FR2927170A1 - MOLECULE CHIP DEVICES AND USES THEREOF. - Google Patents

Abstract

The present invention relates to low molecular weight, especially small molecule, microarray chip devices comprising a substrate of which at least a portion of at least one surface is coated with a layer comprising: - agarose and / or at least one of its derivatives; and at least one polyethylene glycol and / or at least one of its derivatives. The invention also relates to the uses of said devices for the detection of interactions between molecules and targets such as proteins, cells or organisms. .

Description

The present invention relates to micro-molecule chip devices, particularly small molecule, low molecular weight devices, and their uses for the detection of interactions between molecules and targets such as proteins, proteins and proteins. cells or organisms. The invention also includes the use of such devices for determining the presence of a target capable of binding specifically to the molecules present on the surface of said device. The invention also relates to methods for screening and selecting molecules, present on the surface of said device, capable of binding specifically to a given target.

Molecule chips, especially small molecules (low molecular weight molecules) can detect interactions between molecules and different types of targets (Uttamchandani et al., Mol Biosyst., 2006, v.2, 58-68; al., ACS Chem.

Biol. 2007, v.2, 24-30). This technology, which is in full emergence, has the advantage of being able to simultaneously compare and characterize a large number of molecules while replacing many individual tests with one and to evaluate several parameters in parallel. Such chips prove to be a powerful tool for the high-throughput screening of libraries or molecules banks, especially of chemical libraries allowing the identification of molecules capable of binding specifically to a protein, a cell, an organism or any other target. biological interest. These chips are therefore particularly useful for the identification of drug candidates, molecules that can be used for certain biotechnological, biomedical or agronomic applications or tools for research but also for the development of diagnostic methods. In particular, these chips are a particularly useful tool in the development of new antibacterial agents. Infectious diseases are still today one of the most important causes of death. In the absence of the discovery of new antibacterial or antibiotic agents, the progression of multi-resistant germs may lead, in the coming years, to more and more therapeutic failures. After several years of exhaustive studies in the field of medical chemistry, it becomes difficult to modify the structure of known molecules to make them more effective. This is why pharmaceutical research is beginning to focus on the discovery of other protein targets for the development of new types of antibacterial agents and this line of research is encouraged by the growing number of sequenced genomes of pathogenic bacteria. In addition, the advancement in the three-dimensional resolution of the structure of enzymes and multi-protein complexes, already exploited as targets for antibiotics, gives a strong impetus for the creation of new synthesized drugs including from organic molecules, weak molecular weight. In recent years, several approaches, called Small Molecule Microarrays (SMM), have been developed for the discovery of molecules of interest in chemical libraries (Winssinger et al., Proc Natl Acad Sci USA 2002, v.99, 11139-11144 Barnes-Seeman et al., Angew Chem Int Ed Engl 2003, v.42, 2376-2379, Bailey et al., Proc Natl Acad Sci USA 2004, v.101, 16144-16149, Kurosu and Mowers, Bioorg Med Chem; Lett., 2006, v.16, 3392-3395, Urbina et al., Chembiochem, 2006, v.7, 1790-1797). These methods are all based on the covalent attachment of low molecular weight molecules on plane surfaces previously activated by chemical means. These methods thus require chemical protection steps for the molecules and / or surfaces. Indeed, these methods require the creation of linkers (spacers or arms usable to fix molecules to a surface) particular for each type of molecule. Moreover, by orienting the immobilization of the molecules on the support via the linkers, this type of approach does not make it possible to evaluate the interaction capacity of the target, in particular protein-targets, with all the chemical groups of the molecules. of interest, which makes the screening of molecules limited. Moreover, since this approach is dependent on particular linkers for each type of molecule, it may not be compatible with different binding solutions (solution in which the target is located), which makes the screening of the molecules rather tedious and non-universal since little or no suitable for various banks of molecules, including various chemotec. Finally, some devices currently used for the immobilization of molecules, are not compatible with organic solvents (such as DMSO and DMF), used to improve the solubility of the little or not soluble molecules.

It is therefore essential to develop microarray molecules, especially small molecules (low molecular weight) for high throughput screening of various types of molecules (various molecules banks), with significant sensitivity while being inexpensive and adapted processes implemented on an industrial scale. The inventors have now established molecule chip devices to solve all or part of the problems mentioned above. Indeed, the inventors have developed a chip device with molecules, in particular with low molecular weight molecules, which does not require functionalization, neither molecules nor the device used, which makes it possible to avoid the tedious steps of chemical protection. and assures the target, in particular the target protein, the accessibility to all the chemical groups of the immobilized molecules on the device. Such a device makes it possible in particular to bind the molecules, in particular the molecules of low molecular mass, in a non-covalent manner. In addition, such a device is compatible with organic solvents (such as DMSO and DMF), used to improve the solubility of certain molecules.

In addition, the inventors have now established a method for detecting the interaction between molecules, in particular molecules of low molecular mass, linked to said device and different types of targets, in particular proteins. This method allows the high-throughput screening of molecules, in particular of a bank of molecules and in particular of chemical libraries composed of a plurality of different molecules of low molecular weight, with a good sensitivity of detection while being inexpensive, simple, fast and therefore particularly suitable for processes implemented at the industrial level. Thus, according to a first aspect, the subject of the invention is a molecule chip device comprising a substrate of which at least a portion of at least one surface is coated with at least one layer, in particular a layer comprising: agarose and / or at least one of its derivatives; and at least one polyethylene glycol and / or at least one of its derivatives. In order to allow a better understanding of the present invention, certain definitions are provided. Unless otherwise indicated, the other technical terms used in this application must be interpreted in their usual meaning. For the purposes of the present invention, "molecule chips" are understood to mean any device on which molecules are deposited. Generally, the molecules are immobilized on the device by non-covalent or covalent bond, directly or via spacer (or linker). The molecule chips may comprise a small or very large number of molecules and the size of the total surface area of the support occupied by the molecules may be variable.

Non-covalent bonds are here understood to mean ionic bonds, hydrogen bonds, Van der Waals forces or hydrophobic bonds. For the purposes of the present invention, the term "molecules" means the chemical and biological structures existing in nature or artificially created which can be distinguished from one another by their properties. As examples of molecules, mention may be made of proteins, peptides, polynucleotides such as DNAs (deoxyribonucleic acid) and RNAs (ribonucleic acid) and small molecules of natural or synthetic origin, organic whose molecular weight is between 50 and 150,000 Daltons, preferably between 50 and 1000 Daltons and most preferably between 100 and 600 Daltons. According to the invention, a binding is considered specific, when this binding is detected in a medium comprising a non-specific inhibitor, for example a molecule weakly binding to a target protein. According to the invention, a bond between a molecule and a target is considered to be specific, when this binding is carried out with a catalytic site of a target, in particular a target protein, and / or with a functional site of a target. target and / or with a structure that is likely to affect the functionality of a target.

For the purposes of the present invention, the term "target" means an organism or a macromolecular structure or a macromolecule or a mixture of identical or different macromolecules capable of binding to a molecule immobilized on the device according to the invention.

In particular, the target is chosen from the group comprising proteins, polypeptides, cell extracts, cells, organisms, nucleic acids, aptamers, in particular proteins. For the purposes of the present invention, agarose is understood to mean a linear polymer purified from agar, in particular from certain marine algae. The agarose used in the devices according to the invention may be of varied nature and in particular have various degrees of purity. The characteristics of the agarose that can be used in the devices according to the invention will be easily determined by those skilled in the art. In particular, the agarose that can be used in the devices according to the invention can form, after boiling, a transparent solution that is retained or adsorbed on a given substrate so as to form a transparent and relatively flat agar surface. In particular, such an agarose can melt above the temperature of 88 ° C, can be converted into a gel below 39 ° C, can have an electroendosmosis capacity of between 0.1 and 0.2, can contain less than 0.1% of sulfate, may be devoid of DNase and RNase activity and may be characterized by a gelling power greater than 1500 g / cm 2. For example, the agarose that can be used in the device according to the present invention may be the agarose available from Eurogentec under the reference EP-0010-05 (Molecular Biology Grade Agarose). The agarose derivatives that can be used in the layer coating the substrate can be of varied nature. Thus, it can be any agarose derivative adapted to bind non-covalently molecules, especially whose molecular mass is between 50 and 150 000 Daltons, preferably between 50 and 1000 Daltons and most preferably between 100 and 600 Daltons. By way of examples of such agarose derivatives, mention may be made of the agarose derivatives obtained by the addition of amine, carboxyl, thiol, imidazole or phenol radicals. For the purposes of the present invention, the term "polyethylene glycol" means any ethylene glycol polymer of formula HO- (CH2-CH2-O-) n -H. This polymer particularly stimulates the fusion of lipid structures, and is used to facilitate the transfer of nucleic acids, proteins and other molecules through the lipid membrane, ensuring a facilitated absorption of certain molecules with which it is in contact. Said at least one polyethylene glycol and said at least one of its derivatives may have a molecular weight of between 300 and 10,000,000 Daltons, preferably between 6000 and 40,000 Daltons and most preferably between 15,000 and 20,000 Daltons. The polyethylene glycol may be in the form of a branched or linear molecule, preferably linear. The polyethylene glycol may be prepared by polymerizing the ethylene oxide selected from the group consisting of ethylene oxide derivatives.

The polyethylene glycol derivatives that can be used in the layer coating the substrate can be of varied nature. Thus, it can be any polyethylene glycol derivative adapted to non-covalently bind molecules, in particular having a molecular mass of between 300 and 10,000,000 Daltons, preferably between 6,000 and 40,000 Daltons, and most preferably between 15,000 and 20,000 Daltons. The polyethylene glycol derivatives may be chosen from the group comprising polyethylene glycol copolymers, preferably the polyethylene glycol bisphenol A epichlorohydrin copolymer (available in particular from Sigma, under the reference P2263), the derivatives comprising methoxypolyethylene glycol, and the derivatives activated by different radicals such as carboxyls, NHS-esters. The layer coating the substrate may be varied in nature, suitable for non-covalently bonding molecules, especially molecules whose molecular mass is between 50 and 150,000 Daltons, preferably between 50 and 1000 Daltons and most preferably between 100 and 100 Daltons. and 600 Daltons.

In particular, the layer is a mixture, in particular a homogeneous mixture, of agarose and / or of at least one of its derivatives, in particular agarose; and at least one polyethylene glycol and / or at least one of its derivatives, in particular at least one polyethylene glycol. In the sense of the present invention, the term "mixture" means the combination of at least two substances.

For the purposes of the present invention, homogeneous mixing is understood to mean a mixture comprising a single phase. Thus, for example, a mixture of agarose and polyethylene glycol after having been heated to temperatures between 65 ° C and 70 ° C, can be deposited on the substrate to form a layer coating said substrate. Said layer coating the substrate (which is formed after drying of the deposited mixture) may have a thickness of between 1 μm and 200 μm, preferably between 1 μm and 50 μm, and most preferably between 1 μm and 20 μm. According to a particular embodiment of the device according to the invention, the concentration of agarose and / or its derivatives in said layer, in particular in said mixture, can be between 0.002 g / ml and 0.04 g / ml, preferably between 0.005 g / mL and 0.02 g / mL and most preferably 0.01 g / mL. The concentration of polyethylene glycol and / or its derivatives in said layer, in particular in said mixture, may be between 0.0005 g / ml and 0.02 g / ml, preferably between 0.001 g / ml and 0.015 g / ml. and most preferably between 0.002 g / ml and 0.01 g / ml. For the purposes of the present invention, the term "substrate" is intended to mean a material on the surface of which a layer of material is deposited and which serves to support it by retaining it or by adsorbing it. The substrate of the device according to the invention may be of varied nature. Thus it may be any substrate adapted to be coated with an agarose layer and / or its derivatives and at least one polyethylene glycol and / or its derivatives. In particular, the substrate may comprise a flat and / or curved surface, and be solid or semi-solid. Moreover, the substrate can have a shape and variable dimensions. Circular, rectangular, square substrates, etc. can be mentioned in particular. The surface of the substrate may be between 10 mm 2 and 400 000 mm 2, preferably between 100 mm 2 and 400 000 mm 2 and most preferably between 10 000 mm 2 and 20 000 mm 2. Said substrate may be made based on at least one material chosen from the group comprising glasses, silicon, silicone, polymers such as plastics, polyacrylamide, polypyrrole, polyoses and metals such as gold , platinum and a mixture of these. In particular, the substrate may be a glass slide.

The present invention also relates to a method for preparing a device according to the invention comprising the following steps: i) coating at least a portion of at least one surface of a substrate with a layer comprising: agarose and / or at least one of its derivatives; and at least one polyethylene glycol and / or at least one of its derivatives; and optionally ii) hydration by at least one hydration step of said layer obtained in step i).

Thus, according to a particular embodiment of the device according to the invention, said layer can be hydrated. Thus, after the layer has been deposited on the substrate of the device according to step i), the layer may undergo at least one hydration step. This hydration step ii) can be performed by any suitable means known to those skilled in the art. For example, said device may be immersed in water such as distilled and / or deionized water (in particular MilliQ) for a period of a few minutes to a few hours, preferably 30 minutes at room temperature. Said device can then be dried, in particular at 37 ° C, for a period of a few minutes to a few hours, preferably for 45 minutes. A device according to the invention comprising a hydrated layer has the advantage of reducing the intensity of the background noise and optimizing the detection sensitivity of the binding between molecules immobilized on said layer by non-covalent bonding and targets. The subject of the present invention is also the use of a device according to the invention for the immobilization, by non-covalent bonding, of at least one molecule, in particular of a plurality of different molecules and especially of a bank of molecules.

For the purposes of the present invention, the term "molecule library" is intended to mean a library of molecules chemically synthesized or isolated and of natural origin, which contains at least two different molecules. The banks of molecules include chemical libraries obtained chemically, manually or automatically and composed of purified or not, more than two molecules. The molecules that can be used to be immobilized on the device according to the invention may be the molecules that can be used for producing the molecule chips according to the invention.

The subject of the invention is also a molecule chip comprising at least one molecule, preferably a plurality of different molecules, non-covalently linked to said layer of the device according to the invention, in particular at defined positions.

The molecule chips according to the invention has the advantage of immobilizing molecules on the device via said layer, by non-covalent bond. This makes it possible in particular to avoid the tedious steps of chemical protection and allows accessibility to a given target, to all the chemical groups of the immobilized molecules. In particular, the molecule chips according to the invention allow immobilization by non-covalent bonding of molecules whose molecular mass is between 50 and 150 000 Daltons, preferably between 50 and 1000 Daltons and most preferably between 100 and 600 Daltons. . The molecules that can be used in the molecule chips according to the invention can be of varied nature. They may be identical or different molecules, in particular a plurality of different molecules. In particular, the molecules may be derived from libraries of molecules or chemical libraries. Examples of such libraries are the ZINC chemistry library (Irwin and Shoichet, J Chem Inf Model 2005, v.45, 177-182) and other public and commercial libraries. The molecules that can be used for the molecule chips according to the invention can be chosen especially from the group comprising proteins such as antibodies, peptides, polynucleotides such as DNAs (deoxyribonucleic acid) and RNAs (ribonucleic acid), mixtures thereof. DNA and RNA and preferably molecules of natural or synthetic origin, organic whose molecular mass is between 50 and 150 000 Daltons, preferably between 50 and 1000 Daltons and most preferably between 100 and 600 Daltons. The molecules may or may not be purified. For example, the molecules may be brought into contact with the devices according to the invention in order to be immobilized in the form of an extract, such as a crude extract of a plant. The molecules may be of natural or synthetic origin, in particular of synthetic origin.

By molecule of natural origin, in the sense of the present invention, is meant a molecule synthesized by a biological organism or present in nature, on the Earth or in the universe. For the purpose of the present invention, the term synthetic molecule is understood to mean a molecule synthesized chemically by manual or automated means. The molecules that can be used in the molecule chips according to the invention can be both identified molecules whose interaction with a given target is known, as well as test molecules whose binding capacity is to be evaluated on a given target. .

The chips according to the invention can comprise a low or very high number of molecules, between 2 and infinity, and the size of the total surface of the device occupied by the molecules can be variable from 100 to 400,000 mm 2. For the purposes of the present invention, a plurality of molecules is understood to mean a number of molecules greater than 2, in particular greater than 10 and most particularly greater than 50. The number of molecules present in a spot (defined position where the molecules are immobilized on the device) can vary from 2 to 100,000,000,000. The spots can have various forms, including circular or square. For example, the number of circular shaped spots with a diameter of between 50 and 1000 μm can vary from 2 to 100,000 on a device according to the invention. The invention also relates to a method for preparing a chip according to the invention comprising the following steps: i) the preparation of a chip device with molecules according to the invention, preferably hydrated; and ii) contacting at least one molecule, preferably a plurality of different molecules with said device, according to the invention obtained in step i), in particular at defined positions, in a medium and in appropriate conditions to obtain the non-covalent binding of the molecules to said device, in order to obtain a network of molecules; and optionally iii) the elimination by at least one washing step under the appropriate conditions of molecules not linked to said device.

Step ii) of the process for preparing a chip according to the invention can be done according to techniques well known to those skilled in the art. Examples of such techniques for depositing samples comprising molecules on the surface of said layer of the device include manual techniques (for example with the Arrayer Replicator V & P Scientific) or techniques using a contact robot (for example the Affymetrix GMS 417 Arrayer) or using a piezo-electric robot (for example the Arrayjet Arrayjet Sprint Inkjet Microarray Spotter). Preferably, step ii) of the process for preparing a chip according to the invention is carried out by contact means such as the Arrayer Replicator of V & P Scientific or the GMS 417 Arrayer of Affymetrix which make it possible to have the fingerprints molecules deposited on the device according to the invention. According to the invention, the samples comprising the molecules of interest can be deposited in the form of drops with a volume of between 10 picoliters (pL) and 15 nanoliters (nL). The agar structure of the layer of the device according to the invention allows a diffusion of the immobilized molecules as the conservation of the chips according to the invention, which makes it possible to increase the efficiency of the non-covalent immobilization of said molecules. . The intensity of the signals detected (detection of the target-molecule binding, in particular by fluorescence) after several days of conservation of the molecule chips according to the invention, is thus higher compared to the intensity of the signals detected after a day of preservation of said molecule chips. In addition, the molecule chip device according to the invention is tolerant (compatible) with the action of various organic solvents (such as DMSO and DMF) used to improve the solubility of certain molecules, especially of low weight molecules. molecular, chemically synthesized. This property of the device according to the invention is an advantage for the preparation of the chips with molecules having a low solubility. In addition, the device according to the invention is compatible with various reaction solutions, such as solutions prepared based on PBS buffer (Phosphate Buffer Saline or Tris phosphate buffer). Preferably, the solutions (media) used in the present invention are prepared at pH 7.4, to be placed at a physiological pH and thus under the appropriate physiological conditions, in particular for the study of interactions between at least one protein target and at least one molecule of low molecular weight, especially whose molecular mass is between 50 and 150,000 Daltons, preferably between 50 and 1000 Daltons and most preferably between 100 and 600 Daltons. Step iii) can be performed by techniques well known to those skilled in the art (Lam and Renil, Curr Opin Chem Biol 2002, v.6, 353-358). For example, step iii) can be carried out by incubating at least once, in particular three times, the chips for a period of a few seconds to a few minutes, preferably 5 minutes, in PBS (Phosphate Buffer Saline). 0.1% Tween-20 at 4 ° C., with stirring. The method for preparing a chip according to the invention may further comprise a step iv) blocking intended to reduce the non-specific interactions that take place between the target and the device according to the invention or between the target and the solvents ( media) used.

This step can be carried out by techniques well known to those skilled in the art (Lam and Renil, Curr Opin Chem Biol 2002, v.6, 353-358). This step may preferably be performed after step ii). This step can for example be carried out by incubating the chips with polyvinyl alcohol (PVA), in particular at 2%, for a period of a few minutes to a few hours, in particular 1 hour, preferably at 4 ° C. and with stirring.

The invention also relates to a kit or necessary for the detection of interactions between at least one molecule and at least one target selected from the group comprising proteins, polypeptides, cell extracts, cells, organisms, acids nucleic acids, aptamers, in particular proteins, said kit comprising: at least one device according to the invention; and - at least one molecule, preferably a plurality of different molecules; and at least one target chosen from the group comprising proteins, polypeptides, cell extracts, cells, organisms, nucleic acids, aptamers, in particular proteins; and at least one means for detecting the interaction between said at least one molecule and said at least one target. The molecules that can be used for the kits according to the invention may be the molecules that can be used for the chips according to the invention.

The subject of the invention is also a kit or kit for the detection of interactions between at least one molecule and at least one target chosen from the group comprising proteins, polypeptides, cell extracts, cells, organisms, acids nucleic acids, aptamers, in particular proteins, said kit comprising: a chip according to the invention; and at least one target chosen from the group comprising proteins, polypeptides, cell extracts, cells, organisms, nucleic acids, aptamers, in particular proteins; and at least one means for detecting the interaction between said at least one molecule and said at least one target. Said means for detecting the interaction between said at least one molecule and said at least one target may be any means allowing the detection of at least one molecule to which at least one target is specifically bound. In particular, said detection means may be based on the labeling of the target, of the molecule or of a secondary agent (such as an antibody) which recognizes the target or target bound to a molecule. For example, the target may be labeled, directly or indirectly, by a radioactive element or by a fluorophore (fluorescent dye). Examples of such detection means include fluorescence (including FRET Fluorescence Resonance Energy Transfer), chemiluminescence and radioactivity. According to a particular embodiment of the kits according to the invention, said target is a protein and said detection means is a fluorescent dye, preferably in the near infrared, said fluorescent dye being bound to the protein. According to another particular embodiment of the kits according to the invention, said target is a protein and said detection means is a fluorescent dye, preferably in the near infrared, bound to an antibody directed against the protein. The fluorescent dyes that can be used in the present invention and the techniques for binding a fluorescent dye to a protein, in particular an antibody, are well known to those skilled in the art. The fluorescent dye can be bound directly to the protein or antibody or via a spacer (linker). Those skilled in the art will know how to use well known, appropriate marking techniques. As examples of fluorescent dyes that can be used in the present invention, mention may be made of cyanines, fluorophores emitting in the near infrared (IRDyes) and nanocrystals. As examples of fluorescent near-infrared colorants that can be used in the present invention, mention may be made of Alexa Fluor (Invitrogen), IRDye800 CW (LI-COR Biosciences) and DyLight (Pierce).

In particular, the fluorescent dyes in the near infrared which can be used in the present invention have a wavelength of between 700 and 2500 nm, preferably between 700 and 1100 nm and most preferably between 720 and 820 nm.

The near-infrared detection system has the advantage of optimizing the detection sensitivity of the binding between the molecules and the targets. The subject of the invention is also the use of a device or a chip or a kit according to the invention, for the detection of interactions, between at least one molecule and at least one target chosen from the group. comprising proteins, polypeptides, cell extracts, cells, organisms, nucleic acids, aptamers, in particular proteins. The molecules may be identified molecules whose interaction capacity is known to a given target or molecules whose ability to interact with a given target is to be tested.

Thus, they may be identified molecules whose ability to interact with a target is known. Such chips or kits may be useful in particular to determine the presence of a target and possibly its concentration in a sample but also to characterize a target (for example determine the conformation of a protein). Indeed, some targets, including proteins, have the property of binding specifically with small molecules, depending on their conformation. The small molecules immobilized on the device of the chip can bind to the targets, in particular the proteins whose presence, the concentration and the characterization is then possible to determine. Such chips may also be useful for the separation of target proteins in different conformations (such as an active or inactive conformation) present in a sample. Molecule chips, kits or devices according to the invention may also be useful for the screening of molecules whose binding capacity to a given target is to be tested. It can in particular be the screening of a given bank of molecules, in particular a chemical library. In particular, such chips can be used to identify molecules capable of modulating (activating or inhibiting) the enzymatic activity of target proteins and / or binding to the catalytic site of target proteins. Such chips are particularly useful for the screening and selection of antibacterial agents or therapeutic agents for preventing and / or treating pathologies, including cancers, cardiovascular and neuromuscular diseases. Such chips according to the invention are thus particularly useful for the identification of drugs (such as antibiotics directed against bacterial proteins), molecules that can be used for certain biotechnological, biomedical, agronomic applications or as tools for research.

Thus, the devices, the chips and the kits according to the invention find applications in particular for: screening for molecules capable of interacting with said at least one target, in particular the screening of molecules capable of modulating the enzymatic activity of a protein and / or to bind to a catalytic site of a protein; or - screening for targets capable of interacting with said at least one molecule, in particular the screening of proteins which are ligands of said at least one molecule; or - determining the presence and / or concentration and / or characterization of at least one target present in a sample, in particular a protein, in particular with a particular protein conformation; or - the separation of particular forms of said at least one target present in a sample, in particular the separation of the active and inactive forms of at least one protein present in a sample. Said molecules targeted by these applications may be the molecules that can be used for the molecule chips according to the invention. In particular, the molecules can have a molecular weight of between 50 and 150,000 Daltons, preferably between 50 and 1,000 Daltons and most preferably between 100 and 600 Daltons.

The subject of the invention is also a method for detecting interactions between at least one molecule, preferably a plurality of different molecules and at least one target chosen from the group comprising proteins, polypeptides, cell extracts, cells, the organisms, nucleic acids, aptamers, in particular proteins, comprising the following steps: contacting at least one molecule, preferably a plurality of different molecules, with said device according to the invention, in particular with defined positions, in a medium and under conditions suitable for obtaining the non-covalent binding of said molecule to said device, in order to obtain a network of molecules; (Ii) contacting said grating obtained in step (i) with at least one target selected from the group consisting of proteins, polypeptides, (i) cell extracts, cells, organisms, nucleic acids, aptamers, in particular proteins, in a medium and under conditions suitable for allowing the formation of a specific bond between said at least one molecule and said at least one target; (iii) detecting on said device the molecule or molecules to which said at least one target specifically binds.

Step i) of the method for detecting interactions according to the invention may be carried out according to step ii) of the process for preparing a chip according to the invention, namely, according to techniques well known to humans of the Trade, either manually (for example with the Arrayer Replicator from V & P Scientific) or using a robot (for example the GMS 417 Arrayer from Affymetrix). The molecules that can be used in the process for detecting interactions according to the invention may be the molecules that can be used for producing the molecule chips according to the invention. In particular, the molecular weight of said at least one molecule is between 50 and 150,000 Daltons, preferably between 50 and 1000 Daltons and most preferably between 100 and 600 Daltons. The invention also relates to a method for detecting interactions between at least one molecule, preferably a plurality of different molecules and at least one target selected from the group consisting of proteins, polypeptides, cell extracts, cells, organisms, nucleic acids, aptamers, in particular proteins, comprising the following steps: (ii) bringing into contact with a chip according to the invention at least one target chosen from the group comprising proteins, polypeptides, cell extracts, cells, organisms, acids nucleic acids, aptamers, in particular proteins, in a medium and under appropriate conditions to allow the formation of a specific binding e between the molecules and said target; (iii) detecting on said chip the molecule or molecules to which said at least one target specifically binds. The medium and conditions suitable for forming a specific bond between said at least one molecule and said at least one target can be readily determined by those skilled in the art. Those skilled in the art will know how to use the standard conditions and protocols well known for this type of specific interaction that we wish to implement (Lam and Renil, Curr Opin Chem Biol 2002, v.6, 353-358). . In particular, especially when the target is a protein, said appropriate medium of step ii) methods for detecting interactions according to the invention can be adapted as a function of the isoelectric point of the target, in particular of the target protein and the charge of the immobilized molecules on the device according to the invention. Indeed, the inventors have shown that the composition of said medium of step ii) can affect the interaction of a target, in particular a target protein with immobilized molecules as a function of their charge and the charge of the target. , including the target protein. Thus, when the target is a positively charged target protein, said medium of step ii) may be based on salt derivatives such as PBS (phosphate buffered saline). When the target is a negatively charged target protein, said medium of step ii) may be based on Tris (buffer of trishydroxymethylaminomethane or 2-amino-2-hydroxymethyl-1,3-propanediol allowing pH to be understood in a range between 6.5 and 9.7). Tris has labile protons, with a pKa of 8.30 (at 20 ° C, the pKa then decreases by 0.03 units by elevation of a 1 degree Celsius temperature).

Step iii) methods for detecting interactions according to the invention can be carried out using any means for detecting the interaction between said at least one molecule and said at least one target that can be used in the kits according to the invention. 'invention.

In particular, the detection can be done using fluorescence, preferably in the near infrared. According to a particular embodiment of the methods for detecting interactions according to the invention, said target is a protein labeled with a fluorescent dye, preferably in the near infrared, and step (iii) is carried out using a fluorescence imaging device, preferably infrared. According to another particular embodiment of the methods for detecting interactions according to the invention, said target is a protein and the methods further comprise the following step: contacting the protein with an antibody labeled with a fluorescent dye, preferably in the near infrared, said antibody being directed against the protein; and step iii) is performed using a fluorescence imaging device, preferably infrared. Said step iii) can also be implemented by a computer-assisted method, for example by GenePix Pro 4.0 software (Axon Instruments). Examples of fluorescence imaging devices include the Odyssey Imaging System scanner (LI-COR Biosciences). In particular, the step of bringing the protein into contact with a labeled antibody can be carried out simultaneously with step ii) methods for detecting interactions according to the invention. The near-infrared detection system has the advantage of optimizing the detection sensitivity of the binding between the molecules and the targets. Said fluorescent dyes may advantageously be fluorescent dyes in the near infrared having a wavelength of between 700 and 2500 nm, preferably between 700 and 1100 nm and most preferably between 720 and 820 nm. According to a particular embodiment of the interaction detection methods according to the invention, the concentration of said protein in said medium of step (ii) is between 0.01 and 100 pull, preferably between 0.1 and 100. 10 μM, and most preferably between 0.5 and 2 M. A protein concentration of between 0.5 and 2 M, has the advantage of optimizing the detection sensitivity of the binding between the molecules and the targets.

Thus, the methods for detecting interactions according to the invention have the advantage over other methods (such as chemiluminescence and radioactivity) of allowing high-throughput analysis of interactions between molecules and different targets. data, including different given proteins and / or different conditions (such as the concentration of the target), which optimizes the probability of detection of specific interactions.

According to one particular embodiment of the methods for detecting interactions according to the invention, they furthermore comprise, prior to step (i), the following step: hydration, by at least one hydration step of said layer of said device. The hydration of said layer of said device can be carried out according to the hydration used in the process for preparing a device according to the invention. The hydration of said layer has the advantage of reducing the intensity of the background noise and of optimizing the detection sensitivity properties of the bond between molecules immobilized on said layer by non-covalent bonding and targets, in particular proteins. According to another particular embodiment of the interaction detection methods according to the invention, step (i) is carried out at least 1 day, preferably at least 5 days and most preferably at least 8 days before implementation. of step (ii). A duration of at least 8 days makes it possible to optimize the detection sensitivity properties of the binding between molecules immobilized on said layer by non-covalent binding and targets, in particular proteins. Thus, the prolonged storage (at least 1 day, preferably at least 5 days, and most preferably at least 8 days) of the molecule chips according to the invention improves the detection of the binding between the molecules immobilized on said layer by non-contact binding. covalent and targets, especially proteins. According to another particular embodiment of the methods for detecting interactions according to the invention, said medium of step (ii) comprises at least one salt selected from the group comprising sodium chloride, magnesium chloride and chloride potassium, preferably sodium chloride, at a concentration between 10 mM and 300 mM, preferably between 30 mM and 200 mM, and most preferably between 100 mM and 160 mM. The presence of at least one salt, in particular NaCl at a concentration of between 100 mM and 160 mM in said medium of step (ii), has the advantage of optimizing the interaction efficiency between the molecules immobilized on said layer by non-covalent binding and targets, in particular proteins. According to another particular embodiment of the methods for detecting interactions according to the invention, they further comprise, between step (ii) and step (iii), the following step: least one washing step, under appropriate conditions, targets not specifically related to said at least one molecule.

This elimination step can be performed by techniques well known to those skilled in the art. In particular, especially when the target is a protein, the elimination step can be carried out by incubating at least once, in particular three times, the chips for a period of a few seconds to a few minutes, preferably 5 minutes, in PBS (Phosphate Buffer Saline) / 0.1% Tween-20 at 4 ° C., with stirring, then for a period of a few minutes to a few hours, preferably 1 hour, in 20% isopropanol / 500 mM NaCl. 0.5% Tween-20 at 4 ° C., with stirring. This elimination step has the advantage of optimizing the interaction efficiency between the molecules immobilized on said layer by non-covalent binding and targets, in particular proteins. According to another particular embodiment of the interaction detection methods according to the invention, they furthermore comprise the following step, preferably simultaneously with step (ii): contacting said target and the network obtained in step (i) of the method for detecting interactions according to the invention or a chip according to the invention, with at least one molecule capable of binding specifically to said target.

This step may be useful for identifying high-speed molecules immobilized on said device that compete with known molecules capable of binding specifically to said target. In particular, especially in the case where the target is a protein, this step may allow the detection of molecules (immobilized on the device according to the invention) which bind to the same site or to another site (particularly catalytic) on the target, the site to which specifically binds the molecule used in this step (which is known to be able to bind specifically to said target). Thus this step may also allow the detection of molecules that bind specifically to a given target and to know their binding site on the target or to identify new sites (especially catalytic) on a target, in particular a protein. The invention also relates to the use of an interaction detection method according to the invention for the screening and selection of molecules capable of binding specifically to a target, in particular to a protein. In particular, the method for detecting interactions according to the invention may be useful for the screening and the selection of antibacterial agents and / or therapeutic agents intended to prevent and / or treat pathologies, in particular cancers, cardiovascular and neurological diseases. The subject of the invention is also the use of an interaction detection method according to the invention for determining the presence and / or concentration and / or the characterization of at least one target, in particular at least one protein present in a sample, said target being capable of binding specifically to said molecules non-covalently bound to said device.

The characterization of a target such as a protein may, for example, be the determination of its particular protein conformation (active, inactive, etc.).

Other advantages and features of the invention will become apparent from the following examples. It should be understood that these examples are given solely by way of illustration of the subject of the invention and in no way constitute a limitation thereof. FIG. 1 represents the fluorescence detection on the various molecule chips of molecules to which the IRDye CW (A) labeled R6 PBP 2x target protein specifically binds and the comparison of the intensity of the fluorescence signals on the Molecule chip devices tested (B). FIG. 2 represents, in the form of a fluorescent image of chips, the effect of the concentration of the IRDye CW-labeled R6 PBP2x (A) and R39 (B) target proteins on the interaction with the immobilized molecules on the Molecule chip devices according to the invention.

FIG. 3 represents the effect of the storage life of the molecule chips according to the invention on the detection sensitivity of the interactions between the R6 PBP 2x protein and the immobilized antibiotics. FIG. 4 illustrates, in the form of a fluorescent image of chips, the competition between the immobilized antibiotics and the ampicillin included in the reaction medium for the interaction with a protein labeled with IRDye 800 CW, R6PBP2x (A) or R39 (B). FIG. 5 shows, in the form of a fluorescent image of chips, the screening of libraries (DF, BA, SRI, AT) for the detection of interactions with R6 PBP2x (A), 5204 PBP2x S337A (B) proteins and R39 (C) labeled with IRDye CW. Each chip was also competed with ampicillin at a concentration of 6 μM or 7.5 μM.

FIG. 6 illustrates, in the form of histograms, the intensities of the signals coming from the spots of small molecules of low molecular weight belonging to the DF (A), BA (B), SRI (C) and AT (D) chemilibraries which possess a F800 / B800 ratio greater than 2 with at least one of the proteins tested. The experiments were performed in the presence or absence of ampicillin.

EXAMPLES 1. Example 1: Use of Molecule Chip Devices According to the Invention for the Detection of the Interaction Between Known Antibiotics and a Target Protein belonging to the Family of Penicillin Bindinq Proteins (PBP) In Order to Stand at a pH physiological and therefore under physiological conditions suitable for the detection of interaction between molecules of low molecular weight (antibiotics) and a protein (R6 PBP2x), all the solutions (media) were prepared at a pH 7.4.

I.1 Preparation of Microarray Devices According to the Invention Agarose (EP-0010-05, Molecular Biology Grade Agarose, Eurogentec) was dissolved at a concentration of 2% in MilliQ water and carried to boiling. This solution was bottled and incubated at 70 ° C. Different concentrations of a polyethylene glycol (PEG) derivative were prepared by dissolving Polyethylene Glycol Bisphenol A Epichlorohydrin Copolymer 15000-20000 (Sigma) (noted Cop PEG) in MilliQ water and heating the mixture to 70 ° C. vs. Equal volumes of the PEG derivative and agarose were mixed and pre-incubated at 70 ° C for 1 hour before being deposited on a glass slide cleaned with 100% ethanol.

I.2 Preparation of Molecule Chips According to the Invention A step of hydration of the layer of the device produced according to Example I.1 was carried out. Thus, the devices were immersed for 30 minutes in MilliQ water at room temperature without stirring and then dried at 37 ° C for 45 minutes. The molecules used to be immobilized non-covalently on the support according to the invention are 14 antibiotics known to interact with the proteins of the Penicillin Binding Proteins (PBP) family: 8 antibiotics belonging to the penicillin group, 5 to the group of cephalosporins and one to the carbapenem group.

Abbreviations of the antibiotics used are: 6-APA, 6-Amino-phenicillinic acid; Amp, ampicillin; Penicillin; Tem, temocillin; Mth, methicillin; Oxa, oxacillin; 7-ACA, 7-Aminocephalosporanic acid; Carb, carbenicillin; Tic, ticarcillin; Cpht, cephalothin; Cphx, cephalexin; Cftx, cefotaxime; Imp, imipenem; Cfx, cefoxitin. Kanamycin (Kan) was used as the negative control. The antibiotics were deposited from 3 mM solution (dilution in PBS) into 4 replicates (spots or defined positions). Negative controls such as 1X PBS, 55% DMSO (dimethyl sulfoxide) or BSA (bovine serum albumin) were also contacted with the devices of the invention. Anti-PBP2x polyclonal antibody (0.6 μM) was immobilized non-covalently on the devices according to the invention and used as a positive control. The molecules were deposited on the devices by a contact device either manually with the Arrayer Replicator (V & P Scientific) or with the robot GMS 417 Arrayer (Affymetrix). The molecule chips thus obtained were then blocked with 2% PVA (polyvinyl alcohol) (prepared extemporaneously in PBS) for 1 h at 4 ° C. with stirring and then washed 3 times 5 minutes with PBS / 0.1 Tween-20. % at 4 ° C with stirring.

I.3 Contacting Molecular Chips According to the Invention with a Target Protein: R6 PBP2x The molecule chips obtained according to Example I.2 were then incubated for 18 h at 4 ° C. with stirring with 12.5. log / mL of R6 PBP2x protein (the recombinant protein derived from the strain Streptococcus pneumoniae R6) preliminarily labeled with IRDye800 CW (LI COR Biosciences) and purified on a SigmaSpin Post-reaction column (Sigma). The chips were then washed 3 times 5 minutes in PBS / 0.1% Tween-20 at 4 ° C. with stirring, then 1 hour with 20% isopropanol / 500 mM NaCl / 0.5% Tween-20 at 20% strength. 4 ° C with stirring I.4 Detection on the molecule chip according to the invention of molecules or molecules to which the target protein specifically binds: R6 PBP2x The reading of the chips obtained in Example 1.3 was carried out by the Odyssey Imaging System (LI CORC Biosciences) scanner with 21 psi resolution thanks to a 780 nm excitation laser and a filter capable of detecting fluorescence at 820 nm. The images presented in FIG. 1A were then analyzed by the GenePix Pro 4.0 software (Axon Instruments). The software generates digital data corresponding to the median values of the signal emitted by the pixels of each spot (F) (defined position) and the local background noise (B). From these data, the ratio F800 / B800 for each spot (defined position) is calculated, then the average of the ratios for the 4 replicates. This F800 / B800 ratio was used as the quantitative value to compare the interaction of an immobilized molecule with the target protein (Figure 1B).

It should be noted that in Figure 1B only the results with 8 out of the 14 antibiotics tested are shown. Thus, the intensity of the fluorescent signals detected on the chips is a comparative index of the affinity of the given target protein (present in the reaction medium used to obtain the specific binding between the molecules and the target) with respect to different chemical molecules immobilized on the molecule chip device according to the invention, in a single test.

I.5 Comparison of the Background Noise Obtained with Molecule Chip Devices According to the Invention and the Background Noise Obtained with Other Devices Molecule chips were obtained according to the methods described in Examples 1.1 to 1.4, ci -above.

Thus substrates (glass slides) were coated with a mixture of 1% agarose and 0.2% PEG copolymer (Polyethylene glycol Bisphenol A Epichlorohydrin Copolymer 15000-20000, Cop Cop) with or without a step of hydration of said mixture. These molecule chips were compared with devices whose substrate (glass slide) is covered with 1% agarose or a mixture of 1% agarose and 2% PVA and 0.2% Na2B4O.

The results are shown in Figure 1 (A and B). This quantitative comparison in the form of histograms (FIG. 1B) shows that the highest intensity of the signal (except for cephalexin and imipenem) is obtained on the device comprising a mixture of 1% agarose and Cop PEG 0. , 2% that has been hydrated. In order to quantitatively evaluate the intensity of the signals from the spots, the average of the F800 / B800 ratio of the 4 spots was calculated for each antibiotic. The results are shown in Table 1, below. Agarose 1% Agarose 1% / Agarose 1% / Agarose 1% / Hydrated PVA 2% / PEG 0.2% PEG 0.2% No Na2B4O7 0.2 hydrated hydrate Average 2961.53 4087.77 2605.37 3604.78 background noise Table 1. Evaluation of background noise on different agarose media.

Thus, for most of the antibiotics tested, the best detection sensitivity was obtained with the device according to the invention coated with a mixture of 1% agarose and 0.2% Cop PEG hydrate before the setting step. in contact with the molecules with said device.

In addition, the devices according to the invention have been compared, (by evaluation of the background noise) with other microarray devices such as hydrogels prepared on the basis of an agar, polyacrylamide, agarose or such as biofilms composed of sodium alginate whether or not containing crosslinked and esterified copper, or such as polymers (nitrocellulose from the company Whatman). The results show that the devices according to the invention make it possible to reduce the intensity of the background noise in comparison with these other devices.

In addition, the devices according to the invention are tolerant (compatible) with the action of an organic solvent solution, especially DMSO and DMF at least 50%. This is an advantage for the screening of molecules synthesized chemically in these solvents, without affecting the interaction with the target, especially the target protein.

II. Example 2 Effect of the Concentration of a PEG Derivative on the Performance of the Interaction Detection Method According to the Invention The 14 antibiotics of Example 1 as well as the anti-GST antibody, the anti-PBP2x antibody and PBS were immobilized, according to the technique described in Example 1, on microarray devices according to the invention. invention comprising different concentrations of Cop PEG. 0.2%; 0, 5%; 0.8%; 1 and 2%. The chips thus obtained were incubated with 180 μl of the R6 PBP2x protein at 20 log / ml for 18 h at 4 ° C after being blocked for 1 h with 2% PVA. After washing for 1 hour with 20% isopropanol / 500 mM NaCl / 0.5% Tween-20, the chips were analyzed by fluorescence in the near infra-red according to the technique described in Example 1. Signal intensity from the spots, evaluated as the average of the F800 / B800 ratios of the 4 spots, was calculated for each molecule. The results are shown in Table 2, above.

Type of Blister Coating 0.2% 0.5% 0.8% 1% Cop 2% Cop Cop PEG Cop PEG Cop PEG PEG PEG Antibiotic / Control (mg / mL) Ratio F800 / B800 6-APA 0 , 2.25 6.75 6.78 6.64 2.74 6-APA 0.1 2.67 4.25 3.93 4.71 2.13 6-APA 0.05 1.53 1.62 1.83 1.99 1.37 Amp 0.0125 1.60 1.99 1.87 2.21 1.27 Amp 0.00625 1.52 1.60 1.57 1.90 1.17 Amp 0, 00315 1,19 1,24 1,38 1,37 1,11 Amp 0.0125 1.86 1.65 2.07 2.12 1.53 Amp 0.00625 1.53 1.41 1.70 1, 97 1.28 Amp 0.00315 1.15 1.14 1.36 1.36 1.12 Pen 1 0.93 0.99 1.03 1.03 1.02 Tem 1 1.09 1.11 1, 17 1.19 1.09 Tem 0.5 1.02 1.05 1.07 1.14 1.03 Tem 0.25 1.00 1.01 1.02 1.03 1.01 Mth 1.08 0.99 0.99 1.02 1.01 Oxa 1 0.96 1.00 1.04 0.96 0.99 7-ACA 1 1.07 1.20 1.25 1.37 1.12 ACA 0.5 1.04 1.13 1.16 1.15 1.07 7-ACA 0.25 0.95 1.05 1.03 0.94 0.99 Tic 1 0.88 1.08 1, 09 1.09 1.04 Tic 0.5 0.99 1.03 1.05 1.05 1.02 Tic 0.25 0.96 1.04 1.03 0.99 1.01 Cpht 0.2 1 , 81 1.76 2.11 1.91 1.22 Cpht 0.1 1.25 1.26 1.44 1.44 1.11 Cpht 0.05 1.12 1.14 1.21 1.19 1 , 05 Cphx 0.2 1.35 1.2 4 1.39 1.45 1.27 Cphx 0.1 1.17 1.14 1.18 1.21 1.11 Cphx 0.05 1.09 1.05 1.08 1.08 1.04 Cftx 0 , 2.27 2.03 2.44 2.73 1.64 Cftx 0.1 1.77 1.46 1.95 2.04 1.41 Cftx 0.05 1.27 1.27 1.51 1 , 47 1.17 Imp 0.1 1.50 1.42 1.55 1.80 1.23 Imp 0.05 1.15 1.13 1.14 1.25 1.09 Imp 0.025 1.05 1, 05 1.09 1.12 1.03 Carb 1 2.38 3.72 5.07 5.39 4.11 Carb 0.5 1.82 2.58 3.45 3.58 2.62 Carb 0.25 1,53 1,77 2,44 2,31 1,91 Cfx 1,06 1,03 1,03 1,04 1,02 Kan 1 1,00 0.99 0.95 0.97 0.97 Ac anti-GST 0.99 0.96 1.03 1.01 1.00 rabbit Ab anti-PBP2x 1.12 1.64 1.61 1, 96 1.37 rabbit PBS 1.03 0.99 1.02 0 , 99 1.00 PBS 1.04 0.98 0.97 0.98 0.98 Table 2. Interaction of immobilized molecules with R6 PBP2x protein as a function of Cop PEG concentration.

For most antibiotics, the best ratio and thus the best interaction between the antibiotics and the target protein is obtained on the device comprising between 0.5 and 1% Cop PEG mixed with 1% agarose.

However, beyond 0.2% Cop PEG, the polymerization of the agarose is carried out very rapidly making the homogeneous coverage of the surface of glass slides very delicate.

III. Example 3 Effect of the concentration of the target protein on the performance of the interaction detection method according to the invention The optimal target protein concentration was determined on the chips composed of 14 antibiotics described in Example 1 (glass slide coated with a hydrated mixture of 1% agarose and 0.2% PEG Cop), as well as PBS, BSA, anti-PBP2x antibodies, anti-PBP5 antibodies. Various concentrations of labeled proteins were added to the reaction medium (medium suitable for allowing the formation of specific binding between the molecules and the target protein) ranging from 0.3 μM to 1 μM for the R6 PBP2x protein and from 0 25 μM at 2 μM for the R39 protein. The contacting of the target proteins with the molecule chips described in Example 1 was carried out at 4 ° C. for 18 h under 70% constant humidity and very gentle stirring. The other experimental conditions described in Example 1 remain identical.

The results are shown in Figure 2 (A and B) and in Tables 3 and 4, below. Concentration of R6 PBP2x (pM) 0.30 0.50 0.60 0.80 0.90 1.00 Antibiotic / Ratio F800 / B800 Control 6-APA 4.54 5.68 7.01 7.48 6 , 79 7.10 Pen 1.04 1.05 1.05 1.06 1.06 1.06 Amp 15.08 23.62 32.54 30.51 22.90 31.80 Carb 2.22 2.92 3.17 3.13 2.87 3.11 Tic 2.84 3.95 4.65 4.44 3.84 4.20 Tem 1.03 1.06 1.04 1.05 1.04 1.05 Mth 1.02 1.02 1.02 1.00 1.01 1.00 Oxa 1.01 1.04 1.02 1.03 1.04 1.03 7-ACA 1.02 1.04 1.03 1.03 1.04 1.01 Cpht 1.50 1.40 1.93 2.01 1.49 1.95 Cfx 1.05 1.06 1.06 1.09 1.06 1.10 Cphx 1 54 1.70 1.83 2.03 1.77 2.04 Cftx 2.97 3.03 3.86 4, 22 3.56 4.05 Imp 1.17 1.21 1.18 1.29 1, 1.30 1.10 1.13 1.06 1.22 1.24 1.23 DMSO 55% 1.03 1.03 1.04 1.10 1.05 1.08 PBS iX 1.03 1 , 03 1.01 1.01 1.02 1.02 BSA 1.87 2.72 1.90 2.93 3.37 2.77 Anti-PBP2x 23.12 26.46 25.74 13.46 20, 16.90 Anti PBP5 3.66 8.10 6.90 11.51 8.47 8.47 Table 3. Effect of concentration of labeled R6 PBP2x protein on interaction with immobilized antibiotics.

According to the results obtained, it can be seen that the signal intensity, still based on the F800 / B800 ratio, is constantly increasing for all the antibiotics tested reacting with the R6 PBP2x protein in the concentration range from 0.3 to 0. 6 μM (Table 3). When increasing its concentration above 0.8 pM, the ratio value oscillates, showing that a plateau has been reached. Thus, the optimal concentration of R6 PBP2x is between 0.6 and 0.8 μM under the conditions used. To ensure that this R6 PBP2x wild-type protein concentration does not promote the appearance of non-specific signals, the assay was also performed with 0.6 μM of the mutant protein 5204 PBP2x S337A (the recombinant protein mutant Streptococcus pneumoniae 5024 in which serine (position 337) in the active site in is replaced by alanine) which is unable to interact with beta-lactam antibiotics. Indeed, no interaction is detected between the mutated protein and the immobilized antibiotics, which proves that the signals detected with the wild-type protein are specific.

Another PBP protein, R39 from Actinomadura, also interacted with the antibiotics tested, but the highest signals were detected at concentrations of 0.75-1 μM of this protein (Table 4). Concentration of Protein R39 (pM) 0.25 0.50 0.75 1.00 1.50 2.00 Antibiotic / Ratio F800 / B800 Control 6-APA 1.33 1.52 1.83 1.94 1, 67 1.83 Pen 1.01 1.02 1.03 1.05 1.05 1.05 Amp 13.53 19.72 40.58 30.99 26.29 22.28 Carb 1.36 1.62 1 , 88 2.07 1.88 2.01 Tic 1.54 1.90 2.27 2.45 2.24 2.44 Tem 1.10 1.20 1.27 1.30 1.31 1.34 Mth 1.01 1.01 1.01 1.03 1.01 1.02 Oxa 1.01 1.03 1.05 1.05 1.06 1.06 7-ACA 1.05 1.09 1.12 1 , 18 1, 14 1.15 Cpht 1.52 1.81 2.07 1.93 1.79 1.85 Cfx 1.71 1.99 2.22 2.25 1.94 1.90 Cphx 10.32 17.87 37.14 30.09 20.25 21.78 Cftx 1.66 2.30 2.68 2.87 2.41 2.82 Imp 1.08 1.15 1.26 1.25 1.24 1.23 Kan 1.07 1.17 1.20 1.28 1.23 1.33 DMSO 55% 1.01 1.06 1.08 1.08 1.10 1.12 PBS iX 1.01 1, 02 1.03 1.03 1.03 1.04 BSA 2.01 2.18 8.14 6.37 2.99 4.28 Anti-PBP2x 1.85 2.45 3.02 2.81 1, 57 2.48 Anti PBP5 5.12 7.27 15.19 9.42 14.82 21.50 Table 4. Effect of concentration of R39 protein on interaction with immobilized antibiotics.

Thus, the method for detecting interactions between at least one molecule and at least one target, in particular a target protein, according to the invention has the advantage over other approaches of allowing high-throughput analysis. interactions between immobilized molecules and different targets, including different proteins (under different conditions, for example, different concentrations) in parallel assays and thus increase the probability of the detection of specific binding between a molecule and a target. IV. EXAMPLE 4 Effect of the Presence of a Salt on the Performance of the Method for Detecting Interactions According to the Invention Chips composed of 8 beta-lactam antibiotics (6-APA, ampicillin, carbenicillin, ticarcillin, temocillin, cephalothin, cefoxitin, cephalexin and cefotaxime), immobilized PBS, DMSO, anti-PBP5 and anti-PBP2x antibodies were prepared from a 3 mM solution for each molecule and incubated with 0.6 μM R6. PBP2x diluted in a solution of PBS containing 12 mM or 154 mM sodium chloride. both solutions were supplemented with 0.1% Tween-20. The device used is a glass slide coated with a hydrated mixture comprising 1% agarose and 0.2% Cop PEG, prepared according to Example 1. The preliminary stages of blocking, washing, and the reaction conditions are those described in Example 1. The results are shown in Table 5.35 Salt concentration 12 mM NaCl 154 mM NaCl Antibiotic / Ratio F800 / B800 Control 6-APA 43.65 77.45 Amp 55.59 106.06 Carb 25.48 52.64 Tic 28.74 60.58 Tem 2.23 3.16 Cpht 29.33 60.44 Cfx 1.62 2.73 Cphx 4.89 13.47 Cftx 18.11 51.40 Water 0 , 99 1.01 PBS iX 0.98 0.91 DMSO 100% 1.00 1.18 DMSO 30% 1.00 1.00 DMF 100% 0.97 0.90 DMF 20% / Water 0.97 0, 96 Ab anti-PBP5 26.83 12.33 Anti-PBP2x Ab 18.71 18.20 85.31 Mean background noise 118.58 Table 5. Effect of the different NaCl concentrations in the reaction medium on the interaction of R6 PBP2x protein with immobilized antibiotics.

The analysis of the results shows that the presence of a higher concentration of salts in the reaction medium (suitable medium to allow the formation of a specific binding between the antibiotics and the target protein) can increase the intensity of the fluorescent signals. . Indeed, when 154 mM NaCl are added to the reaction medium, the F800 / B800 ratio is increased from 42 to 184%. This increase can only be attributed to the decrease in background noise since it is itself slightly higher in the presence of 154 mM NaCl.

Thus the presence of high concentration salts in the reaction medium (appropriate medium to allow the formation of a specific binding between the antibiotics and the target protein) can increase the efficiency of the interaction between the target protein and the molecules. immobilized and in this way improve the performance of the interaction detection method according to the invention.

V. EXAMPLE 5 Effect of the Shelf Life of the Molecular Chips According to the Invention on the Performance of the Interaction Detection Method According to the Invention The Molecule (Antibiotic) Chips According to the Invention According to the Invention Example 1 (glass slide coated with a hydrated mixture of 1% agarose and 0.2% Cop PEG) were stored at 4 ° C for 1, 8, 15 and 43 days before being contacted with a target protein: R6 PBP2x. The other experimental conditions, in particular for the detection of interactions, are those described in Example 1.

The results are shown in FIG. 3. The comparison of the results with the R6 PBP2x protein shows that, for all the antibiotics studied, a significant difference in the intensity of the signals was detected on the chips kept for eight days compared to those kept only one day. In addition, signal increase was again observed after 43 days of storage. The structural and functional characteristics of the small molecules can thus affect the efficiency of their non-covalent immobilization on the agarose and Cop PEG layer. Thus, the prolonged storage of the molecule chips according to the invention further improves the performance of the interaction detection between a molecule and a target.

VI. Example 6 Comparison of Media Comprising PBS and Tris on the Interaction of R6 PBP2x Protein with Immobilized Antibiotics The R6 PBP2x protein from Streptococcus pneumoniae has an isoelectric point of 5.17. An experiment was performed with this protein present in two media (medium appropriate to allow specific binding between antibiotics and target protein) different: PBS (with 154 mM NaCl / 0.1% Tween-20) at pH 7, 4 or 10 mM Tris (with 154 mM NaCl / 0.1% Tween-20) at pH 7.4 on an antibiotic chip obtained according to Example 1 (glass slide coated with a hydrated mixture of agarose 1% and PEG Cop 0.2%). The other experimental conditions, in particular interaction detection, are those described in Example 1. The results are shown in Table 6. PBS Tris Antibiotic / Ratio F800 / B800 Control buffer Control 6-APA 77, 45 84.80 Amp 106.06 114.48 Carb 52.64 57.12 Tic 60.58 61.16 Tem 3.16 3.01 Cpht 66.50 79.95 Cfx 2.64 2.01 Cphx 13.47 14.93 Cftx 51.40 44.25 Table 6. Effect of reaction media the interaction of R6 PBP2x protein with immobilized antibiotics. A 31.3% change in the F800 / B800 ratio was detected for cefoxitin when the reaction medium used was PBS, while for the other eight antibiotics, the difference was not significant. These results indicate that the composition of the reaction medium (suitable medium to allow the specific binding between the antibiotics and the target protein) can affect the interaction of the target protein with the immobilized molecules according to their charge. VII. Example 7: Study of competition between immobilized antibiotics and an antibiotic added to the reaction medium for interaction with the target protein The interaction of two molecules which bind to the same site on a target protein can be evaluated by following their competition in the reaction medium (appropriate medium to allow the formation of a specific binding between the molecules and the target protein). For this purpose, the competition experiments on the chips according to the invention (according to Example 1, glass slide coated with a hydrated mixture of 1% agarose and 0.2% PEG Cop), between antibiotics immobilized and directed against the active site of two R6 PBP2x and R39 proteins and ampicillin which binds to the same active site of these proteins were performed. Thus, ampicillin was added to the reaction medium with the protein given in the 10: 1 molar ratio and then this mixture (R6 PbP2x or R39) was incubated on chips composed of 14 antibiotics and various positive controls and negative. The other experimental conditions are those described in Example 1. The results are shown in Figure 4 (A and B) and in Table 7, below. The results show that after adding ampicillin as a competitor, a clear decrease in fluorescent signals was detected for all immobilized antibiotics (Fig. 4A and B). Protein R6 PBP2x Protein R39 Concentration in ampicillin (pM) 0 6 0 7.5 Antibiotic / Ratio / B800 Control F800 6-APA 21.96 1.02 3.55 1.01 Pen 1.11 1.02 1.04 1 , 03 Amp 31.80 1.04 33.92 1.02 Carb 7.62 1.01 4.50 1.01 Tic 12.00 1.01 6.11 1.00 Tem 1.15 1.00 1, 98 1.00 Mth 1.02 1.00 1.02 1.00 Oxa 1.05 1.02 1.04 1.00 7-ACA 1.08 1.01 1.29 1.01 Cpht 6.30 1 , 03 7.93 1.02 Cfx 1.23 1, 08 3.69 1.04 Cphx 2.36 1.16 25.99 1.24 Cftx 7.58 1.00 5.29 1.01 Imp 1, 34 1, 24 1.32 1.05 DMSO 55% 1.05 1.04 1.04 1.06 PBS iX 1.40 1.24 1.02 1.03 BSA 9, 53 7.90 10.32 11 , 35 Anti-PBP2x 26,27 24,22 4,73 2,69 Anti PBP5 10,54 12,99 17,61 21,62 Table 7. Competition between ampicillin in the reaction medium and immobilized antibiotics for 10 l interaction with R6 PBP2x and R39 proteins.

Quantitative spot analysis has shown that ampicillin in the solution is as effective in inhibiting the interaction of immobilized antibiotics with R6 PBP2x protein as with R39 protein (Table 7). This approach may be useful for high throughput identification of new molecules (immobilized on said device) that compete with known molecules capable of binding specifically to said target. In particular, this approach may be useful for high throughput identification of novel molecules that compete with beta-lactam antibiotics interacting with the catalytic site of PBPs. VIII. EXAMPLE 8 Screening of Different Chemical Libraries Various chemical libraries (DF, Oxford University, BA, SRI and AT (Grenoble IBS)) were screened according to the method for detecting interactions according to the invention, against the wild type R6 PBP2x, its antibiotic resistant mutant 5204 PBP2x S337A, the wild-type protein R39 but also the antibiotic resistant protein PBP5 from Enterococcus faecium D63r. Molecules capable of binding to the catalytic site of the PBP2x protein belonging to these libraries were selected after virtual screening. The molecules belonging to the different chemical libraries were dissolved in water, or in DMF if they were insoluble in water. The DMF concentrations tested for the solubilization of the molecules vary between 20 and 100%, without affecting either the quality of the device, in particular of said layer or the interactions with the protein targets. It should be noted that the possibility of using the dissolved molecules in a solution of DMF (amide derived from formic acid and dimethylamine) or 100% DMSO and deposited on the device according to the invention is an advantage in view of screening molecules chemically synthesized in these solvents, without affecting the interaction with the protein target. Thus, the device according to the invention is tolerant to the (compatible) action of various solvents, in particular organic solvents and is thus particularly useful for the preparation of chips with molecules, in particular of low molecular weight and which have a low solubility. The experimental conditions are those described in Example 1. A hundred small molecules from four different libraries were immobilized in 4 replicates on a chip device with molecules according to the invention, namely a glass slide covered with a hydrated mixture of 1% agarose and 0.2% CopPEG. Various positive and negative controls (the anti-PBP2x polyclonal antibody, the 14 antibiotics previously described) were also included in these chips which were stored for 8 days at 4 ° C prior to the experiment. The fleas were then incubated with one of the labeled proteins under the various conditions described above. Similar results were obtained in these experiments except that the intensity of the signals was variable according to the condition used. The images obtained after the interaction reaction show that each of the four libraries contains molecules that react with the protein targets (Fig. 5 A, B and C). The quantitative analysis of the results shows that among these molecules there are some that have a strong affinity towards at least one of the tested proteins. A filter of 2 was applied to the values of the ratios obtained to discriminate the molecules able to bind specifically with the proteins (molecules whose value of the ratio F800 / B800 is greater than 2) of those which do not bind specifically with the proteins (molecules whose F800 / B800 ratio is less than 2) (Fig. 6A, B, C and D). The seven molecules DF 26, BA 1, BA 9, SRI 8, SRI 13, AT 2 and AT 39 specifically bind with the four proteins tested. The SRI molecule 17 specifically binds with the R6 PBP2x and R39 wild-type proteins but not with the mutated proteins. It is remarkable that the intensity of the signals for the DF 26, BA 9 and AT 39 molecules was very high, which could indicate their ability to react with the same efficiency to the catalytic site of the wild-type and mutated proteins.

The concurrent competition experiment showed that ampicillin can inhibit the interaction of proteins with certain molecules that have the ability to interact with these targets. Indeed, a significant competition was detected between the immobilized AT 2 molecule and ampicillin in the solution for the interaction with the R39 protein.

A total of 19 molecules were identified after screening the four libraries by comparing their ability to interact with high affinity with at least one of the three R6 PBP2x, 5204 tested proteins.

PBP2x, R39 and PBP5 (Table 8). R6 PBP2x 5204 PBP2x R39 PBP5 Molecules Ratio F800 / B800 DF 8 2.08 2.56 1.34 1.09 DF 14 2.15 1.52 2.53 2.43 DF 21 0.80 0.70 1, 08 2.02 DF 22 0.89 0.81 1.14 2.07 DF 26 3.59 4.66 6.85 2.50 BA 1 4.18 4.39 3.75 2.80 BA 9 24, 56 8.39 35.03 12.90 SRI 8 2.73 2.02 3.11 2.96 SRI 11 1.53 2.42 2.26 1.78 SRI 13 2.61 2.73 3.66 2 , 00 SRI 17 2.04 1.63 2.17 1.11 SRI-01-050 2.93 3.05 3.71 1.52 AT 2.44 3.89 10, 20 4.35 AT 12 2 , 50 1.83 2.86 4.60 AT 16 1.40 1.28 1.67 2.47 AT 22 1.33 1.18 2.00 1.59 AT 27 0.77 0.76 0.73 2.36 AT 34 2.08 1.93 2.64 2.37 AT 39 8.98 22.21 2.44 46.31 * - Ampicillin in the competition experiment was added in a concentration of 6 μM for the R6 PBP2x and 5204 PBP2x S337A proteins and 7.5 pM for the R39 protein. Table 8. The molecules selected following the screening of four libraries against the R6 PBP2x, 5204 PBP2x S337A, R39 and PBP5 proteins.

IX: Example 9: Confirmation of the antibacterial activity of the molecules selected from Example 8 The antibacterial activity of the molecules selected according to Example 8 was evaluated by the antibiogram technique on gram-positive bacteria and gram-negative. It has been found that certain molecules (SRI 11 and AT 12) have significant activity against both types of bacteria. These results demonstrate the effectiveness of the molecule chips and interaction detection methods according to the invention, in particular for the screening of potential anti-bacterial agents. This method can be considered universal because of its ability to detect all types of interaction of molecules with a target, in particular a target protein. In particular, especially in the case where the target is a protein, the step of competition with at least one molecule capable of binding specifically to the target may allow the detection of molecules which, for example, bind to the same or to another site (especially catalytic) on the target that the molecule used in this step (which is known to be able to bind specifically to said target).

Thus this step may also allow the detection of molecules that bind specifically to a given target and to know their binding site on the target or to identify new sites (especially catalytic) on a target, in particular a protein. Thus, the lack of competition of the immobilized molecules with ampicillin may be an indication of the presence of a different site (s) of the catalytic pocket in the PBP proteins that can be used for the development of new agents. anti-bacterial. Finally, the method developed can also be applied for the screening of other therapeutic agents, in particular against cancers, cardiovascular and neuromuscular diseases. 15

Claims (29)

A molecule chip device comprising a substrate of which at least a portion of at least one surface is coated with a layer comprising: - agarose and / or at least one of its derivatives; and at least one polyethylene glycol and / or at least one of its derivatives. 2. Device according to claim 1, characterized in that said layer is a mixture, in particular homogeneous, - agarose and / or at least one of its derivatives, in particular agarose; and at least one polyethylene glycol and / or at least one of its derivatives, in particular at least one polyethylene glycol; the concentration of agarose and / or its derivatives in said mixture is between 0.002 g / ml and 0.04 g / ml, preferably between 0.005 g / ml and 0.02 g / ml and most preferably 0.01 g / ml mL; and the concentration of polyethylene glycol and / or its derivatives in said mixture is between 0.001 g / ml and 0.02 g / ml, preferably between 0.0005 g / ml and 0.02 g / ml, preferably between 0.001 g / mL and 0.015 g / mL and most preferably between 0.002 g / mL and 0.01 g / mL. 10 3. Device according to one of claims 1 or 2, characterized in that said at least one polyethylene glycol and said at least one of its derivatives have a molecular weight between 300 and 10,000,000 Daltons, preferably between 6,000 and 40,000 Daltons and most preferably between 15,000 and 20,000 Daltons. 4. Device according to any one of claims 1 to 3, characterized in that said substrate is a solid or semi-solid support made based on at least one material selected from the group consisting of glasses, silicon, silicone polymers such as plastics, polyacrylamide, polypyrrole, polyoses and metals such as gold, platinum and a mixture thereof. 5. Device according to claim 4, characterized in that said substrate is a glass slide. 6. A molecule chip comprising at least one molecule, preferably a plurality of different molecules, non-covalently bonded to said layer of the device according to any one of claims 1 to 5, in particular at defined positions. 7. A chip according to claim 6, characterized in that the molecular weight of said at least one molecule is between 50 and 150,000 Daltons, preferably between 50 and 1000 Daltons and most preferably between 100 and 600 Daltons. 8. Chip according to one of claims 6 or 7, characterized in that said at least one molecule is of natural or synthetic origin, in particular of synthetic origin. Kit or kit for detecting interactions between at least one molecule and at least one target selected from the group consisting of proteins, polypeptides, cell extracts, cells, organisms, nucleic acids and aptamers, particularly the proteins, said kit comprising: - at least one device according to any one of claims 1 to 5; and - at least one molecule, preferably a plurality of different molecules; and at least one target selected from the group consisting of proteins, polypeptides, cell extracts, cells, organisms, nucleic acids and aptamers, in particular proteins; and at least one means for detecting the interaction between said at least one molecule and said at least one target. Kit or kit for detecting interactions between at least one molecule and at least one target selected from the group consisting of proteins, polypeptides, cell extracts, cells, organisms, nucleic acids and aptamers, particularly the proteins, said kit comprising: - a chip according to any one of claims 6 to 8; and at least one target selected from the group consisting of proteins, polypeptides, cell extracts, cells, organisms, nucleic acids and aptamers, in particular proteins; and at least one means for detecting the interaction between said at least one molecule and said at least one target. 11. Kit according to any one of claims 9 or 10, characterized in that said target is a protein and said detection means is a fluorescent dye, preferably in the near infrared, said fluorescent dye being bound to the protein. 20 12. Kit according to any one of claims 9 or 10, characterized in that said target is a protein and said detection means is a fluorescent dye, preferably in the near infrared, bound to an antibody directed against the protein. 25 13. Use of a device according to any one of claims 1 to 5 or a chip according to any one of claims 6 to 8 or a kit according to any one of claims 9 to 12 for the Detecting interactions between at least one molecule and at least one target selected from the group consisting of proteins, polypeptides, cell extracts, cells, organisms, nucleic acids and aptamers, particularly proteins. 14. The use according to claim 13 for: screening for molecules capable of interacting with said at least one target, in particular the screening of molecules capable of modulating the enzymatic activity of a protein and / or of binding to a protein catalytic site of a protein; or - screening for targets capable of interacting with said at least one molecule, in particular the screening of proteins which are ligands of said at least one molecule; or - determining the presence and / or concentration and / or characterization of at least one target present in a sample, in particular a protein, in particular with a particular protein conformation; or - the separation of particular forms of said at least one target present in a sample, in particular the separation of the active and inactive forms of at least one protein present in a sample. 15. A method of detecting interactions between at least one molecule, preferably a plurality of different molecules and at least one target selected from the group consisting of proteins, polypeptides, cell extracts, cells, organisms nucleic acids and aptamers, in particular proteins, comprising the steps of: contacting at least one molecule, preferably a plurality of different molecules, with said device according to any one of claims 1 to 5, in particular at defined positions, in a medium and under conditions suitable for obtaining the non-covalent binding of said molecule to said device, in order to obtain a network of molecules; (ii) bringing said network obtained in step (i) into contact with at least one target chosen from the group comprising proteins, polypeptides, cell extracts, cells, organisms, nucleic acids and aptamers, in particular the proteins, in a medium and under conditions suitable for allowing the formation of a specific bond between said at least one molecule and said at least one target; (iii) detecting on said device the molecule or molecules to which said at least one target specifically binds. 25 16. The method of claim 15, characterized in that the molecular weight of said at least one molecule is between 50 and 150 000 Daltons, preferably between 50 and 1000 Daltons and most preferably between 100 and 600 Daltons. (i) 5 10 15 20 30 A method of detecting interactions between at least one molecule, preferably a plurality of different molecules and at least one target selected from the group consisting of proteins, polypeptides, cell extracts, cells, organisms, nucleic acids and the like. aptamers, in particular proteins, comprising the following steps: (ii) the contacting with a chip according to any one of claims 6 to 8 of at least one target chosen from the group comprising proteins, polypeptides, cell extracts, cells, organisms, nucleic acids and aptamers, in particular proteins, in a medium and under conditions suitable for allowing the formation of a specific bond between the molecules and said target; (iii) detecting on said chip the molecule or molecules to which said at least one target specifically binds. 20 18. A method according to any one of claims 15 to 17, characterized in that said target is a fluorescent dye-labeled protein, preferably in the near infrared, and that step (iii) is performed at using a fluorescence imaging device, preferably infrared. 19. A method according to any one of claims 15 to 17, characterized in that said target is a protein and in that it further comprises the following step: contacting the protein with a protein antibody labeled with a fluorescent dye, preferably in the near infrared, said antibody being directed against the protein; and in that step (iii) is performed using a fluorescence imaging device, preferably infrared. 20. Method according to one of claims 18 or 19, characterized in that said fluorescent dyes are fluorescent dyes in the near infrared having a wavelength of between 700 and 2500 nm, preferably between 700 and 1100 nm. and most preferably between 720 and 820 nm. 21. Method according to any one of claims 18 to 20, characterized in that the concentration of said protein in said medium of step (ii) is between 0.01 and 100 M, preferably between 0.1 and 10 M, and most preferably between 0.5 and 2 M. 22. Method according to any one of claims 15 to 21, characterized in that it further comprises, prior to step (ii), the following step: - hydration, by at least one step of hydration, said layer of said device. 23. Method according to any one of claims 15 and 17 to 22, characterized in that step (i) is carried out at least 1 day, preferably at least 5 days and most preferably at least 8 days before implementation of step (ii). 24. Process according to any one of claims 15 to 23, characterized in that the said medium of step (ii) comprises at least one salt selected from the group comprising sodium chloride, potassium chloride, sodium chloride and magnesium, preferably sodium chloride, at a concentration of between 10 mM and 300 mM, preferably between 30 mM and 200 mM and most preferably between 100 mM and 160 mM. 25. The method according to claim 15, further comprising, between step (ii) least one washing step, under appropriate conditions, targets not specifically related to said at least one molecule. 26. Method according to any one of claims 15 to 25, characterized in that it further comprises the following step, preferably simultaneously with step (ii): - contacting said target and the network obtained in step (i) according to claim 15 or a chip according to any one of claims 6 to 8, with at least one molecule capable of binding specifically to said target. 25 27. Use of the method according to any one of claims 15 to 26 for the screening and selection of molecules capable of binding specifically to a target, in particular to a protein. 28. Use according to claim 27 for the screening and selection of antibacterial agents and / or therapeutic agents for preventing and / or treating pathologies, including cancers and cardiovascular and neuromuscular diseases. 29. Use of the method according to any one of claims 15 to 26 for the determination of the presence and / or concentration and / or the characterization of at least one target, in particular at least one protein, present in a sample, said target being capable of binding specifically to said molecules non-covalently linked to said device.