontally with their common axis 12 vertical on a suitable plane support.

The two discs are then introduced into the frame 4 as illustrated in FIG. 2 and the latter is closed in a suitable manner. The discs are then vertical with their horizontal axis 12.

The means 38 are actuated to put the two discs forming a rigid assembly in rotation about the axis 12. As can be seen in FIG. 2, the quantity of sample 52 introduced into each chamber 26 is much less than the total volume of the chamber so that the upper level 54 of the sample does not extend to the axis 22 of the chamber. Given the gravity, the sample 52 remains in the lower part of the chamber during the rotation. It is therefore in contact with the corresponding zone of the support element 16. Taking into account the rotation, the sample travels most of the chamber and comes into contact with all of the macromolecules arranged on the support element.

At this stage, the temperature can possibly be adjusted and varied over time if necessary to promote such or such reaction between the sample 52 and the probes 20, for example if one seeks to carry out a hybridization.

After incubation for a suitable period, the temperature is adjusted so that the reaction is blocked.

The entire enclosure 6 is then filled with a washing liquid having a composition suitable for promoting the cessation of the various reactions and for avoiding cross-reactions.

The partitioning disc is then separated from the probe-carrying disc by moving them away from one another to replace them in the configuration of FIG. 1 still inside the enclosure 6 and without interrupting their rotational movement. Under these conditions, the integrity of the mini-reactors formed by the chambers 26 is broken so that the individual samples 52 are mixed in the washing liquid.

This is then eliminated through the conduits 46 and quickly replaced by a clean washing liquid to avoid problems of cross-reactions. At this stage, the level of the washing liquid should be such that the entire surface of the probe carrier disc is immersed in the liquid at one time or another during the rotation. The washing liquid will preferably be supplied in a continuous flow to optimize the dissolution of the residual reactants and facilitate their rapid elimination from the reactor. At the end of this washing, the results of the analysis can be read using the means 42 which detect suitable radiation resulting from the interaction of each macromolecule 20 with one of the compounds present in the corresponding sample or which detect the absence of such interaction.

In the open configuration illustrated in the figure

1, we can also put all of the macromolecules 20 in contact with common reagents

(such as developers) and not just with detergents.

FIG. 7 illustrates an alternative embodiment of the device of the invention. In the description which follows, elements similar to those of FIG. 1 bear numerical references increased by 100. The configuration in this case only varies for the compartmentalization disk 10 and the probe-carrier disk 8.

In the present example, the DPS 108 and the DC 110 are rigidly fixed to each other and are not intended to be made movable relative to each other. In addition, they are constituted by a superposition of sheets. Thus, the DPS is constituted in this case by a simple sheet 108. A second sheet 111 is attached to the latter and has cavities forming the chambers 126. A third sheet 113 covers the second sheet. The sheets 108 and 113 close off each chamber 126 at its two axial ends by sandwiching the sheet 111 interposed therebetween. The sheet 113 has the openings 32 allowing the introduction of the samples into the chambers 26. Finally, a fourth sheet 115 covers the third sheet on the side opposite to the second sheet in order to close these openings. The sheets 111, 113 and 115 form the DC 110 while the sheet 108 constitutes the DPS 108.

The fourth sheet 115 is initially separated from the first three which are supplied to the operator while already being attached to each other. The operator fills the chambers 126 with the samples 52 having previously arranged the assembly horizontally. It then closes the openings 32 with the fourth sheet 115 by fixing the latter to the assembly. He finally introduces this assembly into the frame and performs the analysis as mentioned above.

This alternative embodiment is suitable for situations in which it is not necessary to carry out a washing to carry out the analysis. This is for example the case when the reaction product allows the emission of a fluorescence signal indicating its formation or on the contrary inhibits the emission of fluorescence. Moreover, in this variant, it will generally not be necessary to have the DC-DPS assembly in a particular enclosure 6, washing being often unnecessary. Only the training means and the incubation means can be useful.

An advantage of the invention is that it makes it possible to analyze a large number of samples simultaneously. The invention has the advantage of making the flow of the analysis relatively simple by reducing the manipulation of the test macromolecules and samples. In particular, these do not need to be handled during analysis.

The compound present in the liquid will preferably be labeled in order to be able to detect its presence at a precise point on the support corresponding to a given probe, which provides information on the bond existing between the two molecules. The binding kinetics can then be studied as a function of the intensity of the signal read at a given position. Conversely, the probes 20 can also be marked on the support 8 so that a signal is emitted when a compound binds to the probe.

Of course, many modifications can be made to the invention without departing from the scope thereof.

As illustrated in the variant of FIG. 7, it may be provided more generally (and for example in the embodiment of FIG. 1) that the macromolecules 20 or probes are placed directly on the base 14 without using elements of intermediate support 16. However, the use of the latter introduces more flexibility in the programming of the various analyzes. Thus the user will be able to have stocks of the various support elements 16 associated respectively with conventional analyzes which can be used on demand according to current needs.

Provision may be made for each sample 52 to occupy the entire volume of the associated chamber 26 or at least a sufficient portion of it so that it completely covers the probes. In this case, rotation of the DPS is not necessary during the reaction and provision may be made for the compartmenting disc to be coincident with one of the walls of the frame 4. Under these conditions, only the probe-carrying disc would be movable relative to the frame to allow it to move away from the compartmenting disc and promote washing.

Similarly, if the volume of each sample allows, the device can be placed horizontally, i.e. with its vertical axis 12 during the period of reaction of the samples on the probes, and then be placed vertically just before separation discs and the washes that follow.

Knowing that, in the embodiment of FIGS. 1 and 2, a large part of each chamber 26 does not contain a sample, it is possible during the reaction with the means 42 to analyze the macromolecules 20 carried by the portions of the support elements 16 not covered by the sample 52. However, this requires increasing the possibilities of movement of the reading means 42 relative to the disc.

Although in the preceding examples there has been described a probe-carrying member and a compartmenting member having a disc shape, this is not strictly necessary and other shapes such as regular polygon shapes can be provided .

Likewise, regardless of the shape of these members, the shape of the support elements 16 can be modified, which will not necessarily have a circular shape.

The device may include a single enclosure 26. Finally, the probes may be initially contained in the liquid sample while the or the molecules to be analyzed will initially be attached to the support.

Claims

claims

1. Device for analyzing a molecule, comprising - a support (8; 108) capable of carrying at least one molecule (20) fixed to the support; and a chamber (26; 126) capable of containing a liquid (52) by bringing the liquid into contact with the support, characterized in that the chamber is arranged to be rigidly fixed to the support.

2. Device according to claim 1, characterized in that the support (8; 108) has at least two zones (16) capable of carrying molecules (20) fixed to the zones, the device comprising at least two chambers (26; 126 ) capable of containing two respective liquids (52) by bringing the liquids into contact with the respective zones, the chambers being arranged to be rigidly fixed to the support.

3. Device according to claim 2, characterized in that the molecules (20) of each zone (16) are arranged annularly in the zone.

4 Device according to any one of claims 2 or 3, characterized in that the zones belong to a common room (108) in one piece.

5. Device according to any one of claims 2 to 3, characterized in that the support (8) comprises a base (14) and support elements (16) each forming one of the zones and capable of being fixed to the base.

6. Device according to claim 5, characterized in that the support elements (16) have a disc shape.

7. Device according to any one of claims 1 to 6, characterized in that it comprises a frame (4), the support (8; 108) being mounted movable relative to the frame.

8. Device according to claim 7, characterized in that the support (8; 108) is mounted movable in rotation relative to the frame.

9. Device according to claim 8, characterized in that the support has a horizontal axis of rotation (12).

10. Device according to any one of claims 8 or 9, characterized in that the support has an axis of rotation (12) perpendicular to a thickness of the support (8).

11. Device according to any one of claims 7 to 10, characterized in that it comprises drive means (38) of the support (8) relative to the frame (4).

12. Device according to claim 11, characterized in that the drive means (38) are arranged to cooperate with the contactless support, electromagnetically.

13. Device according to any one of claims 1 to 12, characterized in that the support (8; 108) has a disc shape.

14. Device according to any one of claims 1 to 13, characterized in that the or each chamber (26; 126) has a shape with symmetry of revolution about a main axis (22) of the chamber.

15. Device according to any one of claims 1 to 14, characterized in that the or each chamber (126) is permanently fixed to the support (108).

16. Device according to any one of claims 1 to 14, characterized in that the or each chamber (26) is mounted movable relative to the support (8).

17. Device according to any one of claims 1 to 16, characterized in that it comprises a compartmenting member (10) capable of being brought into contact with the support (8) in a removable manner.

18. Device according to claim 17, characterized in that it comprises a frame (4), the compartmentalization member (10) and the support (8) being arranged to be received in the frame while being movable one by compared to each other.

19. Device according to any one of claims 1 to 18, characterized in that it comprises a frame (4) having an enclosure (6) capable of receiving the support (8) and means (46) for introducing a liquid inside the enclosure and / or extract a liquid from the enclosure.

20. Device according to any one of claims 1 to 19, characterized in that the or each chamber (26; 126) comprises an orifice (32).

21. Device according to claim 20, characterized in that the orifice (32) is obstructed by a compressible element (34) capable of being crossed by a needle (50) so that it opens into the chamber (26).

22. Device according to any one of claims 1 to 21, characterized in that it comprises means (42) for analyzing an interaction of the or each molecule (20) fixed to the support (8; 108) with a molecule present in the liquid (52).

23. Device according to claim 22, characterized in that the interaction comprises a bond between the molecules.

24. Device according to any one of claims 22 or 23, characterized in that the analysis means (42) are arranged to be sensitive to radiation resulting from the interaction.

25. Device according to any one of claims 1 to 24, characterized in that the molecules (20) are nucleic acids, proteins or peptides.

26. Device according to any one of claims 1 to 25, characterized in that it is arranged so that the molecule to be analyzed is present in the liquid before bringing the liquid into contact with the support.

27. Method for analyzing a molecule, characterized in that it comprises the steps consisting in:

- attach at least one molecule (20) to a support (8; 108); and

- Putting a liquid (52) contained in a chamber (26; 126) in contact with the support, the chamber being rigidly fixed to the support.

28. The method of claim 27, characterized in that the molecule to be analyzed is in the liquid before bringing the liquid into contact with the support.