WO2005026219A1

WO2005026219A1 - Novel saccharide-graft polymers and the use thereof for screening processes.

Info

Publication number: WO2005026219A1
Application number: PCT/FR2004/002358
Authority: WO
Inventors: Thierry Livache; Karen Brengel-Pesce; Emilie Mercey; André Roget; Rabia Sadir; Yves Levy; Hugues Lortat-Jacob
Original assignee: Commissariat A L'energie Atomique; Centre National De La Recherche Scientifique
Priority date: 2003-09-18
Filing date: 2004-09-17
Publication date: 2005-03-24
Also published as: FR2859998A1

Abstract

The invention relates to polymers graft by chemical synthetic or natural saccharidic molecules and to a method for the preparation thereof. Said polymers make it possible to fix the saccharidic molecules to a solid support for screening molecules, molecular systems or target micro-organisms in a solution.

Description

The invention relates to polymers grafted by synthetic or natural chemical molecules of saccharide type (mono, oligo or polysaccharides and their derivatives) as well as their preparation process. These polymers allow the fixation of saccharide molecules on a solid support allowing the screening of molecules, molecular complexes or target microorganisms dissolved. The development of DNA microarray technologies has led to a significant advance in programs related to functional genomics. Indeed, the miniaturization of DNA deposition or synthesis techniques has led to parallel analyzes, therefore multiparametric, of DNA on chips [Biochip Technologies ed. J. Cheng and LJ Kricka, Gordon, Breach / Harwood Académie publishers, (2001)]. More recently, the emergence of proteomics has given birth to the concept of protein chips [Zhu and Snyder, Current Op. In Chem. Biol. 2003, 7: 55-63]. These allow parallel analysis of protein / protein or more generally protein / ligand interactions. Previously, Ekins had proposed “antibody microspot immunoassay” allowing simultaneous immunological analyzes on the same support [Ekins, Tibtech Tuesday 1994, 12: 89]. More recently, research in biology has focused on the

"Glycomic", ie the systematic study of carbohydrate / protein interactions. Indeed, glycoconjugates (that is to say any molecule possessing a glycan-type domain, such as glycoproteins, glycolipids, proteoglycans, and more generally any molecule containing carbohydrates have a particularly broad functional repertoire [see for example Roseman J Biol. Chem. 2001, 276, 41527-41542 or Essentials of Glycobiology. Lst ed. Varki, Ajit; Cummings, Richard; Esko, Jeffrey; Freeze, Hudson; Hart, Gerald; Marth, Jamey, editors. Plainview (NY) : Cold Spring Harbor Laboratory Press; cl999] Chemically, these carbohydrates are molecules formed by the assembly of simple monomeric blocks. These assemblies can be of natural origin, and optionally fractionated, or of synthetic origin. molecules belonging to the carbohydrate family are based on the ability of carbohydrate structures to interact with a very large number of molecules. born between carbohydrates and other molecules is an emerging area of research. In particular, it should lead to the design of new therapeutic molecules and a better appreciation of the toxicological risks of certain molecules. At present, there are few systematic methods for producing saccharide molecules. Therefore, the determination of the structural characteristics involved in an interaction between a molecule and a carbohydrate, as well as the characterization of the interaction itself presuppose undertaking long and tedious work, often difficult. It would therefore be useful, in order to progress in the knowledge of the mechanisms of interaction between molecules of saccharide type and their ligands, to be able to screen libraries of molecules of saccharide type with respect to a particular ligand. The methods allowing the study of interactions between saccharide molecules and other molecules, such as for example proteins, are relatively limited: In order to evaluate an interaction between a sugar and a protein, some authors have proposed the use of optical biosensors based on surface plasmon resonance (SPR), according to the Biacore method [Ben Khalifa et al., Anal. Biochem., 2001, 293: 194-203] or on a modulation of an evanescent wave (IASYS process) [Kamei et al., Anal. Chem. 2001, 295, 203-213]. In these two cases, these analysis methods make it possible to obtain fine affinity data but do not allow a comparison of the measurements so as to allow a more systematic and comparative analysis of the sugar / protein interactions. The fixation of different polysaccharides on a surface in the form of spots, so as to form a matrix, or chip, which makes it possible to study the interaction of these polysaccharides with a protein on the whole of this surface has been described by different authors . These substrates, called “carbohydrate array” or “oligosaccharide array” constitute an evolution of the protein chip or the DNA chip described above. They allow parallel analysis of interactions between sugars and other compounds or organisms. More specifically, US patent application 20020127599 describes the manufacture of a library of complex sugars by a combinatorial approach including an enzymatic step; Wang et al., (Nature Biotech. 2002, 20: 275-281) and Fukui et al., (Nature Biotech. 2002, 20: 1011-1017) both describe sugar fleas in which the sugars are simply adsorbed on a nitrocellulose substrate, no chemical reaction being necessary to manufacture the chip. In all these cases, the detection interactions is carried out by measuring fluorescence. An approach based on chemical grafting has been developed by Houseman et al., (Chem. Biol. 2002, 9: 443). This consists of a Diels et Aider reaction between a sugar modified by an arm terminated by a cyclopentadiene group and a benzoquinone present on a self-assembled layer deposited on the substrate. To build the matrix, droplets of different activated sugars are deposited on the substrate and left for 2 hours at 37 ° C so that the reaction is complete. In this example, the detection is also carried out by fluorescence. Another way of making sugar multi-sensors has been described more recently [Smith et al., J. Am. Chem. Soc, 2003, 125, 6140-6148]: it consists in fixing sugars on a gold substrate carrying a layer of self-assembled thiols. The sugars are modified beforehand with a thiol in order to form a di-sulfide bond. The coupling times between the modified sugar and the substrate being very long, the reaction is carried out in channels molded in polydimethylsiloxane (PDMS), which prevents any evaporation. The sugars are therefore deposited in the form of 6 parallel strips spaced 1mm apart. Detection is then carried out by a parallel SPR process. Document US-6, 197,881 describes an electroconductive copolymer formed from electropolymerizable monomers, some of which monomers carry a biotin group or a complex of biotin with another molecule. These polymers are intended for the production of microchips for the detection of affinity between the grafted molecules and the molecules to be tested. The complexes of biotin and another molecule are complexes of biotin with avidin, streptavidin and their derivatives. The processes described above for the manufacture of sugar chips have several drawbacks: in the process according to which the polysaccharides are simply adsorbed on nitrocellulose, the fixing being non-specific, it will be difficult, if not impossible, to immobilize all the sugars in an identical way especially those whose size is small. However, to study the structure / activity relationship of a sample of molecules, measurements of interactions with very short oligosaccharides are necessary. In addition, this support is incompatible with methods allowing detection without tracer and in real time of biological interactions, in particular the SPR technique. It is therefore not possible to obtain kinetic data from this technique. The microchips described by US Pat. No. 6,197,881 are difficult to manufacture and to use because they are based on a complex non-covalent stack. In addition, they are not regenerable. The chips proposed by Houseman et al., Or Smith et al., Allow a specific fixation of the oligosaccharides on a gold substrate well suited for reading in real time and without tracer. However, in the first case, the fixing of the modified sugar involves a chemical reaction of coupling on the substrate which is very slow (2 hours at 37 ° C). It is well known, in the field of DNA chips, that the problems associated with the drying of the droplets during the step of grafting the biological molecule onto the substrate cause the formation of heterogeneous spots. In order to avoid these drying problems, Smith and ah, carry out these couplings in micro-channels. The grafting times are also extremely long (12 hours) and lead to the formation, not of spots, but of parallel bands. In addition, these two methods assume the use of a self-assembled layer of thiols whose stability over time is low. The method described by the company Glycominds (American patent application US2002 / 0127599) is not very explicit and produces a fluorescence response. We have therefore sought to construct sugar chips more quickly and on substrates which are compatible both with fluorescence reading and with optical methods of the plasmon resonance type so as to be able to carry out measurements in real time and at ability to measure the kinetics of the sugar / ligand interaction. The solution to this problem, which is the subject of the present invention, is based on the grafting of sugars onto electropolymerizable polymers. It is known to incorporate DNA molecules directly during the synthesis of a conductive polymer such as polypyrrole while retaining good biological activity [Shimidzu, Reactive polymers 1987, 6: 221-227 or Wang et al.,. Anal. Chim. Acta 1999, 402: 7-12]. Likewise, proteins can be passively incorporated into films of conductive polymers and maintain good activity [Umana et al, Anal. Chem; 1986, 58: 2979-2983 or Hammerle et al, Sens. Actuat., 1992, B6: 106-112]. Enzyme incorporation processes are widely used in the field of glucose biosensors for example (immobilization of glucose oxidase). In accordance with this teaching of the prior art, we therefore tried to incorporate a charged oligosaccharide (heparan sulfate fragment) in a polymer matrix (polypyrrole) then to carry out a specific recognition thanks to an adequate protein and no reactivity was detected. Electropolymerizable polymers grafted with (poly) nucleic acids are already used in the field of DNA chips and allow the grafting or synthesis of DNA strands on solid surfaces. Such polymers have shown good efficacy for the preparation of supports for screening for polynucleic acid ligands (Livache et al ,. Nucleic Acids Res. 1994, 22: 2915-2921; WO94 / 22889). However, the chemistry of saccharides is a chemistry very different from that of nucleic acids, and it was not possible to predict, based on work on polymers grafted by DNA, that the synthesis by electropolymerization of polymers grafted with saccharides would be possible. In addition, the behavior of saccharides vis-à-vis potential ligands is very different from that of DNA molecules vis-à-vis their own ligands: - the mechanism of DNA recognition vis-à-vis its ligands (hybridization) is well known and corresponds to strong affinities (> 10 ¹⁵ M ^" ) while the affinities of sugars for their protein ligands for example are weak and variable (from 10 ³ to 10 ⁹ M--); - interactions between the saccharides (very hydrophilic) and the support polymer could be of the same order of magnitude and make the analysis impossible; - the tests carried out on saccharides included in a polypyrrole matrix gave negative results in recognition tests by ligands while polynucleic acids or proteins, under the same conditions, can associate with their ligands. The present invention therefore relates to polymers consisting of electropolymerizable monomers. grafted with saccharides. The polymers of the invention correspond to the following general formula (I):

(I) in which: - Ai and A _j each represent an electrically polymerizable monomer, the different Aj and A _j being able to be identical or different; - Bj represents a carbohydrate which can be chosen from: a monosaccharide, an oligosaccharide, a natural or synthetic polysaccharide, or one of their natural or synthetic derivatives; - Li represents a covalent bond or a spacer arm connecting the monomer Ai to the group Bj; - x represents an integer greater than or equal to 1; - i is an index varying from 1 to x; - y represents an integer greater than or equal to 0; - j is an index varying from 1 to y when y ≠ 0. Among the electropolymerizable Aj and Aj monomers, mention may be made of acetylene, acrylonitriles, p-phenylene, p-phenylene vinylene, pyrene, pyrrole, thiophene, furan, selenophene, pyridazine, carbazole, aniline, phenol and their derivatives, such as for example pyrroles substituted in position 1 or 3. Advantageously, Ai and A _j are chosen from pyrrole and derivatives substituted with pyrrole such as for example N-methylpyrrole and N-cyano-ethylpyrrole. Preferably Aj and A _j are all identical. According to the present invention, the carbohydrate groups Bj can be chosen from those described for example in the book "Essentials of Glycobiology". lst ed. Varki, Ajit; Cummings, Richard; Esko, Jeffrey; Freeze, Hudson; Hart, Gerald; Marth, Jamey, editors. Plainview (NY): Cold Spring Harbor Laboratory Press; cl 999. Bi can be chosen from the following natural monosaccharides: allose, altrose, arabinose, erythrose, fructose, galactose, glucose, gulose, idose, lyxose, mannose, psicose, ribulose, sorbose, tagatose, threose, talose, xylose, xylulose . It can be chosen from derivatives of these natural monosaccharides. Among these, mention may be made of acid derivatives, such as, for example, glucuronic acid and iduronic acid; amino derivatives such as, for example, glucosamine; substituted amino derivatives such as, for example, N-acetylated glucosamine, N-sulfated glucosamine and O-sulfated glucosamine; Cι-C] ₂ alkyl ester derivatives; Cι-C ₁₂ alkyl ether derivatives; C 1 -C ₁₂ alkyl amide derivatives. Bi can be a polysaccharide comprising at least two units chosen from natural or synthetic monosaccharides and their derivatives, such as for example those cited above. In this case, Bj can be a homosaccharide or a heterosaccharide, it can be linear or branched. Bj can be selected from polysaccharides as defined above, substituted with at least one group selected from peptides, proteins, lipids, such as fatty acids C ₈ -C ₃₀ ^steroids' ides and their derivatives. Bj can be a synthetic compound or a natural product obtained by extraction from an animal or vegetable source. In the polymer of the invention, it can be provided that all the groups Bi are identical or that several distinct groups Bj are present. Preferably, in order to screen for potential ligands, it is chosen to prepare a polymer in which several distinct Bi groups are present. According to a preferred embodiment of the present invention, the ratio x / y is between 1/1 and 1 / 1,000,000, preferably between 1/10 and 1/100,000, and even more preferably between 1/20 and 1 / 50,000. According to the invention, Lj represents a covalent bond between Aj and B; or a spacer arm connecting these two groupings. When Li represents a spacer arm, it is advantageously chosen from the groups corresponding to formula (II): -R [(CH ₂ ) _n -R ₂ ] _a - [(CH ₂ ) _m -R ₃ ] _b - ( CH ₂ ) _P - (II) in which: - n is an integer ranging from 1 to 10; - m is 0 or is an integer ranging from 1 to 10; - p is 0 or is an integer ranging from 1 to 10; - a is equal to 0 or is an integer ranging from 1 to 8; - b is equal to 0 or is an integer ranging from 1 to 8; - Ri, R ₂ and R ₃ which may be identical or different represent a group chosen from: - CH ₂ -; -O-; -S-; -NR'-;-CO-; -CH = CH-; -NR'C (O) -; -C (O) N (R ') -; - NHS (O ₂ ) - and R 'represents a hydrogen atom or a C] to alkyl chain. Within the meaning of the present invention, the word alkyl signifies a linear, branched or cyclic, saturated or unsaturated hydrogen-carbon chain. The present invention also relates to a process for the preparation of a polymer of general formula (I). According to a first variant, this process is characterized in that it comprises at least the following steps: - a first step during which one proceeds to the preparation of a copolymer of general formula (III): - [Ai *] _x - [A _j ] _y - (III) in which A _j , x and y are as defined above, and A; * represents a group Aj comprising a designated function *. a second step during which the fixing, on the polymer of formula (III), of at least one group of general formula (IV):

(IV) in which Bj is as defined above and ** Lj represents a group Lj carrying a function ** capable of reacting with the function * of A; to form an Lj-Aj covalent bond. According to another variant of the invention, the process for the preparation of a polymer of general formula (I) is characterized in that it comprises at least the following steps: - a first step during which the preparation is carried out of the compound of general formula (V): -Ai-

Bi (V) in which Aj, Bj, and Lj are as defined above, - a second step during which one proceeds to the copolymerization of the compound (V) with the monomer A _j as defined above. According to the invention, at least one step of one or other of the variants of the process according to the invention involves at least one electrochemical reaction. This electrochemical copolymerization is advantageously carried out on the surface of an electrode; at the end of the reaction, an electrode is thus obtained, the surface of which is partially or completely covered by a polymer according to the invention. For example, for the implementation of the first variant of the process according to the invention, the step of preparing the copolymer of general formula (III) and / or the step of fixing the group of general formula (IV) can be carried out by electrochemical reaction; in the second variant, the second step is advantageously carried out by electrochemical copolymerization of the compound (V) with the monomers Aj. The group (V) can be manufactured by coupling Bj to an arm Lj and a polymerizable group Aj. Preferably, this reaction can be carried out on the reducing end of the sugar Bj or on another functional group of Bj when the latter is blocked as is the case for many synthetic analogs of saccharides. The electrochemical copolymerization of the mixture is for example carried out by subjecting a mixture of monomers, such as for example the mixture [(V) / A _j ], for a determined period to one or more jumps of potential of sufficient value to cause polymerization by a oxidation or reduction of the monomeric species present in solution. Other conventional methods in electrochemistry such as cyclic voltammetry or chronopotentiometry can also be used for the manufacture of the polymers described in the invention. The addressing of these electrochemical reactions on the substrate can be ensured by various means such as networks of microelectrodes or by localized electrochemical reactions such as those described below. Advantageously, the electrode used for the polymerization reaction can be used later in a resonance imaging process of surface plasmons. It can consist of any conductive material (metallic, semiconductor, plastic or conductive polymer) deposited or not on a solid support. It may, for example, be metal or glass electrodes covered with a layer of gold. Among the electrodes available commercially, there may be mentioned, for example: the plates sold by the company Biacore under the reference 99-1000-01. The quality of the deposit can be controlled by the choice of experimental conditions: the ratio of the monomers Aj and Aj or of the monomers -Ai- Li

^Bi and A _j in solution allow for example to modulate the grafting densities of saccharides on the polymer. The method and the electrochemical conditions make it possible, for example, to modify the thickness and / or the structure of the film formed on the surface of the electrode. In addition, when the electric current necessary for the formation of the polymer is controlled, it makes it possible to measure and possibly limit the thickness of the film formed. Advantageously, it is possible to combine several copolymers carrying possibly different sugars on one or more electrodes. The electrochemical formats used to electrochemically functionalize several points are already described in the literature. When using networks of (micro) electrodes independent of each other, it is easy to successively activate the different electrodes in the presence of the different solutions in which they are successively immersed [solution of compound (V) or of compound Aj or of the compound Aj]. Such electrode networks have already been described [Fiaccabrino et al, Sensors and Actuators 1994, B19, 675; Heller et al, DNA Microarrays. A Practical Approach; 1999 Oxford Press: New York, Livache et al, Biosensors & Bioelectronics 1998, 13, 629]. Thus, a construction diagram of the polymers deposited on each point of the support can be constructed. Another way of proceeding consists, for example, in using the “electrospot” method [Patent WO 00/47317] making it possible to successively and electrochemically synthesize point deposits of polymers on unstructured electrode surfaces. In this case, an electrode can carry several point deposits of possibly different copolymers (I). In this case, a support is obtained comprising deposits of polymers distributed in the form of a matrix or chip, these polymers each comprising one or more sugars covalently attached to said polymer. This chip can be used for biological tests involving recognition between one or more sugars and one or more potential ligands. The term “ligand” means any molecule capable of exhibiting an affinity for the sugars grafted onto the polymer. In particular, the screening of banks of molecules, peptides, proteins, microorganisms, extracts of all kinds, such as cells, ground materials of tissues can be envisaged by this method. Interaction with the saccharides grafted onto the (co) polymer can be detected using fluorescent tracers or directly optically, by resonance imaging of surface plasmons or by any affinity analysis method known to those skilled in the art . The invention therefore also relates to a support in the form of an electrode comprising one or more deposits of at least one polymer grafted with one or more saccharides and corresponding to formula (I). Preferably this electrode has the form of a plate. Advantageously, it can be used in a resonance imaging process of surface plasmons. By way of nonlimiting example illustrating the above, a copolymer (polypyrrole carrying oligosaccharides) can be obtained: 1- by chemical reaction between an activated polypyrrole (carrying for example a hydrazide function linked to the polymer electrosynthesized by a spacer arm) and a oligosaccharide (reaction on the reducing end); 2- by electrochemical copolymerization of pyrrole with an oligosaccharide carrying a spacer arm then a pyrrole nucleus. This method of grafting carbohydrates onto conductive surfaces provides advantages over the techniques used in the field of sugar chips in the prior art. Among these, one can cite the speed of the electrochemical coupling reaction which leads to a covalent fixing of the carbohydrate on the polymer: it lasts less than a second in the process of the invention against several hours in the other processes mentioned. We can also mention the ease of controlling the thickness of the film which, thanks to the process of the invention, can be reduced to a few nanometers, which is essential when using direct optical methods such as SPR imaging. . This thickness can be increased up to several μm if necessary. The spatial resolution of the deposit obtained can be excellent (a few μm) when arrays of microelectrodes are used. The copolymers (I) obtained are chemically very stable and can be dehydrated for storage; moreover when used for carrying out a biological analysis, they can be regenerated within the limit of the stability of the grafted sugars. It is therefore possible to envisage preparing a stock of chips or supports according to the invention carrying deposits of polymers of formula (I), keeping them for a certain time and using them when desired. Furthermore, the supports or chips according to the present invention are the first screening substrates compatible with reading in real time and without a tracer, for example in SPR imaging. This is a fundamental advantage when detailed interaction studies have to be conducted; the use of molecules marked with fluorescent tracers which, by definition, modify the properties of the target molecules studied, do not make it possible to obtain satisfactory objective information. In addition, knowledge of the kinetic behavior of the interaction also makes it possible to characterize the mechanisms brought into play during recognition phenomena [Moulard et al, J. Virol. 2000, 74, 1948-1960, Vives et al, Biochemistry 2002, 41, 14779-14789] and this ability to parallelize measurements allows the comparison of the different behaviors observed. It makes it possible to define the profile of potential ligands of a given target. A further subject of the invention is therefore a method for screening for ligands, characterized in that it comprises bringing a matrix support or chip grafted with saccharides into contact with one or more potential ligands. This contacting can consist, for example, of an immersion of a support as described above in a solution comprising one or more potential ligands. EXPERIMENTAL PART: The tests described below illustrate the manufacture of chips grafted with glycosaminoglycans. Figures: Figure 1 illustrates a gel filtration chromatography of a mixture of oligosaccharides from the heparin digestion with heparinase. FIG. 2 illustrates a gel filtration chromatography of a mixture of oligosaccharides resulting from the digestion of heparan sulfate with heparinase. Figure 3 illustrates the different stages of fluorescence development. Figure 4 illustrates the fluorescence analysis of the affinity of sugars grafted on polypyrroles for SDF. Left photo: photo of the slide under a microscope exposed 0.52s; right: diagram of composition of the blade. Figure 5 illustrates the operating principle of SPR imagery. Figure 6 illustrates the diagram of deposition of polymer grafted by sugars and its visualization in SPR imaging. FIG. 7 represents the gross kinetic curves of interaction between the polymers grafted by saccharides and the SDF. FIG. 8 represents the matrix visualized in SPR imaging. FIG. 9 represents percentages of reflexivity before rinsing. FIG. 10 represents percentages of reflexivity after rinsing. FIG. 11 A represents a matrix seen in SPR imagery. FIG. 11B is a schematic representation of this same matrix. Figure 12 illustrates the matrix processing protocol. Figure 13 shows the raw kinetic curves for different protein concentrations. FIG. 14 represents percentages of reflexivity before rinsing. FIG. 15 represents percentages of reflexivity after rinsing. A: PREPARATION OF OLIGOSACCHARIDES 1-Preparation of oligosaccharides derived from heparin. There are two possible ways (enzymatic or chemical): a- Enzymatic treatment: Six grams of heparin are dissolved in a buffer containing 5 mM TRIS, 2 mM CaCl ₂ , 50 mM NaCl and 0.1 mg / ml albumin. The pH is adjusted to 7.5 with acetic acid. This solution is incubated at 25 ° C, with heparinase I (8 mU / ml) for approximately 50 h. (the enzymatic reaction is followed by the increase in optical density, measured at 232 nm). The mixture is then purified by gel filtration chromatography as illustrated in FIG. 1. The solid phase is Biogel P10, contained in a column of lm50 and 4.4 cm in diameter, eluted at 1 ml / min with NaCl 0 , 25M. The different oligosaccharides obtained (dp2, dp4, ... etc) are dialyzed against water and then lyophilized, (dp = degree of polymerization). The fractions dpl2 and HP6 (that is to say heparin of 6kDa of molecular mass, ie a dp of approximately 24) are used subsequently. Each group of molecules (dp) is then sub-fractionated by ion exchange chromatography, on a Propac PA1 column, sold under the brand Dionex, reference 40137. b- Chemical treatment: One gram of heparin is dissolved in 20 ml of sodium nitrite (NaNO ₂ ) at 2.1 mg / ml. The solution is adjusted to pH 1.5 with sulfuric acid, then incubated at 4 ° C for 3 h. The reaction is stopped, and the oligosaccharides are purified as above. 2-Preparation of oligosaccharides derived from heparan sulfate When the starting material is heparan sulfate, the procedure is as follows: Eight grams of heparan sulfate are dissolved in 40 ml containing 5 mM TRIS, 2 mM CaCl ₂ , 50 mM NaCl and 0.1 mg / ml of albumin. The pH is adjusted to 7.5 with acetic acid. This solution is incubated at 30 ° C with heparinase III (25 mU / ml) for approximately 72 hours. Heparinase III is added again, for a period of 48 h, then the products are purified as described above. The chromatographic profile is illustrated in FIG. 2. B MODIFICATION OF OLIGOSACCHARIDES The oligosaccharides are grafted onto pyrrole groups with arms of variable length and composition on their reducing end according to the following protocol 1. Synthesis of functionalized pyrrole monomers This synthesis is carried out by several stages, the most important of which is the coupling of the activated ester to the hydrazide group of the adipate or to the hydrazine group alone. This synthesis is illustrated by diagram 1 below:

n = 5: caproyle n = 10: undecanoyle

dioxane

a Synthesis of pyrrole acids 5-pyrrolyl-caproic acid (I)

In a flask fitted with a condenser, add 2,5-dimethoxy-THF (490mmol, 64.85mL), 5-amino-caproic acid (430mmol, 56.33g), glacial acetic acid (430mL ) and dioxane (570mL). The mixture is brought to reflux in a balloon heater for 4 hours. The reaction is allowed to continue with magnetic stirring overnight at room temperature. The reaction mixture is then concentrated on a rotary vacuum evaporator. The residue is taken up in ethanol (2 times) and concentrated. The pure product is obtained after chromatography on a silica column. Elution from the column is started with pure dichloromethane, then continued with a mixture of dichloromethane / ethanol (increasing elution in ethanol: 2.5% EtOH then 5% EtOH). The purification is followed by thin layer chromatography (TLC) on a silica plate, using a chloroform 90 / methanol 10 mixture as eluent. 67.41 g is obtained. 5-pyrrolyl-caproic acid, representing a yield of 86%. The dry product is stored at 4 ° C, protected from light. Characterization: Η-NMR (200MHz; CDCl ₃ / TMS) δ (ppm): 1.72 (m, 6H, -CH ₂ - (CH ₂ ) ₃ - CH ₂ -); 2.34 (t, 2H, -CH ₂ -CO ₂ H); 3.87 (t, 2H, -CH ₂ -N); 6.13 (dd, 2H, 3-H and 4-H pyrrole); 6.64 (dd, 2H, 2-H and 5-H of pyrrole). 10-pyrrolyl-undecanoic acid (II):

In a flask equipped with a condenser, add 2,5-dimethoxy-THF (490mmol, 64.85mL), 10-amino-undecanoic acid (430mmol, 86.56g), glacial acetic acid (430mL ) and dioxane (570mL). The mixture is brought to reflux in a balloon heater for 4 hours. The reaction is allowed to continue with magnetic stirring overnight at room temperature. The reaction mixture is then concentrated on a rotary vacuum evaporator. The pure product is obtained after chromatography on a silica column. Elution from the column is started with pure dichloromethane, 10-pyrropyl undecanoic acid being even more nonpolar than 5-pyrropyl caproic acid, there is no need to make a gradient between dichloromethane and ethanol. The purification is followed by thin layer chromatography (TLC) on a silica plate with chloroform 90 / methanol 10 as eluent. 70.0 g of 10-pyrrolyl- undecanoic acid are obtained, ie a yield of 64.8%. The dry product is stored at 4 ° C, protected from light. Characterization: Η-NMR (200MHz; CDCl ₃ / TMS) δ (ppm): 1.64 (m, 14H, -CH ₂ - (CH ₂ ) ₈ - CH ₂ -); 2.34 (t, 2H, -CH ₂ -C0 ₂ H); 3.86 (t, 2H, -CH ₂ -N); 6.13 (dd, 2H, 3-H and 4-H pyrrole); 6.64 (dd, 2H, 2-H and 5-H of pyrrole). b Synthesis of activated pyrrole-esters Pyrrole-f61- N-hydroxysuccinimide: PC-NHS (III):

In a flask, I (144mmol, 26.055g), NHS (144mmol, 16.56g), DCC (159mmol, 32.75g) and DMF (500mL) are added. The mixture is left stirring overnight at room temperature. The reaction is followed by TLC, eluent: choroform 95 / methanol 5. The mixture is then filtered to remove the DCU formed. The filtrate is then dried under vacuum. The product is subsequently used as it is, without further purification. Characterization: Η-NMR (200MHz; CDCl ₃ / TMS) δ (ppm): 1.72 (m, 6H, -CH ₂ - (CH ₂ ) ₃ - CH ₂ -); 2.34 (t, 2H, -CH ₂ -CO); 2.88 (tt, 4H, CH ₂ -CH ₂ NHS); 3.87 (t, 2H, -CH ₂ -N); 6.13 (dd, 2H, 3-H and 4-H pyrrole); 6.64 (dd, 2H, 2-H and 5-H of pyrrole).

Pyrrole-flll- N-hydroxysuccinimide: PU-NHS (IV):

Following the same protocol as above for the product (III), II (95mmol, 23.9g), NHS (95mmol, 10.925g) and DCC (104.5mmol, 21.52g) are mixed in 500mL of DMF. The mixture is left stirring overnight at room temperature. The reaction is followed by TLC in a choroform 95 / methanol 5 mixture. The reaction mixture is then filtered to remove the DCU formed. The filtrate is then dried under vacuum. The product is subsequently used as it is, without additional purification, so as not to risk hydrolyzing. Characterization: Η-NMR (200MHz; CDCl ₃ / TMS) δ (ppm): 1.64 (m, 6H, -CH ₂ - (CH ₂ ) ₈ - CH ₂ -); 2.6 (t, 2H, -CH ₂ -CO); 2.89 (tt, 4H, CH ₂ -CH ₂ NHS); 3.86 (t, 2H, -CEfe-N); 6.13 (dd, 2H, 3-H and 4-H pyrrole); 6.65 (dd, 2H, 2-H and 5-H of pyrrole). c Synthesis of Pyrrole-Hydrazides Pyrrole Undecanoyl Hydrazide: PUH

In a flask, put the PUNHS IV activated ester (20mmol, 6.96g), 1M hydrazine in THF (20mmol, 20mL), in stoichiometric mixture, in DMF (180mL), in order to avoid as much as possible the formation of by-products ("dimers"). The mixture is left stirring overnight at room temperature, following the reaction by TLC (CHC1 ₃ 90 / MeOH 10). Then, the DMF is evaporated using the rotary vacuum evaporator. We observe the presence of several products: PUH, PUHUP, traces of PUNHS IV activated ester. Attempts to separate PUH from other products have been unsuccessful. This was therefore used as a mixture with PUHUP and PUNHS. Characterization: Mass spectrometry: T = 200 ° C, solvents: CH C1 ₂ / DMSO, m / z = 266.3 (M + H) ⁺ PUH; 348.1 (compound IV); 251 (compound II), 225.3 (DCU).

Pyrrole Caproyl Adipate Hydrazide: PCAH

N- (CH ₂ ) ₅ -CO-NH-NH-CO- (CH ₂ ) ₄ -CO-NH-NH ₂

The PCNHS III (5mmol, 1.4g) and the adhydate dihydrazide (5mmol, 0.87g) are placed in a flask in 50mL of DMF. Since the adipate dissolves poorly, 15 ml H ₂ O are added. The mixture is stirred overnight, at ambient temperature; a white deposit is formed. TLC carried out in CHC1 ₃ 95 / MeOH 5, the end of reaction, indicating that all the activated ester PCNHS was consumed. The mixture is filtered on sintered glass, 30 mg of solid corresponding to NHS is recovered, insoluble in a DMF / H ₂ O mixture. The DMF / H ₂ O mixture is evaporated from the filtrate, 2.1 g of crude product is obtained. Characterization of the crude product in mass spectrometry: 3 characteristic peaks: 173.1 (MH) ⁺ or 174.1 for adipate; 336.2 (MH) ⁺ or 337.2 for the PCAH; 399.2 (MH) ⁺ or 400.2 for the PCACP dimer. The reaction product is purified on a silica column, eluted in a DMSO gradient (0-50%) in CH ₂ C1 ₂ . We manage to eliminate adipate as well as a majority of the PCACP dimer. The SM still shows traces of dimer. The mass of product obtained is 750 mg (the mass expected for 100% yield is 1.685 g), ie a yield of approximately 44%. Characterization: ^] H-NMR (200MHz; DMSO / TMS) δ (ppm): 1.42 (tt, 4H, -CH ₂ - (CH ₂ ) ₂ - CH ₂ - adipate); 1.95 (t, 2H, -CH ₂ -CO-NH-NH ₂ ); 2.04 (t, 2H, -CH ₂ -CO-NH-NH- of adipate); 2.46 (t, 2H, -CH ₂ -CO-NH-NH- of caproyl); 3.78 (t, 2H, -CH ₂ -N); 4.12 (si, 2H, -NH-NH ₂ ); 5.91 (dd, 2H, 3-H and 4-H of pyrrole); 6.67 (dd, 2H, 2-H and 5-H of pyrrole); 8.9 (s, 3H, -NH-NH- and NH-NH ₂ ).

Pyrrole Undecanoyl Adipate Hydrazide: PUAH

N— (CH ₂ ) ₁₀ -CO-NH-NH-CO- (CH ₂ ) ₄ -CO-NH-NH2

The PUNHS IV activated ester (5mmol, 1.74g) is reacted in a flask with the dihydrazide adipate (5mmol, 0.87g) in 50mL of DMSO, overnight, at room temperature. The reaction is followed by TLC in CHCl ₃ 90 / MeOH 10. The DMSO is then evaporated, with a vacuum pump with stirring and heating. 6.2 g of crude product are obtained. Characterization of the crude product in mass spectrometry: 3 characteristic peaks: 175.1 (M + H) ⁺ or 174.1 for adipate; 408.2 (M + H) ⁺ or 407.2 for the PUAH; 641.2 (M + H) ⁺ or 640.2 for the PUAUP dimer. The mixture resulting from the reaction is purified on a silica column, eluted in a DMSO gradient (from 0 to 20%) in CH ₂ C1 ₂ . We manage to eliminate adipate as well as a majority of the PCACP dimer. The SM still shows traces of dimer. The mass obtained of product is 840 mg, ie a yield of approximately 41%. Characterization: ^l H-NMR (200MHz; DMSO / TMS) δ (ppm): 1.43 (tt, 4H, -CH ₂ - (CH ₂ ) ₂ -

CH ₂ - adipate); 1.94 (t, 2H, -CH ₂ -CO-NH-NH ₂ ); 2.03 (t, 2H, -CH ₂ -CO-NH-NH- of adipate); 2.49 (t, 2H, -CH ₂ -CO-NH-NH- of undecanoyl); 3.79 (t, 2H, -CH ₂ -N); 4.12 (si, 2H, -NH-NH ₂ ); 5.9 (dd, 2H, 3-H and 4-H of pyrrole); 6.66 (dd, 2H, 2-H and 5-H of pyrrole); 8.9 (s, 3H, -NH-NH- and NH-NH ₂ ). 2 Coupling of the spacer-pyrrole arm with sugar. The pyrrole-spacer arm groups are PUH, PCAH and PUAH previously synthesized. The sugars used are the heparin or heparin sulfate fragments prepared above. HP6 (~ 6kDa) and dpl2 (~ 3kDa). a Coupling reaction This spacer fixes the spacer arm by its hydrazide function to the aldehyde function of sugar, the sugar being in equilibrium between a pyranose form and an open form comprising an aldehyde function. After synthesizing the pyrrolized spacer arms, these are assembled with sugar to obtain the final molecule, ready for copolymerization. We operate by a coupling reaction, in two stages. The reaction is carried out in 48 hours at 56 ° C., in a DMSO 20 / H ₂ O 1 mixture. The coupling steps are as follows: first the pyrrole spacer arm (PUH, PUAH or PCAH) is coupled to the HP6 sugar by a hydrazone function, which hydrolyzes easily in water. This is why, in a second step, this hydrazone function is reduced to a stable hydrazide by treatment with sodium cyano-borohydride. The general principle of coupling is summarized in diagram 2 below.

Coupling principle

Diagram 2 In an Eppendorf, the reagent "Pyrrole-spacer arm - Hydrazide" (50mM in DMSO, 250μL) is mixed with HP6 sugar (20mM in 2M acetate buffer pH 4.2, 12.5μL), the samples are placed at the oven at 56 ° C for 48 hours, after 6-8 hours of reaction, the sodium cyanoborohydride reducing agent (4M in EtOH, 25 μL) is added. At the end of the reaction, the salts are removed by passage over a PD10 column (mini exclusion column of Pharmacia brand). A reading is then carried out with a UV-visible spectrometer, at the wavelength λ = 232nm, to check the presence of the sugar. Then the collected fractions are combined and lyophilized. C: MANUFACTURE OF SUGAR CHIP The technique of "electrospotting" [Livache et al, Synthetic Metals, 2001, 121, 1443-1444] is transposed to the grafting of pyrrolized sugars, and is deposited by successive copolymerizations of the pyrolysed sugar HP6 grafted onto one of the monomers PUH, PUAH or PCAH. Electropolymerization The slides are made of glass with a refractive index n = 1.776, covered with chromium (2 nm) and gold (49 nm). The reaction medium contains 20 mM of pyrrole, 25mM phosphate buffer pH = 6.8 and the sugar-pyrrole at the concentrations specified below. The construction of the chip is carried out according to the “electrospot” method using an x / y / z robot (Newport-microcontrol); the polymerization time is 125 ms under a potential difference of 2.4V. After synthesis, the deposits are rinsed with distilled water and then dried. D: STUDY OF CHIMIOKINE / SUGAR HP6 INTERACTIONS Chemokines are a family of protein molecules recognizing all heparin-type glycoaminoglycans [Lortat-Jacob et al, Proc Natl Acad Sci USA, 2002, 99, 1229-1234]. 1 Study of interactions by fluorescence The principle of revelation in fluorescence of the biotinylated HP6 / SDF complexation is represented in FIG. 3, SDF being a commercial chemokine taken as a model. The studs of the chip are placed in the presence of a rinsing buffer solution; this buffer contains the BSA protein, used to “block” the bare gold of the chip, in order to avoid non-specific fluorescence. Once the blocking is done, the chip is rinsed, then lOOμL of biotinylated SDF is added to the concentration of lμM diluted in the rinsing buffer for 30 minutes. Then rinsed again with the buffer, then put the R-streptavidin phycoerythrin (SAPE-molecular probes) 15 minutes, protected from light. It is rinsed again, then a glass slide is placed on the chip, and the fluorescence intensity (IF or gray levels) is read using the epifluorescence microscope. For this study, the deposits were made on a glass slide covered with chromium (2 nm) and gold (49 nm). Figure 4 illustrates the polymer deposition scheme and the fluorescence intensity measured. 25 polypyrrole pads were placed on the slide • Species 1: HP6 sugar + PCAH spacer arm (PCAH-HP6) • Species 2: HP6 sugar + PUAH spacer arm (PUAH-HP6) • Species 3: HP6 sugar + PUH spacer arm (PUH-HP6) • Species 4: HP6 sugar without spacer arm and not grafted with pyrrole (HP6 alone) • Species 5: Chondroitin Sulfate sugar (CS) does not recognize the protein + PCAH spacer arm (PCAH-CS) • Species 6: pyrrole (no grafted biological compound called Milieu

Reaction (MR) These plots were deposited at 6 different concentrations of monomer grafted with a sugar: A = 100 μM, B = 50 μM, C = 25 μM, D = 10 μM, E = 1 μM, F = 500 nM. The gray plot of the diagram is a MR plot on which no deposit has been made. These concentrations represent the total sugar concentration (sugar - pyrrole and sugar alone). The Chondroitin Sulfate (CS) sugar was modified by a pyrrole like the other sugars and was used as a negative control, for its structure similar to heparin HP6 and its non-recognition of the SDF protein. Results: The objective of this experiment is: to verify the recognition of HP6 sugar by the biotinylated SDF protein, to determine if the spacer arms play an important role in recognition, to determine from which concentration recognition can be detected if it takes place . The photo obtained under an epifluorescence microscope (cf. fig. 4) illustrates the recognition of SDF by sugars. The fluorescence intensity measurements taken on the photo are summarized in Table 1:

Table 1: summary of the fluorescence intensity measurements. The 3 MR pads, in accordance with our expectations, do not exhibit fluorescence, which shows the absence of non-specific adsorption on the surface of the polypyrrole film. The negative control PC AH-C S grafted with polypyrrole, has only a very weak fluorescence, the protein does not adsorb on sugar. The HP6 control not grafted with pyrrole but immobilized in the polypyrrole, shows a much lower fluorescence than the HP6 studs grafted with pyrrole. This demonstrates that the detection of protein / sugar recognition requires that the sugar be grafted onto a monomer participating in the copolymerization. By comparing the fluorescence intensities of the three spacer arms, it can be seen that there is better fluorescence for the PUH arm, followed by the PCAH arm and finally by the PUAH arm. We can already conclude that the immobilization technique of sugar HP6 is favorable to the recognition of the protein, moreover, we can determine the range of optimal concentrations: between 10 μM and 50 μM, since there is no fluorescence for the 0.1 μM and 1 μM plots, and it seems that the 1 OO μM plots exhibit a saturated fluorescence intensity . 2. Study in SPRi Interactions can be studied by SPR imaging directly on the chip and in real time [Guedon et al. Anal Chem. 2000, 72 (24): 6003-9]. This technique does not require the use of labeled molecules. The operating principle of the SPR analysis is illustrated in FIG. 5. Objective of the experiment: The primary objective of the experiment is to verify the recognition, in real time, of the HP6 sugar by the SDF protein as a function of the nature of the spacer arm and the second is to optimize the measurable signal by varying the sugar concentration on the pads as well as that of injected target protein. For this kinetic study, the deposits were made on a prism covered with chromium (2 nm) and gold (49 nm). 16 polypyrrole pads were placed on the prism. The duration of electrocopolymerization was 125 ms. These studs are detailed as follows (see fig. 6):

• Species 1: HP6 sugar + PCAH spacer arm (PCAH-HP6) • Species 2: HP6 sugar + PUAH spacer arm (PUAH-HP6) • Species 3: HP6 sugar + PUH spacer arm (PUH-HP6) • Species 4: sugar HP6 without spacer arm and not grafted with pyrrole (HP6 alone) • Species 5: Chondroitin Sulfate sugar (CS) does not recognize the protein + spacer arm PCAH (PCAH-CS) • Species 6: pyrrole (no grafted biological compound, called Reaction Medium (MR) These plots were deposited at 4 different concentrations: A = 100 μM, B = 25 μM, C = 6.25 μM, D = 1.56 μM. These concentrations represent the concentration of total sugar (pyrrole sugar and sugar alone ). We used the Chondroitin Sulfate (CS) sugar as a negative control, for its structure similar to heparin HP6 and its non-recognition of the SDF protein. In FIG. 4, the image on the left represents the prism seen in SPR imaging, when it is in rinsing buffer solution; the image on the right is a diagram of the arrangement of the studs on the golden surface of the prism. Measurement of interactions We have positioned ourselves at 54 °, to be in the right flank of the absorption peak of the reflectivity curve. All measurements were made with a flow rate of 30 μL / min. On this prism, the protocol was as follows (examples fig. 7): -passing the rinsing buffer, solution containing 1% BSA, serving as a blocker for the experiment. „, - measurement and recording of the reflectivity as a function of the angle of incidence - passage of a solution containing the SDF protein diluted in the rinsing buffer, for 15 minutes. - return to the rinsing buffer alone in order to measure the difference in level in the same medium before and after the interaction (rinsing for more than 15 minutes). - passage of a 1M NaCl solution in the rinsing buffer (120 μL / min for 30 seconds) to regenerate the pads: protein / sugar dissociation. - return to buffer in order to measure the difference in level in the same medium between before and after regeneration. This protocol was carried out for 7 different concentrations: 1 nM,

10 nM, 50 nM, 100 nM, 250 nM, 500 nM and 1 μM.

FIG. 7 represents the raw kinetic curves for the 100 nM (left) and 250nM (right) concentrations of SDF protein. For information, the different injections are separated by a vertical line: buffer / Protein / buffer / NaCl (large peak) / buffer). Images of the matrix are shown in Figure 8 after each protein injection, before rinsing, to observe the ignition of the pads specific (we notice the change in gain increasing in parallel with the concentration of protein injected). FIG. 8 illustrates the variations in reflectivity before rinsing due to the fixation of SDF on the HP6 sugar. For better readability of the images, only the areas taken into account in the measurements have been represented (artificially black background). The graphs shown in Figures 9 and 10 represent the variations in reflectivity before rinsing (fig. 9) and after rinsing (fig. 10) due to the fixation of the SDF protein on the HP6 sugar fixed to the pyrrole by different spacer arms in function of the concentration injected. FIG. 9 illustrates measurements taken in% of reflectivity, corresponding to the recognition between the SDF protein and the HP6 sugar, before rinsing. The HP6 1.56μM studs are not shown, because the taps are not significant. FIG. 10 illustrates measurements taken in% of reflectivity, corresponding to the recognition between the SDF protein and the HP6 sugar, after rinsing. The 1.56μM HP6 pads are not shown, because the intakes are not significant Before the rinsing in buffer, we can detect a significant signal of fixation of the SDF protein at injection concentrations as low as 10 nM, for the pads functionalized containing 100 or 25 μM of HP6 sugar. It can be seen that when the amount of protein injected is increased, the corresponding reflectivity percentage increases fairly linearly. At the same time, the more grafted HP6 sugar the stud has, the stronger the biological recognition of SDF protein / HP6 sugar. We reach saturation around 500nM in SDF protein, since by injecting 1 μM of protein, the percentage hardly increases any more. Rinsing serves, at the very beginning, to “unhook” the surplus of biological molecules fixed by adsorption on the polymer as on the sugar; its second role is to observe if the complex dissociates and at what speed; from the data obtained during injection and rinsing, we can calculate the Kd.

After rinsing, a specific fixation signal is observed from lOnM in SDF for the blocks of lOOμM in sugar, and from 50 nM for the pads of 25μM of sugar. It is extremely important to note that chondroitin sulfate-pyrrole does not generate any signal (negative control) and that HP6 unmodified by a pyrrole does not generate any response either; sugar pyrrolylation is therefore necessary to ensure correct recognition between the sugar and the SDF protein. 3. Study in SPR Imaging This experiment will make it possible to observe the optimization carried out in terms of the concentrations of deposited sugar and proteins, during the sugar / protein interaction followed in SPRi. For this kinetic study, the deposits were made on a prism covered with chromium (2 nm) and gold (49 nm). 25 polypyrrole pads were placed on the prism. The duration of electrocopolymerization was 250 ms. These studs are detailed as follows (see fig. 11 A and 11B): ^s • Species 1: HP6 sugar + PCAH spacer arm (PCAH-HP6) • Species 2: HP6 sugar + PUAH spacer arm (PUAH-HP6) • Species 3: HP6 sugar + PUH spacer arm (PUH-HP6) • Species 4: HP6 sugar without spacer arm and not grafted with pyrrole (HP6 alone) • Species 5: Chondroitin Sulfate sugar (CS) does not recognize the protein + arm PCAH spacer (PCAH-CS) • Species 6: pyrrole (no grafted biological compound, called Reaction Medium (MR) • Species 7: dp sugar 12 + PUAH spacer arm (PUAH-dpl2) These plots were deposited at 5 different concentrations : A = 25 μM, B = 12.5 μM, C = 6.25 μM, D = 3, l μM, E = 1.5 μM. These concentrations represent the concentration of total sugar (pyrrole sugar and sugar alone). is a fragment of HP6 sugar from an enzymatic digestion, it only has one protein binding site while HP6 has two. Read the Chondroitin Sulfate (CS) sugar as a negative control, for its structure similar to heparin HP6 and its non-recognition of the protein SDF-1a. FIG. 11A represents the prism seen in SPR imaging, when it is in rinsing buffer solution. FIG. 11B represents a diagram of arrangement of the studs on the golden surface of the prism. Measurement of interactions: We have positioned ourselves at around 53 °, to be in the right flank of the absorption peak of the reflectivity curve. All the measurements were made with a flow rate of 45 μL / min. On this prism, the protocol was as follows as illustrated in FIG. 12: - Passage of the rinsing buffer. - Measurement and recording of reflectivity as a function of the angle of incidence. - 2 successive injections of 1% BSA solution in rinsing buffer, serving as a blocker for the experiment. - Passage of a 1M NaCl solution in the rinsing buffer (100 μL / min), to regenerate the studs, only the gold surface is blocked for the rest of the experiment. - Passage of a solution containing the SDF protein diluted in the rinsing buffer, for 7 minutes. - Return to rinse buffer with free HP6 lμM sugar, in order to measure the dissociation of the sugar / protein complex. - Passage of a 1M NaCl solution in the rinsing buffer (100 μL / min), to regenerate the pads. - Return to buffer in order to measure the difference in level in the same medium between before and after regeneration. This protocol was carried out for 6 different concentrations: 15.6 nM, 31.2nM, 62.5nM, 125nM, 250nM and 500nM. FIG. 13 represents all the raw kinetic curves for the different concentrations of SDF protein. In Figure 13, the different SDF injections are marked by an arrow, the dissociation by a vertical line: buffer / Protein / Buffer with free HP6 / NaCl (large peak) / buffer. In Figure 13, species 1 has been shown, these curves were obtained after a first treatment of the experiment. It is noted that the uptake in% during the protein injection increases in proportion to the quantity of sugar deposited, likewise, the higher the protein concentration, the higher the uptake until reaching saturation; here, the saturation of all the binding sites available for the 25 μM sugar plots is reached for 250 nM of SDF, the 6.25 μM plots saturate from 62.5 nM of protein. The protein concentration at 500nM hardly increases the signal anymore. The graphs represented in FIGS. 14 and 15 represent the variations in reflectivity before rinsing (fig. 14) and after rinsing (fig. 15) due to the fixing of the SDF protein on the HP6 or dp 12 sugar fixed to the pyrrole by different arms spacers, depending on the concentration injected. FIG. 14 illustrates measurements taken in% of reflectivity, corresponding to the recognition between the SDF protein and the HP6 or dp 12 sugar, before rinsing. The 3.1nM and 1.5nM plots are not shown, because the catches in% are not significant. FIG. 15 illustrates measurements taken in% of reflectivity, corresponding to the recognition between the SDF protein and the HP6 or dpl2 sugar, after rinsing. The 3.1nM and 1.5nM plots are not shown, because the catches in% are not significant. Before rinsing in buffer with free HP6, we can detect a significant signal of fixation of the SDF protein at injection concentrations as low as 31nM, for the functionalized pads containing 25 μM of HP6 or dpl2 sugar. Rinsing with free sugar makes it possible, first of all to “unhook” the excess of proteins fixed by adsorption on the polymer on the sugar and to observe a real dissociation due to the competition of association between the deposited sugar and the sugar. free with protein in solution. After rinsing, a specific fixation signal is observed from 3 lnM in SDF for the 6.25 μM sugar pads. It is important to note that unlike the previous experience, the 100 μM sugar pads have been removed, it is useless to deposit as many sugars on the surface to obtain correct sugar / protein interactions, which also makes it possible to consume a less protein. The negative control chondroitin sulfate, like the unmodified HP6, gives no fixation signal; specific recognition between sugar and protein requires that the sugar be covalently linked to the polypyrrole polymer. Note that the regeneration of the NaCl plots allows the signal to return to the baseline, as before the protein injection. This regeneration is non-destructive and reproducible, whatever the plot considered. Unlike the previous experiment, the rinsing buffer no longer contains BSA, which makes it possible to obtain a return to the baseline after regeneration much faster (cf. fig. 7 and fig. 13).

Claims

CLAIMS 1. Polymer corresponding to the following general formula (I):

(I) in which: Aj and A _j each represent an electrically polymerizable monomer, the various A; and A _j can be identical or different; - Bj represents a carbohydrate which can be chosen from: the following natural monosaccharides: allose, altrose, arabinose, erythrose, fructose, galactose, glucose, gulose, idose, lyxose, mannose, psicose, ribulose, sorbose, tagatose, threose, talose, xylose, xylulose; from the derivatives of these natural monosaccharides such as acid derivatives, amine derivatives, amino-substituted derivatives, derivatives alkyl esters Cι _Cι-2 alkyl ethers derivatives Cι _Cι-2 alkyl amides derivatives C ₁ -C ₁₂ of these monosaccharides; among the polysaccharides comprising at least two units chosen from natural or synthetic monosaccharides and their derivatives; among the polysaccharides substituted by at least one group chosen from: peptides, proteins, lipids, such as for example C ₈ -C ₃₀ fatty acids, steroids and their derivatives; - Li represents a covalent bond or a spacer arm connecting the monomer Aj to group B; chosen from the groups corresponding to formula (II): -Ri- [(CH ₂ ) _n -R ₂ ] _a - [(CH ₂ ) _m -R ₃ ] _b - (CH ₂ ) _P - (II) in which : -n is an integer ranging from 1 to 10; -m is equal to 0 or is an integer ranging from 1 to 10; -p is 0 or is an integer ranging from 1 to 10; -a is equal to 0 or is an integer ranging from 1 to 8; -b is 0 or is an integer ranging from 1 to 8; -Ri, R ₂ and R ₃ which may be identical or different represent a group chosen from: -CH ₂ -; -O-; -S-; -NR'-;-CO-;-CH≈CH-;-NR'C (O) -; -C (O) N (R ') -; - NHS (O ₂ ) - and R 'represents a hydrogen atom or a Ci to Cι ₂ alkyl chain; - x represents an integer greater than or equal to 1; - i is an index varying from 1 to x; - y represents an integer greater than or equal to 0; - j is an index varying from 1 to y when y ≠ 0; it being understood that all of the monomers Ai and Aj cannot be p-phenylene vinylene.

2. Polymer according to claim 1, characterized in that the monomers A; and A _{j which} are electropolymerizable are selected from: acetylene, acrylonitriles, p-phenylene, p-phenylene vinylene, pyrene, pyrrole, thiophene, furan, selenophene, pyridazine, carbazole, aniline , phenol and their derivatives.

3. Polymer according to claim 2, characterized in that Aj and A _j are chosen from pyrrole and substituted pyrrole derivatives such as N-methylpyrrole and N-cyano-ethylpyrrole.

4. Polymer according to any one of claims 1 to 3, characterized in that the monomers Aj and A _j are all identical.

5. Polymer according to any one of claims 1 to 4, characterized in that several distinct Bj groups are present.

6. Polymer according to any one of claims 1 to 5, characterized in that the ratio x / y is between 1/1 and 1 / 1,000,000, preferably between 1/10 and 1/100,000, and even more preferably between 1/20 and 1/50 000.

7. A method of preparing a polymer of general formula (I) according to any one of claims 1 to 6, characterized in that it comprises at least the following steps: - a first step during which one proceeds to the preparation of a copolymer of general formula (III): - [Ai *] _x - [A _j ] _r (III) in which Aj * represents a group Ai comprising a designated function *. a second step during which the fixing, on the polymer of formula (III), of at least one group of general formula (IV):

(IV) in which ** Li represents an L group; carrying a function ** capable of reacting with the function * of A; to form an Lj-Aj covalent bond.

8. Process for the preparation of a polymer of general formula (I) according to any one of claims 1 to 6, characterized in that it comprises at least the following steps: - a first step during which one proceeds to the preparation of the compound of general formula (V): -Ai- Li

Bi (V) - a second stage during which the copolymerization of compound (V) is carried out with the monomer A _j .

9. Method according to claim 7 or claim 8, characterized in that at least one step of the method involves at least one electrochemical reaction.

10. Method according to claim 9, characterized in that the electrochemical copolymerization is carried out on the surface of an electrode.

11. Method according to claim 9 or claim 10, characterized in that an electrochemical copolymerization of the mixture is carried out by subjecting a mixture of monomers to a process chosen from: one or more jumps of potential; cyclic voltammetry; chronopotentiometry.

12. Method according to any one of claims 9 to 11, characterized in that it comprises the use of means chosen from microelectrode arrays or localized electrochemical reactions.

13. An electrode comprising one or more deposits of at least one polymer grafted with one or more saccharides and corresponding to formula (I) according to any one of claims 1 to 6.

14. An electrode according to claim 13, characterized in that it is capable of being used in a resonance imaging process of surface plasmons.

15. An electrode according to claim 13 or claim 14, characterized in that it consists of a conductive material deposited or not on a solid support.

16. An electrode according to any one of claims 13 to 15, characterized in that it comprises deposits of polymers distributed in the form of a matrix.

17. A screening method characterized in that it comprises contacting an electrode according to any one of claims 13 to 16 with one or more potential ligands.

18. The method of claim 17, characterized in that it comprises at least one step of resonance imaging of the surface plasmons.