FR2858852A1 - ORGANIZED PARTICLE ASSEMBLY AND USE THEREOF AS A LABELING SYSTEM - Google Patents

Abstract

The present invention relates to an assembly of particles linked together linearly or in a branched manner, characterized in that said particles belong to at least two subsets identifiable by at least one property specific to each subset, and follow each other in a sequence. predetermined constituting a specific code.

Description

The present invention relates primarily to a particle assembly

  particularly useful as a labeling or coding system for objects or natural or artificial products.

  With the development of an ever more complex world, traceability and identification are playing an increasingly important role. In particular, traceability, which makes it possible to unambiguously identify the origin of a product represents an important issue in terms of safety. For example, it is important to know the origin of a food. It may also be important to unambiguously identify an object or material to detect counterfeits, whether for products, official documents, checks or bank notes. Similarly, it may be important to uniquely identify a material, for example to allow its recycling or to know its manufacturer.

  Various labeling schemes have already been developed for this purpose. The most common are patterns printed or engraved on the surface of the object. As an illustration of this mode of surface labeling, mention may in particular be made of bar codes, patterns printed on banknotes or official documents. However, whatever care is taken to make these documents unfalsifiable, counterfeiters equipped with adequate printing tools always end up making counterfeits difficult to detect. Moreover, these impressions concerning the surface of the objects, they are relatively fragile and generally support poor abrasion or a fusion of the object, if it is for example a plastic material.

  As an alternative and to compensate in part for these limits of use, it has been proposed to incorporate identification criteria in the mass, and not only on the surface, objects to mark. These are, for example, fluorescent markers or watermarks for banknotes. However, here again some counterfeiters manage to overcome these obstacles and for example, to obtain fluorescent markers of spectrum comparable to that of the notes.

  Another alternative from microelectronics consists in integrating, for example magnetic domains or chips, into the considered objects. This type of marking is widely used for example in magnetic cards and smart cards. However, numerous facts have shown that it is possible for well-equipped counterfeiters to program or reprogram magnetic or chip cards. Moreover, these technologies are quite expensive and cumbersome, and their applications therefore remain limited to multi-purpose cards. Finally, they are quite fragile and do not withstand, for example, strong deformations.

  Thus, there remains to date a need for a method of labeling that can be integrated into an object and that makes it possible to identify its source in a safe manner even if the latter has been reduced to pieces or partially destroyed.

  In addition to the fields of use mentioned above, the field of science and more particularly that of life sciences, is also likely to benefit from such a labeling system.

  In fact, the development of genomics and proteomics, or that of combinatorial chemistry generally involves the parallel analysis of thousands or even millions of products. For obvious reasons, it would be particularly advantageous to be able to identify in a simple way, during these analysis operations, the products in question. However, this monitoring analysis is all the more difficult as these analyzes are performed generally with sample and reagent quantities as reduced as possible. Under these conditions, it is very difficult to perform an analysis of a large number of different species dispersed in a mixture.

  A solution that can be used for biomolecules such as nucleic acids or proteins consists in using DNA chips, nucleic acid chips or protein chips. In nucleic acid chips, DNA molecules or oligonucleotides (the probes) are deposited or synthesized in situ at predetermined spots on a surface. Each spot has a typical surface area of 100 microns per 100 microns or less, and contains a unique type of nucleic acid. This surface is placed in the presence of the sample to be analyzed under conditions allowing the formation of a nucleic acid double helix. If the sample contains a nucleic acid complementary to that fixed on a spot, this target nucleic acid will hybridize and this hybridization may be detected, most often by fluorescence, or by other techniques. This method makes it possible to have a large number of recognition sites on a limited surface and thus to carry out a large number of molecular analyzes with a relatively small sample quantity. However, these chip-type systems, also called hybridization networks, have certain drawbacks. Thus, such a system implies that a different nucleic acid is deposited or synthesized on each spot, and this for each chip which makes the manufacture expensive and therefore the price of these chips very high. Finally, the kinetics of hybridization is significantly slowed compared to conventional hybridization in solution.

  In WO 00/71243, it is proposed to test analytes, by employing an array of microspheres arranged on a surface, the microspheres carrying different functionalities. However, this mode of identification involves depositing beforehand at predetermined locations each type of sphere, in order to then identify with which probe a target has interacted.

  A variation proposed in Kohara et al, Micro Total Analysis Systems 2002, Voll, pp 227-229, Y. Baba et al. Eds, Kluwer Academic Publishers consists of linearly organizing individualized beads in a capillary. Each ball has a different type of oligonucleotide on its surface, thus making it possible to compare, in a single channel, a sample with several types of oligonucleotides. However, as in WO 00/71243, it is necessary to introduce one by one the balls in the channel. In addition, the presence of a microchannel to catalyze the balls and maintain their order is essential. All this raises many constraints in terms of microfluidics.

  To compensate in part for this disadvantage, it has been proposed in US Pat. No. 5,981,180 (Luminex) to firstly constitute a set of balls comprising several subsets of balls. In each subset, the beads, individualized relative to each other, carry a specific ligand of a species to be analyzed as well as a unique combination of fluorescent markers. The set of beads is then placed in the presence of the sample containing the species to be analyzed for a time sufficient for each complementary species of a ligand characterizing one of the sub-families of beads to hybridize to the beads of this sub-group. family. The whole of the beads is then analyzed by flow cytometry, according to a mode for simultaneously measuring for each ball the fluorescence light emitted in several wavelengths and to uniquely identify the beads belonging to each of the subspecies and the amount of analytes bound to said beads. This method is elegant and powerful for analytical applications, but its multiplexing capabilities are limited: indeed, the emission spectra of fluorescent molecules are quite large and it is difficult or impossible to use a large number if one wishes to prevent any recovery in these spectra or to overcome any uncertainty on the identification of the corresponding ball.

  More recently, WO 01/25002 describes colloidal particles in the form of rods that is to say of linear form, consisting of several segments of different and contiguous conducting metals. Each metal segment may be characterized by a specific property such as its dimension or its reflectivity properties. The set of segments thus constitutes a code which makes it possible to identify it with a much richer combination than that of the systems proposed in document US Pat. No. 5,981,180. However, such systems also have disadvantages. In particular, the density of metals being high, these metal rods tend to sediment quickly when suspended in water or in aqueous solutions. In addition, very small metal particles other than those made of noble metals are rapidly corroded in water and in most biological buffers. Therefore, to effectively implement the invention proposed in WO 01/25002 it is most often used particles made from gold, platinum or silver, and therefore expensive. Finally, said particles are limited to a small size, typically not more than fifty microns, by their manufacturing process and by their rigidity which makes them fragile for larger dimensions.

  As a result, for the reasons mentioned above, there is currently no life science system in the field of life sciences that is completely satisfactory in terms of cost, simplicity of implementation and reliability. .

  The object of the invention is precisely to propose a new labeling system, useful for unequivocally marking all types of objects, whether natural or artificial biological materials or compounds.

  More precisely, the present invention relates, according to a first of its aspects, to an assembly of particles which are connected to one another linearly or in a branched manner, characterized in that said particles belong to at least two subsets identifiable by at least one property specific to each subset, and follow each other in a predetermined sequence constituting a specific code.

  The invention aims in particular at a linear or branched assembly of particles permanently bonded to one another, said assembly having along its length a succession of predetermined properties carried by the particles, said succession constituting a characteristic code of said assembly.

  This code is said to be characteristic insofar as it makes it possible to distinguish the assembly considered vis-à-vis other assemblies comprising either other properties or the same set of properties but with those arranged in a different order of succession.

  More particularly, the particles are permanently or irreversibly bonded and in a linear form in the assemblies according to the invention.

  Within the meaning of the invention, the particle chain, or linear assembly of particles, will be indifferently referred to as an assembly of particles bonded to each other so as to constitute an object having at least locally an elongated shape. Different examples of such particle chains according to the invention are presented in FIGS.

  They can thus be in the form of a pearl necklace, in which the width of the chain is essentially that of a particle, as shown in Figures 1a, 1c, or 1d. They can also be in columns, in which each section of the chain comprises several particles.

  In all cases, the linear assemblies of particles according to the invention have an aspect ratio (ratio of the length to the largest dimension of a section) significantly greater than 1, typically greater than 3, and especially greater than 5. However, for many applications, significantly higher aspect ratios of 10 or more, or even greater than 100 and up to 10,000 may also be advantageous.

  Also within the scope of the invention, a branched assembly of particles comprising several linear chains connected together, provided that at least one of said chains has an aspect ratio greater than 3, and in particular greater than 5. A An example of such a branched chain according to the invention is shown in FIG.

  Whether in a form of linear chain (s) or branched chain (s), the assemblies according to the invention are clearly distinguishable from the compact three-dimensional aggregates of particles in which the organization of particles between them is totally arbitrary and therefore not reproducible.

  The assemblies according to the invention may have dimensions, in terms of average diameter and length, which are very variable. Typically, their average diameter varies from 10 nm to 1 mm, in particular from 10 nm to 100 g, in particular from 100 nm to 5 gm. Their length typically varies from 200 nm to 5 mm, in particular from 200 nm to 500 μm, and in particular from 1 μm to 100 μm.

  In general, the section of the particle assemblies according to the invention is essentially circular. However, it can also be of any other form, insofar as the largest dimension of this section remains smaller than the greatest length of the colloidal chain by a factor of at least 3.

  The particle assemblies according to the invention can be relatively rigid (essentially in the form of a rod), as shown in FIG. 1a, semi-rigid (capable of reversibly having a radius of curvature comparable to their length). as shown in FIG. 1 d, or flexible (capable of reversibly having a radius of curvature much shorter than their length), as shown in FIG. 1c. In the case of flexible chains, the length in the description above is along the curvilinear abscissa of said chain.

  According to a preferred variant, said particle assemblies according to the invention are flexible or semi-rigid. Indeed, such assemblies are advantageous for several reasons. On the one hand, they slide more easily towards each other. On the other hand, they are at rest in a folded form and as such occupy less space and are more easily held in suspension without them entangled. Finally, they can be easily aligned in a flow, for example in the flow chamber of a flow cytometry device. This latter property is particularly significant for semirigid assemblages, which therefore constitute a particularly privileged compromise.

  For the purposes of the invention, the term "particle" is understood to mean any compact three-dimensional object consisting of a multitude of atoms or molecules and capable of being suspended in a fluid.

  The dimensions of a particle according to the invention are typically between a few tens of nanometers and a few hundred microns, in particular between a hundred nanometers and ten microns. By way of example, latex spheres, microgels or magnetic beads of micron or submicron size, nanocrystals or isolated microcrystals constitute particles within the meaning of the invention, provided that their method of obtaining them makes it possible to obtain or suspend them in a fluid.

  The particles used to form the assemblies according to the invention are preferably of substantially spherical shape, for example in the form of beads.

  By essentially spherical form is meant an object that visually appears as a sphere with little or no deformation, typically having spherical defects of less than 30%, and preferably less than 15%.

  This form has several advantages. On the one hand, it is easy to obtain commercially spherical colloidal particles, generally called microbeads or nanobeads, or even latex, already carrying different properties such as different colors or different spectra of absorption or fluorescence emission. , and also carrying on their surface chemical or biological functions useful for applications. On the other hand, spherical particles tend generally to form, when aligned in an electric or magnetic field, a more regular chain than anisotropic particles, which can associate with each other in different orientations.

  Advantageously, the particles used for the preparation of assemblies according to the invention are of homogeneous composition. According to one variant, they may, however, be surface-coated with one or more layers of different compositions. In all cases, however, that is to say if one abstracts from this possible coating, the particles used for the preparation of assemblies according to the invention are not multisegmented in terms of composition. More specifically, they do not consist of a sequence of several segments of distinct compositions.

  In a preferred manner, the particles used to form the assemblies according to the invention have a low polydispersity in size, in particular less than 2 and in particular less than 1.5. As previously stated, this allows for more regular assemblies.

  Within the meaning of the invention, the term "property" refers to a characteristic in particular physical, chemical, optical, electrical, magnetic or biological, measurable or detectable, which makes it possible to differentiate the particles from one another. By way of example, these properties may be optical properties (absorption or emission of light, fluorescence, or luminescence (color), or more generally properties relating to interaction with electromagnetic or radioactive radiation, magnetic properties, properties of dimension, shape, or density.

  According to a first variant, these properties are properties pertaining to interaction with electromagnetic radiation.

  According to a second variant, the properties held by the particles are optical properties.

  According to a third variant, these properties concern the light absorption spectrum.

  According to another variant, said optical properties are fluorescence emission properties at one or more predetermined wavelengths, under the action of an excitation at one or more predetermined wavelengths.

  According to another also preferred variant, said optical properties relate to the spectrum of emission of luminescence, or properties of luminescence at one or more predetermined wavelengths, spontaneously or under the action of a stimulus such as the presence of a substrate or the application of an electric field.

  For the purposes of the invention, the term "code" refers to the sequence of all the properties present within the assembly of the particles and in particular along the said linear chain of particles.

  By way of example, and according to a so-called digital coding mode, the chain of FIG. 1a represents an assembly of particles characterized by the property code ABBACAB, where A, B and C represent different properties, for example different colors. . With the exception of the branched chains, the codes within the meaning of the invention are reversible along the chain, that is to say that the ABBACAB code for example, is equivalent to the BACABBA code. As another example, the chain of Figure ld represents the ABBCABCA code.

  It is also possible, in the context of the invention, to use another variant of so-called global coding using the succession of properties along the chain, without taking into account the number of particles having said property. According to this reading mode, for example, the assembly presented in FIG. 1 has the code ABACAB, and that presented in ld meets the code ABCABCA. This more global coding mode is particularly advantageous when the detection method used to read the code is not very precise, when the particles are very small and / or when the method of manufacturing the assemblies does not allow ensure a certain fixed number of consecutive particles with a given property.

  One can likewise use an intermediate coding mode, said analog, taking into account the succession of different properties carried, not by each particle, but jointly by several particles over a certain length. In this variant, the coding can relate both to the order of succession of the properties, and to the length of the chain assigned to one or more of the properties under consideration. For example, the chain shown in FIG. 1b is characterized by the code A (11) B (12) C (13) A (14), where 11, 12, 13 and 14 are, respectively, the lengths on which the chain is composed of particles bearing the properties A, B, C and A. This coding mode is particularly suitable for columnar chains, as shown schematically in Figure lb, or for small particles.

  In the case of branched chains, the code may have a more complex structure. The branched chain of FIG. 1 e, for example, carries, according to a so-called digital coding mode, the code ABC (AB) BAAB (CCA) CC, according to a notation in which the first parenthesis represents the code carried by a sub-code. chain carried by the third particle of the main chain presenting the property C, and the second parenthesis contains the code carried by a second substring, carried by the seventh particle of the main chain, carrying the property B. The branches advantageously allow to considerably multiply the number of possible combinations.

  The particles used to form the chains according to the invention may be organic, inorganic or organomineral. As already stated above, many types of organic or organomineral particles are available commercially or known to those skilled in the art. They make it possible to construct colloidal chains according to the invention with very diverse properties.

  In the case of the present invention, the particles may advantageously be non-conductive dielectric particles compatible with an electric field alignment. It can also be magnetic particles compatible with alignment in a magnetic field.

  Also suitable for the invention are particles consisting at least in part of organic polymer such as polymers and macromolecular materials.

  According to a preferred variant, they are wholly or partly of organic nature, or of an organomineral nature, that is to say having both organic constituents and mineral constituents.

  Many types of organomineral particles are commercially available or known to those skilled in the art. They advantageously make it possible to combine properties derived from the organic part and properties derived from the mineral part, and thus to construct assemblies of particles according to the invention with very diverse properties.

  Regarding the chemical nature of the mineral part (or the whole of the particle if it is essentially mineral), it can also be very varied, and include in particular quantum dots with specific properties of fluorescence or absorption light, and / or dielectric materials such as metal oxides or semiconductor metals. Magnetic materials, such as superparamagnetic, ferrimagnetic, ferromagnetic materials are particularly suitable for the invention.

  As the semiconductor oxides, the particles consisting essentially of silicon oxide or silica or having a silica shell are particularly advantageous.

  With regard to the chemical nature of the organic part of said particles, it can also be very diverse, and include in particular, for example, vegetable oils, of petroleum or synthetic origin, various polymers such as derivatives of acrylamide, polystyrene, polycarbonate, crosslinked or not, and more generally all the materials used to form latexes.

  In particular, particles having a mineral core coated with an organic polymer-type layer, such as, for example, polymers of the polystyrene or polycarbonate type, or derived from acrylic-type monomers such as N-isopropylacrylamide, are suitable for the invention. glycidyl acrylate or methacrylate, 2-hydroxyethyl methacrylate (HEMA), or ethylene dimethacrylate (EDMA). It may also be polymethyl methacrylate.

  An organic envelope is particularly advantageous insofar as it provides, via the presence of its organic surface functions, grafting possibilities for recognition sites and / or additional compounds and facilities for the linear organization of said particles. All kinds of reactive functions, well known to those skilled in the art, can be used as surface reactive functions. It may be, by way of example and in a nonlimiting manner, carboxylic, amine, alcohol, thiol functions, polymerizable functions such as double or triple bonds, in particular the allyl or acrylic functions, or even polyols, hydrazines and / or epoxides. They may also be strong binding sites for transition metals, such as histidine cages for nickel.

  Of course, it will advantageously be possible to use, in the context of the invention, particles having any combination of the preferred characteristics stated above, such as, for example, particles that are at the same time organomineral, dielectric and magnetic, and having various functions. of surface.

  Advantageously, the particles are chosen so as to give the linear assemblies according to the invention a density of less than 2. In particular, this density is less than 1.8 and more particularly less than 1.6. Ideally, this density can range from 0.7 to 1.4.

  These different variants are particularly advantageous when said assemblies must be, for a certain time, kept in suspension in a fluid, and in particular an aqueous fluid, as is the case for biological applications. Indeed, in these applications, a low density prevents assemblies from sedimentation, promotes their interaction with the targets with which they must react, and makes their subsequent analysis, for example by flow cytometry, easier.

  As specified above, the particles are organized within the assemblies according to the invention so as to form a predetermined sequence characteristic of a specific code. More particularly, these particles are organized in the form of at least one linear chain, where appropriate carrying one or more branches. Such an organization involves maintaining a cohesion between said particles. This cohesion allows the particle chains to be held in suspension, deposited on a surface, or dispersed in a matrix, without altering the sequence of properties contained in the code that makes it possible to identify them.

  In the particle chains according to the invention, the cohesion between the particles can be ensured by covalent bonds, in particular established directly or indirectly between said particles. Indeed, these links can also result from bridging with molecules or macromolecules.

  More generally, this covalent bond involves specific interactions either directly between said particles or between the particles and molecules or macromolecules, via reactive functions present on the surface of these particles. The reactive functions may be amine, carboxylic, alcohol, aldehyde, thiol, epoxide, hydrazine, halogen and / or double bond functions. However, the constitution and maintenance of a linear organization between the particles may also involve electrostatic, hydrophobic or Van der Waals interactions. They can also be obtained by interpenetration between polymers initially carried by different particles.

  For certain applications, in particular for the analysis of species or biological fluids, it may be advantageous for the particle chains according to the invention to also carry on their surface molecules capable of avoiding non-specific adsorption phenomena. . Such molecules are well known to those skilled in the art. It may be in particular hydrophilic polymers such as polyxyethylene, polypropylene glycol, polysaccharides and in particular dextran or polyacrylamide, or hydrophilic polymers of acrylamide, such as Duramide, poly-N-acryloylamino propanol, poly-N-acryloylaminoethanol, polyvinyl alcohol, polyvinylpyrrolidone, polydimethylacrylamide or copolymers of dimethylacrylamide and allyl-glycidyl ether. Such polymers may be grafted onto the surface of the particles either during the preparation of said particles, or after the formation of said chains, by using reactive functions integrated on the surface of said particles, or by direct adsorption.

  The presence of a bond between the particles, preferably irreversible, and in all cases sufficient to ensure cohesion between said particles for a time sufficient for the use of the assemblies according to the invention, The absence of an external field or a channel confining said particles gives the invention significant advantages, for example compared to the balls of the prior art discussed above. Indeed, the assemblies according to the invention are perfectly compatible with use both in volume and in a channel. Moreover, if one chooses to use said assemblies in a microchannel, one can in the same microchannel implement a large number of these assemblies, which is advantageous in terms of the number of analyzes.

  According to a preferred variant, which is particularly advantageous for species analysis, the particle assembly according to the invention may also carry at least one type of recognition site (s) for a species.

  For the purposes of the invention, the term "species" means molecules or macromolecules, particles, atoms, ions, objects of natural or artificial organic origin, such as nucleic acids, proteins, enzymes, antibodies, antigens, peptides, polypeptides, haptens, polysaccharides, proteoglycans, organelles, viruses, cells, cell sets, microorganisms and colloids. It may also be nano- or micro-particles of natural or artificial origin, organic or organomineral molecules, drugs, drugs, food, veterinary or cosmetic products and pollutants.

  By recognition site, or probe is meant a molecule, an ion, a surface element, or a specific portion of a molecule or ion, capable of giving rise to an attractive interaction or a chemical reaction with a particular species or category of species.

  The recognition sites present on the particle chains according to the invention may also be chosen from chemical functions capable of specifically recognizing other chemical species, for example by binding to them (as, for example, crown ethers capable of binding transition metals or vice versa). They may also consist of metal-specific ligands, molecular fingerprints, catalytic sites, hydrophobic groups or, more generally, the functionalities used in chromatography to give columns specific affinity for certain species. In particular, the recognition sites present on the assemblies and in particular the particle chains according to the invention may be chosen from compounds comprising aromatic or heterocyclic chemical functions, or sites capable of giving rise to hydrogen bonds.

  Several distinct types of recognition sites may be present at a particle assembly according to the invention and in particular carried by a linear chain of particles constituting or composing said assembly. However, in the majority of applications, a given assembly of particles according to the invention carries a unique type of recognition site, which can be uniquely identified by the code consisting of the sequence of specific properties otherwise carried by said assembly. This is a decisive advantage for species analysis. Indeed, it is possible to prepare in a solution a set of assemblies of particles according to the invention, comprising several subsets of assemblies. Each subset is characterized by a particular code associated with a specific sequence of properties along its length, and the constituent assemblies of said subset also carry a particular type of probe. We can then put this set of particle assemblies, in the presence of a sample containing a set of species (targets), which we want to know if they interact with the probes. Thanks to the presence of a multiplicity of microscopic chains, one can thus test a sample with respect to a large number of probes, as in the so-called techniques of microarrays, of chips with proteins, or chips with RNA, or more generally in so-called hybridization network techniques, but with, compared to these chips or networks, the following advantages: On the one hand, as the particle assemblies according to the invention occupy the space in a three-dimensional manner, can work with the same average number of probes in a much smaller volume, which reduces sample and reagent requirements, increases target concentration for a given sample quantity and therefore increases kinetics . The kinetics can be further increased by the fact that the particle assemblies according to the invention can be suspended in a liquid, and can therefore be stirred more efficiently than, for example, probes fixed on a surface.

  Fixation of a target species on a particle chain according to the invention can be detected by means of a technique known to those skilled in the art, and in particular by fluorescence, if the targets with a fluorescent marker, or by a chemiluminescence reaction, or by light absorption properties, this list being given by way of example and without limitation. We can then uniquely identify the probes with which a given target interacted, thanks to the unambiguous association existing between said probes and the code characterizing the assembly of particles carrying the said probes. There are many ways to prevent this detection of target fixation on the chains according to the invention from interfering with the recognition of the code: According to a preferred variant, it is possible for example to use for the coding and for the marking of fluorescent or luminescent markers emitting at different wavelengths or with different time constants, or absorbing light absorption markers at different wavelengths.

  According to another preferred variant, compatible with the preceding, the code characterizing the chain may be carried by a certain portion of the length of the chains according to the invention, the probes being carried by another portion. Several examples of such assemblies are shown in FIG. 2, which shows: A first assembly carrying the code AABBCCDDAA and the recognition site V, a second assembly carrying the code BBAAAACCBB and the recognition site T, and a third carrier assembly of the code BBBBDDAABB and the recognition site I Of course, for the sake of simplicity only a number of preferred variants have been described above in which only one type of probe can be uniquely associated with a single code. However, for other applications, and for example for the development of multiplexing, or to rely on redundancy in order to reduce the uncertainties, it is possible to use in the context of the invention a more complex combinatorial between the probes. and the codes.

  The subject of the invention is also a set of assemblies of particles bonded to each other linearly or in a branched manner, composed of several assembling subassemblies according to the invention, the assemblies belonging to a given subset carrying a specific code specific to said subset, in particular defined by a specific sequence of properties along their length, and further carrying at least one type of specific recognition site (s) of said subset.

  An assembly assembly joining three assemblies according to the invention belonging to three different subassemblies is illustrated in FIG.

  The present invention further relates to a process for preparing at least one particularly linear assembly of particles bonded together, having in its length a succession of predetermined properties constituting a specific code, comprising at least the steps consisting in: suspending, in a fluid medium, particles belonging to at least two subsets identifiable by at least one property specific to each subset, and capable of binding together, B / activate at least one means leading to their organization in said medium in a substantially linear form, and C / irreversibly bridging between the particles during their alignment state.

  The invention also relates to a method for constituting at least one assembly of particles bonded together in a linear fashion, comprising at least the steps of: A1 preparing a fluid medium comprising monomers capable of binding by polymerization or polycondensation, B / put suspended in said medium, particles carrying functions capable of reacting with said monomers, C / activate at least one means leading to their organization in said medium in a linear form, and D / obtain irreversible bridging between the particles during their state of alignment.

  As stated above, the methods according to the invention may comprise a preliminary step comprising introducing into the medium, molecules capable of binding by polymerization or polycondensation them and react with said particles.

  According to this variant, the starting medium may also comprise a priming system such that the polymerization reaction is not completed at the time of step C but gives rise to a bridging between the particles during their maintenance in an aligned form. . According to a variant also preferred, the priming system is a redox couple.

  According to a preferred variant, the monomers are of the acrylic type. Among the monomers that can be used to bridge the particles in the context of the processes described above, acrylamide is particularly suitable, acrylamide derivatives carrying acid functions and particularly acrylic acid, acrylamide derivatives carrying amine functions and more particularly N- (3aminopropyl) methacrylamide hydrochloride, and the copolymers of these different monomers.

  According to a preferred variant of the methods described above, said bridging is obtained using a stimulus. This stimulus may correspond to a temperature change, in particular an increase in temperature and / or activation using light, an electric field, a magnetic field, an electromagnetic field, and / or radioactive radiation.

  In the case where the ignition is carried out by an electromagnetic field, the medium may advantageously contain in addition to monomers a photoinitiator such as, for example, AIBN (Azobisisobutyronitryle), or methylene blue. For example, methylene blue may be used in the presence of a redox system, as described in Righetti and Caglio, Electrophoresis, 1993, 14, 573.

  According to a preferred variant, the processes described above further comprise a step intended to recover at the end of the bridging operation, said assembly of particles in linear form.

  When the particles are particles made up of all or part of silica, acrylic monomers can advantageously be used for bridging these particles between the particles, and functionalizing the surface of the particles with double bonds, or by using macromonomers with surface adsorption, such as, for example, poly-dimethylacrylamide molecules carrying at their end a double bond, or by grafting onto the surface of said particles a silane having a polymerizable function, such as trimethoxyxilyl 3-propyl methacrylate (Bind silane); , Aldrich).

  When the particles are latex particles bearing amine functional groups, acrylic or allylic monomers, optionally containing a redox initiator pair and / or a photoinitiator, and may be advantageously used in the suspension of step A. a photoactivator, and previously activate the particles with N-hydroxysulfosuccinimide (sodium salt, Fluka), optionally supplemented with 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (sigma)).

  A particularly advantageous process for preparing large quantities or a multiplicity of particle assemblies bonded together, said assemblies having, according to their length, a succession of predetermined properties according to the invention, comprises at least the steps of A / preparing in a first container of the particles identifiable by at least one property specific to these particles, or a composition comprising particles and at least one reagent making it possible to confer over time or under the action of a stimulus to said particles such a property, B in at least one second container, to prepare particles that are identifiable by at least one specific property, or a composition comprising particles and at least one reagent for conferring over time or under the action of a stimulus to said particles such property, the said property being different from that characterized the particles or reagents contained in the first container, C / introduce into a microchannel, by at least two separate arrival channels, the solutions prepared in steps A and B, and possibly other solutions by other channels of arrival, so that said solutions form in the main channel liquid veins essentially laminar and parallel, D / activate, within said channel, at least one means inducing the organization of particles in linear chains, E / link between them the particles in their linear state, and F / recover said assembly thus obtained.

  Of course, the method can be applied to a larger number of particle types and to a larger number of arrival channels. The possibility of having several modes of succession of the particles defining the code characterizing the assemblies, of a greater number of types of particles and of channels allows a more complex combinatorics.

  According to a preferred variant, said means comprise an external field substantially perpendicular to the planes separating the different liquid veins. This variant is particularly advantageous since it makes it possible to obtain directly, along the particle chain, a sequence of properties, predetermined by the properties of the particles or the markers contained in the different liquids, used to constitute the different liquid veins in the main channel.

  According to a preferred variant of the methods described above, the means for organizing the linear chain particles comprise the application of an electric field. According to another preferred variant, said means comprise the action of a magnetic field. According to a third preferred variant, said means comprise the action of an electromagnetic field or of a radioactive radiation. According to a fourth preferred variant, said means comprise a hydrodynamic flow.

  According to a preferred variant, the particles are magnetic and said means comprise the application of a magnetic field.

  According to another preferred variant, the particles are dielectric and said means comprise the application of an electric field. Preferably, this electric field is alternating, and even more preferably of frequency between 200 kHz and 5 MHz.

  To align dielectric particles in an electric field, it may also be advantageous to use a suspension of said particles in an organic solvent such as, for example, ethanol, methanol, acetonitrile or in a mixture of water and an organic solvent miscible with water. This provides a less conductive medium than water, and can be applied without heating higher electric fields which facilitates the setting of the particles in columns.

  By analogy with the methods defined above, step E is generally carried out according to the protocols proposed above. In particular, step E aims to permanently fix the particles in a linear form.

  As stated above, the assembly according to the invention proves particularly advantageous for marking objects, in particular fluids or materials. These assemblies are effective marking systems at the microscopic scale carrying a code extremely difficult to counterfeit.

  The present invention thus also relates, in another of its aspects, to a marking system for an object, characterized in that it comprises at least one assembly or set of assemblies according to the invention.

  As can be seen from the following description, these objects may be solid or fluid, in particular liquid and of any kind.

  By ordering particles carrying these markers, in a predetermined order, within assemblies according to the invention, it is possible to constitute a code having an extremely large number of combinations (10 billion with only 10 different markers and 10 zones along the assembly), whereas conventional fluorescence labeling techniques have only a much smaller number of markers for the same family of markers because of the superposition of the spectra. The ability to spatially distinguish the different types of markers is a key advantage, which can be easily exploited with an epifluorescence microscope, a confocal microscope, or scanner-like systems.

  On the other hand, contrary for example to bar codes, the particle chains according to the invention can be very small, typically of microscopic dimensions. Thus, they can be dispersed within or on the surface of a material such as a plastic or paper, and allow its identification even after the object has been broken or even melted. This offers new and advantageous tools for industrial and food traceability, waste recycling, police and military applications, or the prevention of counterfeiting.

  Similarly, the subject of the invention is also a method useful for marking an object, in particular a fluid, or a solid of the commodity type or a material, characterized in that it comprises the association with said object, of at least one linear or branched assembly of particles, having in its length a succession of predetermined properties carried by the particles, said succession constituting a code specific to said assembly. More specifically, this method can implement as a marking system, an assembly or a set of assemblies according to the invention.

  The code represented by said assembly makes it possible to differentiate it from other assemblies comprising either other properties or the same property set, but with these arranged in a different order of succession.

  This association with the object to be marked may be achieved by dispersing said assembly either at the surface or within said object.

  Of course, it is also possible to use in the implementation of the assemblies according to the invention, a multiplicity of different assemblies to increase the number of possible combinations, or to perform a coding according to several criteria. However, in many applications concerning marking, the combinatorics allowed by the assemblies according to the invention is sufficient to allow the use of a single type of assembly per product.

  The invention also relates to a method for the detection or analysis of particle assemblies as described above, as well as the recognition of species by said assemblies.

  These methods can implement various means known to those skilled in the art. According to a preferred variant, it is carried out by conventional or confocal microscopy, after deposition of the chains according to the invention on a surface. According to an even more preferred variant, said chains are deposited in the presence of a flow or by passage of a meniscus, as in Bensimon et al, Phys Rev Lett. 1995, 74, 4754-4757. They are all aligned in the same direction, and thus easier to analyze automatically.

  According to another preferred variant, this detection can be carried out by flow cytometry. The assemblies and in particular the chains according to the invention are particularly interesting in combination with flow cytometry. Indeed, on the one hand the flow tends to align the chains, and on the other hand the various particles constituting a chain, pass in front of the detector sequentially. Thus, for the analysis, a double information is available both on the light emission properties and on the temporal sequence of these properties, which constitutes a major advantage over the techniques of the prior art discussed above. .

  The subject of the present invention is also, according to another of its aspects, a method for analyzing, identifying, assaying, purifying or preparing a sample, implementing at least one step of bringing said sample into contact with at least one assembly of particles according to the invention.

  Optionally, this method further comprises a washing step in the course of which certain products contained in the sample are eliminated and / or during which certain products are recovered.

  Another object of the invention is, according to another of its aspects, a method for analyzing the interaction of species contained in a sample with respect to a multiplicity of recognition sites or probes, comprising at least the steps of : A / bringing said sample into contact with a set of linearly or branched assemblies of particles that are composed of several subsets, each subset being characterized by a code and by at least one type of sites of recognition specific to it, and B / seek and possibly quantify the fixation of species on at least some of said subsets of assemblies.

  By way of example and in a nonlimiting manner, the particle assemblies 10 according to the invention, and the various components and devices implementing said assemblies, can be used for: the diagnosis in vivo or in vitro, - the identification and / or the preparation of molecules, macromolecules, particles, atoms, ions, objects of natural or artificial origin, such as nucleic acids, proteins, enzymes, antibodies, peptides, polypeptides, polysaccharides, proteoglycans, organelles, cancer cells, rare cells, epithelial cells, endothelial cells, fetal cells, genetically modified organisms or products derived from genetically modified organisms, organisms or agents pathogens, viruses, microorganisms; - identification and / or preparation of chemical active ingredients such as toxic products, drugs, pollutants; medicines, dietary supplements.

  - recognition of plant animal varieties or microorganisms; detection of mutations; - the search for allergies; - genotyping; the search for genes involved in diseases; - Research and / or the preparation of reaction products from combinatorial chemistry protocols.

  By way of example and also without limitation, the particle assemblies according to the invention may be used for identification purposes for: - marking industrial, strategic or food products, packaging, liquids, materials , plastics, paper and cardboard, weapons, vehicles for traceability purposes, and to be able to identify their origin after the fact, and to mark monetary and fiduciary tools such as banknotes, credit cards or checks, official documents such as passports, laissez-passer, identity cards, products covered by trademarks and copyrights such as clothing, leather goods, audio or audiovisual CD or DVD, to identify possible counterfeits.

  The examples and figures which follow are presented as an illustration and not a limitation of the field of the invention.

  FIG. 1: Variants of particle assemblies according to the invention.

  1 a: rigid linear assembly, of the pearl necklace type, holder of the ABBACAB digital code; lb: rigid linear assembly, of the column type, carrying a code combining a property information of the particles and a length information assigned to said property. The code carried by this specific example is A (11) B (12) C (13) A (14), where 11, 12, 13 and 14 are, respectively, the lengths on which the string consists of particles bearing the properties A, B, C and A. lc: assembly of the bead necklace type, flexible.

  1 d: assembly of the pearl necklace type, semi-rigid.

  1 st: Assembly of the type of pearl necklace, branched. The particular assembly represented bears the digital code ABC (AB) BAAB (CCA) CC, according to a notation in which the first parenthesis represents the code carried by a substring carried by the third particle of the main chain presenting the property C, and the second parenthesis contains the code carried by a second substring, carried by the seventh particle of the main chain, carrying the property B. Figure 2: Schematic representation of three particle assemblies according to the invention, combining a code and a type of recognition site for one or more species. We present, from top to bottom: A first assembly, bearing the digital code AABBCCDDAA (or global code ABCDA) and the recognition site V, a second assembly bearing the digital code BBAAAACCBB (or global code BACB) and the site of recognition T, and a third assembly carrying the code BBBBDDAABB and the recognition site I Figure 3: Schematic diagram of a microfluidic cell for manufacturing in large quantities of assemblies of particles carrying a particular code, from particles Magnetic carriers carrying specific properties A, B, C, D in a predetermined order and a type of recognition site T. FIGS. 4 and 5: Different assemblies of particles carrying specific absorption and fluorescence emission properties prepared according to Example 1 and visualized according to Example 2.

Example 1

  Preparation of magnetic particle chains, carrying a code constituted by a succession of different colors in fluorescence.

  A / Preliminarily, 100 microliters of a suspension containing 1% of a sample of fluorescent magnetic beads carrying amine functional groups and having predetermined fluorescence properties are selected: In this example, a 1: 1: 1 mixture is more particularly chosen. of 2.6 m beads, amino, yellow, ref PFAM-2052, 2, m, amino, pink, ref PFAM-2058, and 2.5 m, amino, non-fluorescent, ref PMN-2.5 (Kisker GbR) . The beads are washed 5 times with 0.1 M phosphate buffer at pH 7.3 by sedimenting them with a magnetic separator (Dynal) and resuspending them in the buffer. The beads are resuspended in 100 microliters of a phosphate buffer solution of pH 7.3 at 0.1 M, containing EDC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (sigma)), at a ratio of 15 mg per 10 ml and S-NHS (N-hydroxysulfosuccinimide (Sodium salt, Fluka)), 2.5 mg / 10 ml, prepared immediately before use. Stir vigorously and incubate, with gentle stirring, overnight at 4 C.

  B / a solution B, at 1% by weight of acrylamide, is prepared in phosphate buffer at pH 7.3, which is degassed for at least 2 hours. The beads prepared in A are sedimented and resuspended in 800 microliters of solution B. The solution containing the beads is degassed for two hours, while bubbling nitrogen.

  In addition, a solution C, of 10% ammonium persulphate, and a solution D, of TEMED at 10% E are prepared, and to a total of 100 microliters of the suspension prepared in C, 14 microliters of solution C and 14 microliters of solution D. The whole is stirred vigorously.

  F / the corresponding solution is placed immediately in a cell with 10 parallel faces, in which a magnetic field is created, perpendicular to the faces of the cell, of at least 20 mTesla.

  G / after one hour of incubation, the magnetic field is interrupted. A large number of linear assemblies of particles according to the invention are obtained, such as those presented in FIG. 2, bridged by polyacrylamide polymers. They can be observed as described in Example 2, or sorted according to the color sequence for later use, by flow cytometry.

Example 2

  Observation and determination of the code of assemblies of particles according to the invention, prepared according to Example 1.

  The assemblages are deposited by sedimentation on a microscope slide, and observed in epifluorescence, in a Zeiss Axiovert 100 inverted microscope equipped with a 100X immersion objective, an epifluorescence device and an intensified CCD camera (LHESA). , (the Ulis). Two fluorescence observation conditions are used: a set of Carl Zeiss filters 09, with excitation between 450 and 490 nm (blue), and emission above 512 nm (green and beyond), and a set of filters 15 from Carl Zeiss, with excitation at 546 nm (green) and emission beyond 590 nm. Transmission lighting is also used to view the particles independently of their fluorescence and can be combined with fluorescence.

  The three types of particles, non-fluorescent, pink and yellow, are visible in transmission. With the set of filters 09, yellow particles, which is the optimal absorption, are bright, and pink particles, fluorescent more weakly. Finally, with the set of filters 15, only the pink particles are visible. For the sake of brevity, the letter P will be attributed to the pink particles, the letter Y to the yellow particles and the letter N to the non-fluorescent particles.

  Figures 4a and 4b show the same assembly, in transmission (4a) and with the set of filters 09 (4b). Figure 4a gives us the number of particles, 6, and Figure 4b makes it easy to distinguish the two particles Y, very bright on the left, and the four weakly bright particles, on the right: the code of this assembly is YYPPP.

  Figures 4c to 4d show another transmission assembly (4c), with the filter 09 (4d) and with a combination of the filter 15 and the transmission.

  Figures 4c and 4e, there are easily 7 particles. Figure 4e indicates that only the second, starting from the left, is P, and we see on 4d that the 3rd and the seventh are also D, since it does not give a signal on 4th. The code is NPYNNNY.

  FIGS. 4f to 4h show a set of two assemblies having a different code, in lighting respectively in transmission and filter 15, in filter 09 alone, and in transmission plus filter 15. The assembly of the top has 5 particles, and responds to the code NNNPY, while the bottom assembly has three particles, and responds to the NNP code. (we can note on the right an isolated particle, of the N type, and at the bottom an isolated particle, of the P type, and to note that they do not interfere with the analysis).

  It is thus seen that by combining a limited number of observation conditions, it is possible to unambiguously identify the code of a large number of assemblies according to the invention. The same identification was also made, even easier, using a color camera installed on the microscope.

Example 3

  Observation of a branched type assembly.

  The conditions are identical to those of Example 2, Figure 5d corresponds to transmission lighting, and the figure to the right to illumination with the filter 09. The assembly has a main chain of 6 particles, and a branching of two particles carried by the third particle. The code is NNN (YY) NNN. .

Example 4

  Series Manufacturing in a Microfluidic Cell This example describes a method for preparing assemblies of particles according to the invention, carrying a code and a type of recognition sites on a separate area of the code. This method of preparation is particularly advantageous for obtaining a large amount of assemblies all having the same code.

  A / it operates as in Example 1 to step E, but is prepared four containers, respectively containing fluorescent magnetic beads P, Y and N type, and non-fluorescent magnetic beads bearing streptavidin PMSt-2.0 (kisker, Steinfurt), The different inlet channels 1, 2, 3, 4, 5, 10 are respectively connected to an eppendorf tube containing the Y, N, P, Y, P and streptavidin particle solutions. The other difference with Example 1 is that the acrylamide solution (solution B in Example 1) further contains methylene blue at the concentration of (100 M) in the presence of a redox system (1). mM sodium toluene sulfinic acid, and 50 M diphenyliodonium chloride (Fluka)), B / immediately connect each of the containers to one of the inlet channels (1-4) of a microfluidic system such as that shown in Figure 3 , prepared in PDMS according to Xia, Y. & Whitesides, GM Soft lithography. Angew. Chem. Int. Ed. 37, 550-575 (1998) and a permanent flow of the contents of these containers in the form of a laminar liquid vein is induced in the main channel 12, to the collection tank 14 by exerting a depression above the reservoir. An inverted microscope, identical to that used in Example 2, is advantageously used to control the smooth running of operations and the formation of assemblies. A magnetic field substantially uniform and perpendicular to the interfaces between the liquid veins, at least 10 mTESLA, is created using an electromagnet whose pole pieces are machined so as not to create a large magnetic field gradient in the main channel 12 and in the conduit connecting said channel with the collection tank 14.

  C / once the steady state is established, the main channel 12 is intensely illuminated by a UV lamp, in order to induce the rapid photopolymerization of the acrylamide and the bridging of the particles. Thus collecting in the tank 14 assemblies of particles according to the invention, whose color code is given by the order of the colors of the particles contained in the containers respectively connected to the channels 1 to 5, or in the example proposed here YNPYP (in this embodiment, the liquid veins do not allow to control the number of particles in an exact manner, and therefore a coding of the global type). The presence of streptavidin on the upper particle zone is easily verified by incubating the particles with fluorescent biotin (Molecular Probes).

  D / by repeating the operation by inverting the containers, and changing the collection tank 14, one can thus obtain a large number of particle assemblies according to the invention, without having to resort to sorting after their synthesis.

Claims (26)

CLAIMS,   1. Assembly of particles linked together linearly or in a branched manner, characterized in that said particles belong to at least two subsets identifiable by at least one property specific to each subset, and follow one another in a predetermined sequence constituting 'a specific code.   2. Assembly according to claim 1, characterized in that the particles are bonded to each other permanently and in a linear form.   3. Assembly according to claim 1 or 2, characterized in that it is flexible or semi-rigid.   4. Assembly according to any one of the preceding claims, characterized in that the particles are in substantially spherical form.   5. Assembly according to any one of the preceding claims, characterized in that it has a mean diameter ranging from 10 nm to 1 mm, in particular from 10 minutes to 100 m, in particular from 100 nm to 5 m and a length varying from 200 nm to 5 mm in particular from 200 nm to 500 m, and in particular from 1 m to 100 m.   6. Assembly according to any one of the preceding claims, characterized in that it has a density less than 2, in particular less than 1.6, and more particularly ranging from 0.7 to 1, 4.   7. Assembly according to any one of the preceding claims, characterized in that said particles are bonded together by covalent bonds.   8. Assembly according to any one of the preceding claims, characterized in that the particles are bonded to each other by bridging with molecules or macromolecules.   9. Assembly according to any one of the preceding claims, characterized in that said particles are dielectric or magnetic particles.   10. Assembly according to any one of the preceding claims, characterized in that the particles are of organic, mineral or organomineral nature.   11. Assembly according to any one of the preceding claims, characterized in that the properties held by the particles are interaction properties with electromagnetic radiation, optical properties of fluorescence emission and / or luminescence type or properties concerning the light absorption spectrum.   12. A particle assembly according to any one of the preceding claims, characterized in that it is further carrier of at least one type of site (s) of recognition for a species.   13. Set of assemblies of particles bonded together linearly or branched composed of several subassemblies of assemblies according to any one of claims 1 to 12, said assemblies belonging to a given subset being carriers at a time. at least one type of recognition site (s) specific to said subset and a specific code also specific to said subset.   14. Process useful for preparing at least one assembly of particles bonded together, said assembly having in its length a succession of predetermined properties constituting a specific code, characterized in that it comprises at least the steps consisting of: suspending in a medium, particles belonging to at least two subsets identifiable by at least one property specific to each subset, and capable of binding together, B / activate at least one means leading to the organization of said particles within said medium in a linear form, and C / obtain irreversible bridging between the particles during their alignment state.   15. A method useful for constituting at least one assembly of particles bonded together in a linear fashion, comprising at least the steps of: A / preparing a fluid medium comprising monomers capable of binding by polymerization or polycondensation, B / implement suspension in said medium, particles themselves carrying functions capable of reacting with said monomers, C / activate at least one means leading to the organization of said particles in said medium, in a linear form and D / bridging irreversible between the particles during their alignment state.   16. A method useful for preparing a plurality of interconnected particle assemblies, said assemblies having along their length a succession of predetermined properties, characterized in that it comprises at least the steps of:   A / preparing in a first container particles that are identifiable by at least one property specific to these particles, or a composition comprising particles and at least one reagent for conferring over time or under the action of a stimulus to said particles such a property, B / preparing in at least one second container particles identifiable by at least one specific property, or a composition comprising particles and at least one reagent for conferring over time or under the action of a stimulus to said particles such a property, said property being different from that characterizing the particles or reagents contained in the first container, C / introducing into a microchannel, by at least two separate arrival channels, the solutions prepared in steps A and B, and possibly other solutions by other arrival channels, so that said solutions formen t, in the main channel liquid veins essentially laminar and parallel, D / activate, within said channel, at least one means inducing the organization of said particles in a linear form, E / bind together the particles in their state linear, and F / recover said assemblies thus obtained.   17. Method according to any one of claims 14 to 16, characterized in that said particles are as defined in claims 9 to 11.   18. Method according to any one of claims 14 to 17, characterized in that the assemblies are as defined in claims 1 to 12.   19. Method according to one of claims 14 to 18, characterized in that said particles bind together directly or through a coupling agent, in response to a stimulus corresponding to a temperature change and / or the action of light, an electric field, a magnetic field, an electromagnetic field, and / or radioactive radiation 20. Process according to any one of claims 14 or 16 and 17 to 19, characterized in that it further comprises a preliminary step comprising the introduction into the medium of molecules capable of binding by polymerization or polycondensation between them and to react with said particles.   21. The method of claim 16 or 20, characterized in that said molecules are monomers of acrylic type.   22. Method according to any one of claims 14 to 21, characterized in that the means inducing the organization of said particles in a linear form are selected from a magnetic field and an electric field.   23. Object marking system, characterized in that it comprises at least one assembly according to any one of claims 1 to 12 or a set of assemblies according to claim 13.   24. A method for marking an object, characterized in that it comprises at least one step of associating with said object, at least one particle assembly according to any one of claims 1 to 12 or a set of assemblies according to the claim 13.   A method for analyzing, identifying, assaying, purifying or preparing a sample, employing at least one step of contacting said sample with at least one particle assembly according to any one of claims 1 to 12, or assembly assembly according to claim 13.   26. A method for analyzing the interaction of species contained in a sample with respect to a multiplicity of recognition sites or probes, comprising at least the steps of: A / bringing said sample into contact with a set of assemblies according to claim 13, and B / seek and possibly quantify the fixation of species on at least one of said subsets of assemblies identified by their code.