FR2835614A1 - Device for analyzing molecules, useful for nucleic acid or protein interaction assays, comprises fluid-containing chamber in which binding carrier is rotated - Google Patents

Abstract

Device for analyzing a molecule (I) comprises a chamber (4); support (6) for a molecule (II), and system for contacting (6) with liquid able to interact with (II). The liquid forms a bath in (4) and (6) is mobile so that part of it is alternately immersed in the bath and outside the bath. An Independent claim is also included for a method of analyzing (I) by fixing (II) to (6) and moving this, as described, in a bath of liquid that can interact with (II).

Description

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 The present invention relates to methods for measuring the binding of compounds with biological macromolecules fixed on a support, as well as to devices making it possible to carry out these methods.

 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 in 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 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 DNA studied is then observed, and indicates the presence or absence of expression of a gene, in the case of use of complementary DNA derived from RNA retrotranscription. 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, for example, antibodies or antigens are fixed and the presence of, respectively, the antigen or the complementary antibody is detected in the biological sample.

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

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le polypropylène, le polycarbonate... DNA chips have a major advantage over conventional hybridization methods: 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 cm2. 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, inexpensive, inert and mechanically stable material. The small glass plate, substrate of the chip, can in particular be covered with a Teflon grid (which determines hydrophilic and hydrophobic zones), - the polymers which are the basis of other techniques, and more precisely 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,

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 microtips, by inkjet type devices 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 DNA bases 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.

With this process, used in particular by the company AFFYMETRIX, the probes contain a maximum of 30 bases. However, the company PROTOGENE has developed a system allowing the four desired bases to be deposited on the teflon grid of the biochip, using piezoelectric injectors, according to a technology which is similar to a conventional DNA synthesis. It is the equivalent of a four-color inkjet printer (each color in this case being a base: A, G, T, C). This process is extremely flexible: it makes it possible to manufacture such and such a kind of biochip, just as a printer indifferently prints this or that page, whatever the words of the text.

 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 by fluorescence, prepared in solution and brought into contact with the biochip.

 Other marking methods can be used. There are generally three families: a)

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 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 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 electrical 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 target.

 The possibilities of using DNA chips, proteins and other microsystems (also called microarrays) are multiple, and cover several fields: - diagnosis, in particular immunoassays (hormone, cancer), microbiology field (detection of DNA pathogens or 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) ...

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 - 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 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 by means of 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 expressing themselves specifically in the brain tissue of children, or appearing in association with inflammatory rheumatic or intestinal pathologies. Biochips can therefore contribute to the identification of therapeutic targets for pharmaceutical research. They can also

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 used to determine the resistance to antibiotics 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 potential. It also makes it possible to identify the side effects of a product and, during clinical trials, to make toxicity measurements. 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: - Food industry: for example monitoring bacteria producing lactic ferments, or detecting sequences from genetically modified organisms in seeds.

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 - 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 (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 initial phase. 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, we can therefore predict which structures will have to present the molecules for

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 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 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 tracking phase, called "screening", had not been so improved. Long and limited trials of experimental pharmacology

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 classic, succeeded a technique which allows to test in the minimum time thousands of molecules.

 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 are not indifferent: they are those that 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 bringing them into contact with the biological target. These targets can for example be proteins which have been identified

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 experimentally the implication in such or such pathological process.

 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 because the tests are "miniaturized" and use very small sample volumes.

 High-throughput screening is characterized by the use of robots capable of simultaneously managing large numbers of microplates, easily manipulated and storable, 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 are used

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 based on radioactivity or, very often, fluorescence. 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 to be able to carry out in a simple and rapid manner an analysis of bond kinetics between molecules in solution and molecules on solid support.

 The present invention proposes to meet the needs thus demonstrated by providing a device and a method making it possible to carry out studies of binding of compounds on a large scale, by increasing the speed and by 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 preferably defined below, and in particular the supports, it is easy to substitute supports made with materials as described above.

The foregoing description is intended only to recall the context and the field of application of the invention as well as the knowledge of a person skilled in the art.

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 concerning the manufacture, use and methods of analysis of biological micro-supports.

 In order to achieve this goal, there is provided according to the invention a device for analyzing a macromolecule, comprising: - an enclosure; - a support capable of receiving the macromolecule; and - means for bringing the support into contact with a fluid capable of interacting with the macromolecule, the device being arranged so that the fluid forms a bath in the enclosure, the support being mounted mobile in the enclosure so that at least certain portions of the support extend into the bath and then out of the bath according to a time alternation.

 Thus, the analysis of the interactions of the macromolecule with a compound present in the fluid can be carried out when the portion of the support carrying the macromolecule is located outside the bath and this without interrupting the implementation of the process. In addition, one can modify the composition of the fluid over time and follow the evolution of the analysis live, which also gives much more precise results.

 Advantageously, the support is movable so that at each instant, during the movement of the support, certain portions of the support extend in the bath while other portions of the support extend out of the bath.

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 Thus, the analysis can take place without interrupting the interaction with the fluid of the various macromolecules located on the support.

 The device may also have at least one of the following characteristics: - the support is movable in rotation in the enclosure; - The support has an axis of rotation extending outside the bath; and the support is flat and has an axis of rotation perpendicular to its thickness.

 Advantageously, the axis of rotation is inclined relative to the vertical direction and, in particular, is horizontal.

 Thus, when the fluid is a liquid, it flows by gravity along each fraction of the support at the outlet of the bath so that the corresponding surface of the support is essentially or completely free of liquid, once it reaches the upper part of its trajectory, for analysis for example. The analysis can therefore take place immediately, right out of the bath of the portion to be analyzed and without interrupting the movement of the support.

 Advantageously, the axis of rotation is inclined relative to the horizontal direction, and is in particular vertical.

 In this case also, it can be obtained under certain conditions that the support portion leaving the bath is at least essentially free of fluid.

This will be the case, for example, when using the capillary effect described below or else if sealing means are provided at the outlet of the bath.

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 The device may also have at least one of the following characteristics: - The fluid is a liquid; - The enclosure has at least one wall extending opposite at least one face of the support and delimiting the bath, the wall extending at a distance from this face such that the liquid located between the wall and the face is maintained in the bath by capillary action in contact with the wall and the face; - It comprises means for introducing the fluid into the enclosure and / or extracting the fluid from the enclosure; - The means for introducing or extracting a fluid comprise a single orifice opening into the interior of the enclosure; the means for introducing or extracting the fluid comprise an element and a needle capable of opening into the enclosure through the element; - The device is able to put the support in contact with several fluids according to a time alternation; - It includes means different from the support for setting the fluid in motion in the enclosure; - It includes means for analyzing an interaction of the macromolecule attached to the support with the fluid, these means extending opposite an area of the support located outside the bath;

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- The interaction includes a binding of the macromolecule with a compound present in the fluid; and - The analysis means are sensitive to radiation, in particular optical, electromagnetic or radioactive, resulting from the interaction;
A method of analyzing a macromolecule is also provided according to the invention, comprising the steps consisting in fixing a macromolecule on a support, then setting the support in motion in a bath of a fluid capable of interacting with the macromolecule so that 'at least some portions of the support extend into the bath and then out of the bath according to a time rotation.

 The method could also have at least one of the following characteristics: - The support is put in movement so that at any instant, during the movement of the support, certain portions of the support extend into the bath while other portions of the support extend out of the bath; - The fluid is introduced into the enclosure and / or the fluid is extracted from the enclosure during the movement of the support; - The fluid is put in movement in the enclosure during the movement of the support; - We analyze an interaction of the macromolecule in a support area outside the bath; - The analysis is carried out during the movement of the support;

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- We analyze the interaction, then we modify the composition of the fluid, then we analyze again an interaction of the macromolecule with the fluid; - A washing liquid is introduced into the enclosure and the support is set in motion in the liquid; and
The macromolecule is a nucleic acid, a protein or a peptide.

 The present invention makes it possible in particular to measure the kinetics of binding between the compounds present in the fluid and a macromolecule fixed on the support. The support will generally present on its surface a large number of different macromolecules.

 An advantage of the method according to the invention, compared to known methods, is that the measurement can be carried out dynamically, during the binding reactions. Indeed, the support being in rotation, part of it is then immersed in the fluid containing said compound, then allowing the different bonding reactions with the macromolecules fixed on the support, while reading is carried out on the non-submerged part of the support.

 It appears that the support in question is preferably a disc, and that said rotation is carried out with respect to an axis passing through the center of said disc. A disc in CD format (compact disc or compact disc) or a DVD disc (digitaJ versati disc) is also preferably used. We can call it later in this CircArray request.

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The use of the device which is the subject of the invention, for implementing the method according to the invention has several advantages, among which: - handling of the support
The big problem of membrane arrays is indeed the difficulty of their manipulation, especially when they are soaked and they must be handled behind a protective screen if the marker used is radioactive. The disk is here in rotation, and does not need to be manipulated during the analysis.

annulaire d'environ 90 cm2 utile, qui est à comparer avec celle des membranes dites Atlas , de 8x11=88 cm2. Le polycarbonate lui-même ou tout autre polymère que l'on peut coller à la surface peut servir d'amorce pour la fixation covalente de bio-molécules. Des films fins de matière peuvent être obtenus, par exemple par aspersion du support pendant qu'il est en rotation pour profiter de la force centrifuge. - ease of production of the support
The choice in favor of CircArray is oriented by the fact that the discs are able to withstand high temperatures (polycarbonate) and that they carry ah hoc addressing signals for the location of the probes. The surfaces of the discs have a region

annular of about 90 cm2 useful, which is to be compared with that of the so-called Atlas membranes, of 8x11 = 88 cm2. The polycarbonate itself or any other polymer that can be bonded to the surface can serve as a primer for the covalent attachment of bio-molecules. Thin films of material can be obtained, for example by spraying the support while it is rotating to take advantage of the centrifugal force.

- low cost of support
The price of commercial CDs is almost negligible compared to the price of membranes that can be affixed to it.

- ease of depositing probes
The CircArray can be considered in the same way as the current supports for the realization of the deposits and the fixing of the probes. The techniques of

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 currently available deposits can be used, whether direct deposit on CircArray or direct by joining other supports (membranes, glass arrays).

 The support envisaged in the present invention is preferably a rigid support, having a porous or untreated surface to facilitate fixing of the probes or targets according to the specific needs of an application, of circular format, of the CD type, with a base in polycarbonate. These discs are commercially available and the deposition of macromolecules is facilitated by the rigidity of the support and the possibility of covering the surface with a flexible material.

 The compound present in the fluid will preferably be labeled, in order to be able to detect its presence at a precise point of the support, corresponding to a given macromolecule, which provides information on the bond existing between the two molecules. The binding kinetics are studied as a function of the intensity of the signal read at a given position.

 Conversely, one can also mark the molecules fixed on the support so that a signal is emitted when a compound binds to them.

 The measurement is then carried out by means of an optical (for example fluorescence), radioactive or magnetic signal, one of the molecules involved in the bonding being marked appropriately.

 In order to reduce the background noise and the risk of false positives, the method according to the invention can be implemented by introducing one or more washing steps, with appropriate buffers.

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 The reactor or device according to the present invention will advantageously have the following properties: - allow circulation of fluid, in particular in its lower part. To do this, we prefer a reactor having one or more orifices, in order to penetrate the fluids inside and to be able to evacuate them. These ports can be connected to pipes and / or pumps to improve circulation. The reactor according to the present invention therefore facilitates the exchange of solutions, insofar as orifices can be made allowing the entry and evacuation of the reactants. Furthermore, the rotation of the support in the reactor ensures permanent mixing of the reagents, which is an advantage in order to be able to carry out binding studies. It is also possible, thanks to the orifices present in the reactor, to access the reagent during the reaction.

 - allow reading of the signal emitted during the connection between the compound present in the fluid and the macromolecule fixed on the surface. For this purpose, one can either read through the wall (possibly after having made a blank passage to define a baseline), or leave an orifice in the wall.

 - allow the surface of the support, at the place of reading, to be dry or relatively dry, in order to reduce reading errors due to

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 presence of fluid carrying labeled molecules.

 - allow the volume of fluid in which the support soaks to be as low as possible, in order to reduce the volumes of fluid to be used. An attempt is made in particular to use a reactor in which the lower part (containing the various fluids) is capillary, and the upper part wider, in order to allow the fluid to be maintained in the volume of the reactor intended for the bonding reaction.

 As has been said, the reactor according to the invention allows partial immersion of the support in the reaction medium. However, if one wishes to achieve total recovery of the support, one can define a reactor which leaves only a minimal space between the support and the walls of the reactor. Thus, and even by minimizing the volume of the reagents used, the rotation of the support on a horizontal axis still guarantees total recovery of the disc.

 Reading can be done during the actual reaction of binding of the compounds present in the fluid to the macromolecules fixed on the support. It is extremely interesting to have access to the binding results, but also to the measurement of the reaction products during the experiment.

 Preferably, the support will be rigid and circular, with a face dedicated to the positioning of elements useful for locating regions for depositing reagents immobilized on the support, on the same or on the other face. The possible treatment of the surface makes it possible to reveal there the physical-

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 chemicals required to immobilize probes or targets. The rigidity of the support facilitates its automated processing.

 - The enclosure may be generally cylindrical with a cylinder height close to that of the height of the support. The design of the reactor takes into account the need to avoid the recesses which can create zones of retention of the reagents likely to cause heterogeneities of stirring or washing. Part of the reactor, that intended to accommodate the reactants with the smallest volume, is of lower cylinder height, in order to minimize the volume of reagent required. Thus, the preferred cylindrical reactor according to the invention has no dead volume.

 Preferably, the cylindrical axes of the reactor and of the support coincide and are horizontal. Thus, the lower part of the reactor can be used to receive the reagents which thus cover a portion of the useful annular surface of the support. The volume of reagent required is reduced since it must not cover the entire surface of the support and this part of the reactor has a lower height.

 Preferably, the reactor is stationary relative to the reference frame that the device constitutes. This makes it easy to attach the tubes necessary for the exchange of solutions which come into contact with the support. The support is mobile in the reactor.

 Permanent stirring of the reagents is advantageous in order to allow the entire surface area of the support to be covered by the reagents almost permanently. The rotation of the support around its axis and therefore its relative movement relative to the

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 reactor and reagents provide the desired effect. The stirring is obtained on the one hand by the circular movement of the support (common axis with the cylindrical reactor) and on the other hand by a permanent circulation of the reagents thanks to a pumping system on a circuit parallel to the reactor.

 To improve on the one hand the stirring of the reagents, on the other hand the recovery of part of the support at a time t and this by simplifying the control devices, the reagents circulate on a circuit parallel to that of the reactor, which can receive a series of sensors allowing the analysis of the characteristics of the reagent continuously and a series of points of admission of reagents allowing the adjustment of the properties of the reagent in contact with the support. We can therefore analyze the reagents at any time.

 As we have seen, part of the support is preferably permanently outside the reagent and available for carrying out measurements. The movement which animates the support means that the entire useful annular surface is presented opposite a line perpendicular to the axis of rotation, per period. This line (reading axis) can receive the ad hoc sensor for carrying out the measurements (of a nature adapted to the type of marker used), in the absence of covering the surface with an excess of reagent carrying the marker. This gives access to the measurement of the reaction products.

 In this case, the background noise constituted by the remains of the reagent not specifically bound to the support cannot be evaluated on a static measurement

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 during the reaction. Therefore, two measurement options are possible.

 The first consists in carrying out a measurement at the end of the reaction, after removal of the marker not attached to the support by washing. The mobility of the support relative to the reagent administered in continuous flow greatly improves the washing efficiency by minimizing the volume of the washing solution. The centrifugal force generated by the rotation of the support facilitates the removal of the washing solution contaminated by the marker from the surface of the support. The composition of the washing solution can vary continuously or discontinuously to vary the effectiveness of the washing, which can thus affect the weak fixations of the labeled reagent, even if these are specific.

 The second consists of a measurement during the reaction, thanks to a dynamic evaluation of the relevance of the measured signals, in terms of specific signal, by comparison of their evolution over time with that of positive and negative controls. The part of the support not immersed in the reagent is covered with a film of weak reaction medium, the weaker the nature of the support will allow (hydrophobic and non-porous support for example) and the speed of rotation will be high ( increased centrifugal force). Even if the background noise is much higher than the signals that we want to measure, the dynamic comparison of their evolution compared to those collected at a location on the disc does not allow to specifically fix the marked molecules and those that are collected in a place allowing to fix marked molecules

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 witness makes it possible to distinguish the products of the reaction by subtraction of the background noise. This measurement is preferred for studying the bond characteristics as a function of time, or by varying other parameters, in particular the temperature.

 Other characteristics and advantages of the invention will appear in the following description of a preferred embodiment and variants of the analysis device given by way of nonlimiting examples. In the accompanying drawings: - Figure 1 is a vertical sectional view perpendicular to the axis of rotation of the support of the preferred embodiment of the device; - Figure 2 is a view in axial vertical section along the plane II-II of the device of Figure 1; - Figure 3 is a view similar to Figure 1 illustrating a first variant of the embodiment of the invention; - Figure 4 is a view similar to Figure 1 illustrating a second variant of the embodiment of the invention; - Figure 5 is a detailed cross-sectional view along the plane V-V of the device of Figure 4; and - Figure 6 is a detail view of the device of Figure 5 when in use.

 FIGS. 1 and 2 illustrate a preferred embodiment of the device for analyzing a macromolecule.

It could, for example, as explained above, be a device for the analysis of a genome.

 The device 2 has an enclosure 4 essentially closed in the general shape of

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 rectangular parallelepiped. This enclosure contains a support 6 intended to receive the macromolecule to be analyzed and in this case several hundred sequences corresponding to DNA fragments to be analyzed. The support 6 has in this case the form of a flat disc and it is here a disc.

 The disc 6 is mounted movable in rotation relative to the enclosure 4 around a central axis 8 of the disc. The device 2 includes, with reference to FIG. 2, a means for driving the disc 6 in rotation about its axis, comprising in particular a shaft 12 sealingly passing through a wall 14 of the enclosure 4.

 The enclosure 4 has a generally flat shape, the thickness of the enclosure extending in correspondence with that of the disc. The enclosure 4 has in particular two large vertical walls 14 extending opposite the two faces 16 of the disc. The interior of the enclosure 4 in this case essentially has an upper zone 18 and a lower zone 20. These two zones are separated from each other by a border 22 which is essentially rectilinear and horizontal, visible in FIGS. 1 and 2.

 The border 22 extends above the axis of rotation 8. In this way, the lower zone 20 notably includes all of the lower half of the enclosure extending under the axis of rotation 8 as well as all of the half bottom of disc 6.

 In the upper zone 18, the vertical faces 14 of the enclosure have internal faces 24 extending opposite the faces of the disc and at a relatively great distance from them. In this case, the disc 6 having the thickness of a CD

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 conventional, this distance is equal to at least 3 or 4 times the thickness of the disc.

 In the lower zone 20, the faces 24 of the walls 14 extending opposite the faces 16 of the disc are at a relatively small distance from these faces. This distance is much less than the distance separating these same faces in the upper zone of the enclosure. Preferably, this distance d is less than the thickness of the disc itself and is sufficiently reduced to correspond to a capillary thickness with respect to the fluid intended to fill the lower zone 20 as will be seen below.

 The transition between the two zones 18 and 20 is effected by the horizontal edge materializing the border 22.

 The device comprises means 26 in fluid communication with a fluid inlet orifice 28 opening into the enclosure and making it possible to introduce into the lower zone 20 one or more fluids which in this case are liquids. This introduction can be made, on the order of the user or of a control computer, discontinuously between the analysis phases or continuously during the analysis. The device 26 is of a conventional type. It will allow the composition of the introduced fluid (s) to be varied at will and in a discrete or continuous manner.

Likewise, the enclosure 4 has a fluid outlet orifice 30 associated with evacuation means which have not been illustrated. The fluid inlet and outlet devices in the enclosure will be of a conventional type and may include pumps, valves, etc. They will allow the choice to cause the liquid to stagnate in

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 the enclosure, or to circulate it only once through the enclosure in open circuit. The device also comprises means 40 for stirring the fluid in the enclosure, in particular making it possible to circulate it in a closed circuit. The means for stirring the liquid 40 are distinct in themselves from the disc 6 which, due to its movement, itself ensures a certain stirring of the liquid.

 The enclosure is arranged so that most of the lower zone 20 can be filled with liquid in order to constitute a bath 32 whose upper level 34 extends above the axis of rotation 8 of the disc and just below the border 22. In this way, the entire lower half of the disc as well as a portion of the upper half contiguous to the lower half is immersed in the bath 32 of liquid.

 The device 2 is arranged so that the disc can be rotated while it is immersed in the liquid. During the rotation of the disc 6, the liquid flows rapidly along the portions of the disc leaving the bath under the effect of gravity.

 In addition, the thickness d separating the faces 16 and 24 at the level of the lower zone 20 and which is of a capillary order is chosen so that, by capillary effect, at the level of the portions of the disc leaving the bath, the liquid tends to remain in the area 20, in contact with the walls 24 and the faces 16, without rising in the upper area 18 of the enclosure. This capillary effect is added to gravity to ensure that each portion of the disc leaving the bath is essentially or even completely free of liquid,

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 that is to say dry, when, due to the rotation, it arrives in the highest part of its trajectory. Furthermore, the centrifugal force can also help to evacuate the liquid from this portion.

 The device includes analysis means 36 capable of detecting a signal resulting from the interaction of a strand of DNA attached to the surface of the disc with a compound initially present in the fluid and now linked to this strand on the disc. This signal can be optical, electromagnetic or radioactive as indicated above. This reading can for example be carried out through a window 38 formed in one of the vertical walls 14 of the enclosure and extending in the upper zone 18 of the enclosure, opposite the highest zone of the disc. Typically, the reading means 36 make it possible to perform a reading along a vertical radius of the disc in the upper zone with reference to the axis 8. These means 36 will in particular be mounted mobile in a suitable manner in the same way as reading means associated with an audio CD.

 The device which has just been described can for example be used in the following manner to implement the analysis method according to the invention.

 In a first step, hundreds of DNA fragments to be studied are installed on a disk 6.

This implementation is carried out in a conventional manner by means of an insertion machine such as that traditionally used for the analysis of DNA fragments on a chip. The disc is then installed in enclosure 4.

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 Then introduced by means of the device 26 a liquid into the lower zone 20 of the enclosure to form the bath 32. This liquid comprises in particular synthetic DNA fragments generally obtained by RT-PCR and of a known type capable of s '' hybridize with probes representative of the genome to be studied installed on the disc. These fragments present in the fluid were marked by the use of fluorescent primers.

 Once the bath 32 is formed, the disc 6 is rotated, and simultaneously the analysis is started by means of the device 36.

 During the rotation of the disc, when a DNA probe attached to the disc traverses the bath, it is likely to interact with a complementary strand present in the fluid and then to carry it with it in the upper part emerged from the disc. The characteristic signal which results therefrom is then detected by the analysis means 36.

 As can be seen, thanks to this device, most of the portions of the disc extend into the bath then out of the bath and so on according to a time alternation. In addition, some portions of the disc extend into the bath while other portions of the disc extend out of the bath. The probes to be studied are thus brought into contact with the compounds in the lower part of the device at the same time as the analysis of possible hybridizations is carried out in the upper part of the device due to the permanent rotation of the disc. In other words, part of the disc is permanently outside the reagent and available for carrying out measurements. Movement

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 which animates the support means that the entire useful annular surface is presented per period in front of a line perpendicular to the axis of rotation. This line is the reading axis of the sensor for carrying out the measurements, in the absence of covering the surface with an excess of reagent carrying the marker.

This gives immediate access to the measurement of hybridization products.

 Depending on the results of the analysis, it is possible to modify all or part of the composition of the fluid to refine this analysis, the initial liquid being able to be evacuated by the orifice 30. The introduction and evacuation of the liquid can be made of discreet or continuous as well as the modification of its composition.

 The analysis means 36 can easily locate each of the DNA fragments placed on the surface of the disc 6. The analysis can be carried out with a very small margin of error so that it becomes essentially useless to multiply by identical the probes to be analyzed in order to obtain a redundant analysis as was the practice before.

The increase in the precision of the analysis, making it possible to get rid of this redundancy, is also allowed because the composition of the liquid can be modified during the analysis allowing a dynamic follow-up of the results of the analysis and an exploitation much finer of these.

 One of the liquids introduced into the enclosure may be a washing liquid, for example prior to the introduction of a new liquid intended to interact with the fragments attached to the disc.

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 In accordance with the most current CD technology, the analysis will be carried out on one side of the disk 6. However, provision may be made to work on both sides simultaneously, adapting the analysis means 36 if necessary, in particular by placing them on each side of the enclosure.

 The disc 6 will preferably be a polymer disc of the polycarbonate or glass type. This surface will be treated in a known manner to allow fixation of the samples (nucleic acid, protein, tissue section).

 The enclosure may be designed to be for single use and allow the archiving of the bio-CD. In another version, the speaker can be reusable and allow the disc to be changed.

 The device will preferably comprise means for regulating the temperature. It will thus be slaved to a control computer allowing the management of the temperature of the volume of the reagent used in the enclosure, the duration of the reactions and the speed of rotation of the disc.

 This makes it possible in particular to measure binding constants and to determine the presence or absence of polymorphisms in an individual (SNP or single nucleotid polymorphisms).

 Due to the fact that the reactor is stationary relative to the reference frame, it is easy to fix there the pipes necessary for the exchange of the solutions which come into contact with the support. The circulation of the reagents between the inlet and the outlet of the enclosure makes it possible on the one hand to improve the stirring of the reagents and on the other hand to optimize the recovery of the disc to

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 every moment and by simplifying the control devices. The enclosure can receive a series of sensors allowing the analysis of the characteristics of the reagent continuously, and a series of reagent admission points allowing the adjustment of the properties of the reagent in contact with the support.

 A first variant 102 of the device according to the invention has been illustrated in FIG. 3. This variant differs from the device of FIG. 1 by the configuration of the means 126 for introducing the fluid into the enclosure and extracting it.

 The maximum horizontal level that can be reached by the bath 32 is indicated by line 142 in phantom. This level extends under the axis 8. The means 126 comprise a liquid intake conduit 144 opening through its orifice 128 in against 104 and a level control conduit 146 having its orifice 130 in the enclosure.

 The two orifices 128 and 130 are tangent by their edge lower than the level 142, the orifice 130 acting as an overflow.

 The means 126 finally comprise a discharge duct 148, the orifice 150 of which opens into the enclosure at the lowest point thereof. Thus, the enclosure can be emptied under the effect of gravity.

 Not shown, the conduits 144, 146 and 148 can be connected together, for example temporarily to ensure circulation of the fluid in a closed circuit, thanks to the stirring means.

 One can provide an orifice 149 or a conduit opening into the enclosure at its highest point for

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 avoid overpressure or evacuate an air bubble in the event of complete filling of the enclosure, for example for washing it.

 A second variant is illustrated in FIGS. 4 to 6. In this device, the fluid inlet and outlet means 226 communicate with the enclosure 204 by a single orifice 230. The latter is occupied coaxially by a finger 250, by example in elastomer.

 The orifice 230 extends in one of the main vertical walls 214 of the device which it passes right through. The orifice 230 extends near the axis of rotation 8 with its geometric axis parallel to the latter. The orifice extends lower than the axis 8 but higher than the level 242 provided for the bath.

 The fluid entry means 226 comprise a needle 252 of a syringe which the user introduces coaxially into the finger 250 by piercing the latter which therefore becomes a sleeve.

 It is thus possible to introduce through the wall 214 the necessary reagent (s) 254. This variant will be particularly suited to the case where the disc is of small dimensions and brought into contact with a small volume of reagent and to the case where the device is disposable and Disposable.

 As can be seen in FIG. 5, the enclosure is formed by two half-enclosures 260 each carrying a main wall 214. Between the two half-enclosures is interposed a seal 262 of planar shape extending in the plane of the disc 6 or in a plane parallel to it.

FIG. 4 illustrates the line 264 along which the disk 6 is read. This line is inclined. It extends radially in the upper half of the

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 disc, in the quarter thereof starting its descent towards the reagent bath, taking into account the direction of rotation 266. Here again, the lower part of the disc forms with the walls of the enclosure a capillary thickness for the bath of reactive, the thickness being greater at the top of the disc.

 In these various devices, to avoid possible sealing problems, the disc can be driven in rotation by magnetic coupling through one of the walls of the enclosure, without consequently requiring a through shaft 12. The disc must then comprise at least a part made of material suitable for such coupling.

ci. Of course, many modifications can be made to the invention without departing from the scope of this one.

this.

 Provision may be made for the disc 6 to extend with its axis 8 inclined relative to the horizontal direction and possibly with this vertical axis 8. In the latter case, the location of the bath at only part of the disc will be obtained either exclusively by the effect of capillarity and centrifugation, or partially by this effect and partially by means of sealing means isolating the area of the disc bathing in the bath of the area outside the bath.

 In the inclined configuration, centrifugal force will be used mainly or exclusively to evacuate the reagent from disk 1.

 In certain types of analysis or in certain phases of the analysis, the fluid used may be a gas. The bath is then a gas bath.

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 We can modify the shape of the enclosure 4 as well as that of the support 6.

 The movement of the support in the enclosure may be an alternative movement and not a continuous movement.

It may be a translational movement and not a rotational movement. As an example, one could imagine placing the macromolecules to be analyzed on a strip shaped as an endless loop whose lower part is immersed and the upper part emerged, this strip being driven by means of two rollers respectively arranged in the upper parts and lower.

Claims (25)

claims 1. Device (2) for analyzing a macromolecule, comprising: - an enclosure (4; 104; 204); - a support (6) capable of receiving the macromolecule; and - means for bringing the support into contact with a fluid capable of interacting with the macromolecule, characterized in that it is arranged so that the fluid forms a bath (32) in the enclosure, the support (6) being mounted mobile in the enclosure so that at least certain portions of the support extend into the bath and then out of the bath according to a time rotation.  2. Device according to claim 1, characterized in that the support (6) is movable so that at all times, during the movement of the support, certain portions of the support extend into the bath (32) while d other portions of the support extend out of the bath.  3. Device according to any one of claims 1 and 2, characterized in that the support (6) is rotatable in the enclosure (4; 104; 204).  4. Device according to any one of claims 1 to 3 characterized in that the support (6) has an axis of rotation (8) extending out of the bath (32).  5. Device according to any one of claims 1 to 4 characterized in that the support <Desc / Clms Page number 37>  (6) is flat and has an axis of rotation (8) perpendicular to its thickness.  6. Device according to any one of claims 3 to 5 characterized in that the axis of rotation (8) is inclined relative to the vertical direction and in particular is horizontal.  7. Device according to any one of claims 1 to 6 characterized in that the fluid is a liquid.  8. Device according to claim 7 characterized in that the enclosure (4; 104; 204) has at least one wall (14; 214) delimiting the bath (32) and extending opposite at least one face ( 16) from the support (6) at a distance (d) from this face such that the liquid situated between the wall and the face is kept in the bath by capillary action in contact with the wall and the face.  9. Device according to any one of claims 1 to 8 characterized in that it comprises means (26; 126; 226) for introducing the fluid into the enclosure and / or extracting the fluid from the enclosure (4) .  10. Device according to claim 9, characterized in that the means (226) for introducing or extracting a fluid comprise a single orifice opening out inside the enclosure (230).  11. Device according to any one of claims 9 or 10, characterized in that the means (226) for introducing or extracting the fluid comprise an element (250) and a needle (252) capable of opening into the enclosure through the element (250). <Desc / Clms Page number 38>  12. Device according to any one of claims 1 to 11 characterized in that the device is able to put the support (6) in contact with several fluids in a temporal succession.  13. Device according to any one of claims 1 to 12 characterized in that it comprises means (40) different from the support (6) for setting the fluid in motion in the enclosure.  14. Device according to any one of claims 1 to 13 characterized in that it comprises means (36) for analyzing an interaction of the macromolecule fixed to the support (6) with the fluid, these means being understood opposite an area of the support located outside the bath.  15. Device according to claim 14 characterized in that the interaction comprises a binding of the macromolecule with a compound initially present in the fluid.  16. Device according to any one of claims 14 and 15 characterized in that the analysis means (36) are sensitive to radiation, in particular optical, electromagnetic or radioactive, resulting from the interaction.  17. A method of analyzing a macromolecule, characterized in that it comprises the steps consisting in fixing a macromolecule on a support (6), then setting the support (6) in motion in a bath (32) of a fluid capable of interacting with the macromolecule so that at least certain portions of the support extend into the bath and then out of the bath in alternation over time. <Desc / Clms Page number 39>  18. The method of claim 17 characterized in that the support (6) is moved so that at any time certain portions of the support extend into the bath (32) while other portions of the support s 'extend out of the bath.  19. Method according to any one of claims 17 and 18 characterized in that, the bath being formed in an enclosure (4; 104; 204), the fluid is introduced into the enclosure and / or the fluid is extracted from the enclosure during the movement of the support (6).  20. Method according to any one of claims 17 to 19 characterized in that the fluid is moved in the enclosure during the movement of the support (6).  21. Method according to any one of claims 17 to 20 characterized in that an interaction of the macromolecule is analyzed at a region of the support located outside the bath (32).  22. Method according to claim 21, characterized in that the analysis is carried out during the movement of the support.  23. Method according to any one of claims 21 to 22 characterized in that the interaction is analyzed, then the composition of the fluid is modified, then an interaction of the macromolecule with the fluid is again analyzed.  24. Method according to any one of claims 17 to 23 characterized in that the support is moved in a washing liquid.  25. Method according to any one of claims 17 to 24 characterized in that the <Desc / Clms Page number 40>  macromolecule is a nucleic acid, protein or peptide.