WO2012028825A1

WO2012028825A1 - Ultra-thin films of molecularly imprinted polymers confined to the surface of a substrate

Info

Publication number: WO2012028825A1
Application number: PCT/FR2011/051992
Authority: WO
Inventors: Mohamed Mehdi Chehimi; Sarra Gam-Derouiche; Ahmed Madani
Original assignee: Universite Paris Diderot-Paris 7
Priority date: 2010-09-01
Filing date: 2011-08-31
Publication date: 2012-03-08
Also published as: FR2964104A1; FR2964104B1

Abstract

The present invention relates to ultra-thin films of molecularly imprinted polymers confined to the surface of a substrate, to methods for preparing same, and to the uses thereof.

Description

ULTRAMINAL LAYERS OF POLYMERS WITH CONFINED MOLECULAR IMPRESSIONS IN SURFACE OF A SUPPORT

The present invention relates to ultrathin layers of molecular fingerprint polymers confined to the surface of a support, their methods of preparation and their uses.

Molecularly Imprinted Polymers (MIPs) have grown considerably since the 1990s. Their synthesis is carried out by crosslinking around an ionic, molecular or (bio) macromolecular model species, the elimination of which is possible. creating fingerprints having the shape and size of the model species used in their design. MIPs then become artificial receptors vis-à-vis the model species that has been used to nanostructure and confer on them this "biomimetic" character.

Among the published works relating to molecular imprinted polymers, mention may first be made of US Pat. No. 5,630,978, which discloses a method for the preparation of mimetics of a large variety of drugs, thanks to the use of MIPs whose Footprints were used for the polymerization of organic compounds with properties of similar size and shape to those of the model molecule.

US5959050 discloses a method for preparing MIPs formed by two different acrylic monomers and at least one model molecule, in the form of uniform beads used in various screening techniques.

Thus, the MIPs can be used in analytical chemistry for the detection and quantification of molecules of interest present in the trace state, the fixation of said analyte on said polymer resulting in the variation of a physical quantity (for example an electrical potential ) or chemical (for example a pH) whose measurement can be analyzed by a transducer and reported at an analyte concentration. The transducers employed are generally selected according to the type and concentration of analyte sought and are, for example, selected from quartz crystal microbalances, surface plasmon resonance ("plasmon resonance", SPR) sensors, optical and acoustic sensors. Most of these systems and exemplary uses are described in Haupt K., Mosbach, K. (Molecularly Imprinted Polymers and Their Use in Biomimetic Sensors, Chem Rev. 2000, 100: 2495-2504). US Pat. No. 6,888,486 discloses MIP-coated quartz crystal microbalances and methods for their preparation, which are useful for on-line control of floating organic contaminants. However, these MIP-coated surface preparation techniques include tedious organic synthesis steps, otherwise inaccessible to the non-specialist, relatively long or that produce MIPs insufficiently confined to said surfaces. Confinement can however be improved by pretreatment of its surface, which consists in grafting on said surface functional groups initiating polymerization of MIPs or able to play this role after chemical transformation.

For example, several publications disclose processes for preparing MIPs confined to gold surfaces by aromatic thiol groups grafted onto said surfaces. These aromatic thiol groups carry N0 ₂ groups which can be reduced to NH ₂ groups and subsequently attacked by the malonodinitrile of formula CH ₃ - CH (CN) ₂ , in order to obtain a ΓΑΙΒΝ grafted derivative at the surface and capable of to initiate an atom transfer radical polymerization ("Atom Transfer Radical Polymerization", ATRP). The thiol grafting step is however carried out in at least 72 hours. The publications of Geyer et al., Schmelmer et al., Wei et al., And that of Steenackers et al., Deal with this subject (Geyer et al., Electron-induced crosslinking of aromatic self-assembled monolayers: Negative resists for Applied Physics Letters, 1999, 75: 2401-2403, Schmelmer et al., Surface-Initiated Polymerization on Self-Assembled Monolayers: Amplification of Patterns on the Micrometer and Nanometer Scale, Angew Chem Int, Ed 2003, 42: 559-563, Wei et al., Molecular Imprinting by Atom Transfer Radical Polymerization, Biomacromolecules, 2005, 6 (2): 1113-1121, Steenackers et al., Morphology Control of Structured Polymer Brushes, Small 2007, 3: 1764-1773 ).

Another technique consists in using coupling silanes carrying an AIBN type photoinitiator group, as described in the publication by Prucker et al. (Synthesis of Poly (styrene) Monolayers Attached to High Surface Area Silica Gels through Self-Assembled Monolayers of Azo Initiators, Macromolecules, 1998, 31 (3): 592-601), or directly carriers of the model molecule around which will be synthesized MIP, as described in the publication of Zhang et al. (Novel surface modified molecularly imprinted polymer using acryloyl-beta-cyclodextrin and acrylamide as monomers for selective recognition of lysozyme in aqueous solution, Journal of Chromatography, A. 2009, 1216: 4560).

The application FR 2921065 describes a method of manufacturing molecular fingerprint copolymers in which the attachment of the multifunctional groups is carried out in particular by depositing a liquid solution containing them. Pre-treatment of the surface of the support consists in producing a layer of an alkoxysilane-based dielectric material and functional groups fixed by reduction of diazonium salts which carry them are never implemented.

International application WO 01/19886 discloses a method of manufacturing molecular imprint copolymers based on silane chemistry and the use of an AIBN type free radical initiator.

Application FR 2 935 705 describes a process for producing molecular imprint copolymers in which the initiators can be attached directly to the raw substrate or to a previously silanized substrate. It is never mentioned the implementation of a prior step of grafting functional groups on the surface of the support by chemical or electrochemical reduction of diazonium salts carrying said functional groups.

However, these confinements are not yet satisfactory and there is still a need for new methods to improve the confinement of the MIPs.

The use of diazonium salts is known for surface modification (Pinson et al., Attachment of organic layers to conductive or semiconductive surfaces by reduction of diazonium salts, Chem Soc Rev. 2005, 34: 429-439). photografting of preformed polymers (Adenier et al., Attachment of Polymers to Organic Moieties Covalently Bonded to Iron Surfaces, Chem., Mater., 2002, 14 (11): 4576-4585), surface photopolymerization leading to conventional polymers (WO2006000692 ), surface-controlled radical polymerizations (WO2006000692, Matrab et al., Novel approach for metallic surface-initiated atom transfer radical polymerization using electrografted initiators based on aryl diazonium salts, Langmuir, 2005, 21 (10): 4686-4694; Grafting densely-packed poly (n-butyl methacrylate) chains from an iron substrate by aryl diazonium surface-initiated ATRP: XPS monitoring, Surface Science, 2007, 601: 2357-2366, Matrab et al. of polymer brushes by atom transfer radical polymerization on glassy carbon modified by electro-grafted initiators based on aryl diazonium salts. Surface and Interface Analysis. 2006, 38 (4): 565-568; Matrab et al. Surface functionalization of ultrananocrystalline diamond using atomic transfer radical polymerization (ATRP) initiated by electro - grafted aryldiazonium salts. Diamond & Related Materials. 2006, 15: 639-644; Matrab et al. Aryl Diazonium Salts for Carbon Fiber Surface-Initiated Atom Transfer Radical Polymerization. Journal of Membership. 2008, 84 (8): 684-701; Minh Ngoc Nguyen, et al. Protein-functionalized hairy diamond nanoparticles. Langmuir. 2009, 25: 9633-9636), as well as for electropolymerizations (Santos et al., Electrografting Polyaniline on Carbon through the Electroreduction of Diazonium Salts and the Electrochemical Polymerization of Aniline. Journal of Physics Chemistry C. 2008, 112 (41): 16103-16109).

However, none of these publications mention the use of diazonium salts in combination with a surface-confined photopolymerization, especially a transducer surface, for the preparation of ultra-thin layers of molecular imprinted polymers, let alone the simplicity of implementation of such a system.

The solution proposed by the inventors uses diazonium salts. which make it possible to very simply modify substrates (or supports), in particular transducers, with surface-initiating photopolymerization functional groups or capable of playing this role after chemical transformation. This method makes it possible in particular to detect molecules in the form of traces, that is to say concentrated at 1 nanomole / L, or even 0.1 nanomole / L. It also has an industrial advantage because of its simplicity of implementation and this at each stage of preparation of the sensitive layers of MIPs. For example, the cleaning of the plates prepared by this technique, carried out with the UV in the presence of ozone, lasts only a few minutes, while the chemical or electrochemical treatment for the grafting of the photoinitiators from the diazonium salts is carried out in a few minutes. minutes electrochemically, or in 20 to 30 minutes at most by chemical means. The electrochemical pretreatment of the substrates will therefore be preferred. Finally, it has been verified that polymer layers of 200 to 300 nm thick can be obtained after only 10 minutes of photopolymerization. For example, the methacrylic acid system (MAA, used as a functional monomer), ethylene glycol dimethacrylate (EGDMA, used as crosslinking monomer) and dopamine (model molecule), makes it possible to manufacture a dopamine-imprinted polymer in about 13 minutes.

These sensitive layers on the other hand have high stability, are easily regenerated and adhere to the surfaces without any sign of rupture.

Also, the technique developed by the inventors makes it possible to detect (macro) molecules or ions of agri-food, biological, pharmaceutical and environmental interest. It can for example be exploited in the field of sport and the detection of doping products. It will also interest manufacturers wishing to provide custom prepared substrates for the specific detection of an analyte. This technique can also be exploited for the controlled release of drug substances by microelectrodes. To carry out said MIPs, a functional monomer capable of producing covalent or non-covalent bonds (of the van der Waals and / or hydrogen bonding type) must be brought into contact with the model molecule studied ("template"), a monomer crosslinking for forming the three-dimensional network around the model species and a photoinitiator to initiate the photopolymerization reaction, and optionally a co-initiator complementary to the photoinitiator chosen. In order for the films to be generated on the surface of a support (for example an electrode), the photoinitiator must, according to the invention, be grafted onto the surface (directly or indirectly), which the diazonium salts make possible with a very strong membership.

Thus, the present invention relates to a process for preparing a molecular fingerprint polymer confined to the surface of a support, said method comprising the following steps:

a) a step of preparing said grafting surface on said surface is polymerization initiator functional groups or able to play this role after chemical transformation, or co-initiator functional groups, including hydrogen donors, grafting said functional groups occurring by chemical or electrochemical reduction of the diazonium salts by which said functional groups were initially carried,

b) if appropriate a step of chemical transformation of said graft groups on said surface to make them able to initiate a photopolymerization,

c) a step of bringing the resulting surface into contact with a polymerization composition comprising at least one functional monomer, at least one crosslinking monomer, at least one model molecule, optionally a co-initiator hydrogen donor in a polymerization medium ,

d) a radical polymerization step induced by U.V. excitation of said composition from the grafts previously fixed on said surface,

e) a step of obtaining an ultra-thin layer molecular imprinted polymer with a thickness less than or equal to 1 μιη, preferably less than or equal to 100 nm, more preferably less than or equal to 5 nm,

f) a step of removing the at least one model molecule from the matrix of the molecular imprinted polymer, g) optionally repeating steps a) to d) using a composition comprising at least one identical or different functional monomer, at least one identical or different crosslinking monomer and at least one identical or different model molecule.

Thus, according to one embodiment of the process of the invention, the polymerization initiator functional groups are used in the form of diazonium salts which are grafted onto the surface of a support by chemical or electrochemical reduction of said diazonium salts. After fixing these functional groups, these are optionally converted, then the bearing surface of these optionally converted groups is subjected to a photopolymerization reaction in the presence of a polymerization solution optionally containing a co-initiator, for example a donor of 'hydrogen.

In another embodiment of the invention, the co-initiator functional groups are used in the form of diazonium salts which are grafted onto the surface of a support by chemical or electrochemical reduction of said diazonium salts. After fixing these functional groups, the surface is subjected to a polymerization reaction in the presence of a free polymerization initiator in the polymerization solution.

By "surface of a support" is meant the surface of a conductive substrate, or non-conductive but activated by ultrasound, photons, microwaves or by heat treatment. All these methods are well known to those skilled in the art and described for example by Bélanger, J. Pinson, (Electrografting: a powerful method for surface modification J. Chem Soc Rev. 40 (2011) 3995-4048).

"Conductive substrate" means a conductive, semiconductor or conductive substrate. By way of nonlimiting examples of conducting substrates, mention may be made of pure metals or alloys, in particular iron, nickel, platinum, gold, copper, zinc, cobalt, titanium, chromium, silver, tantalum, especially stainless steels, titanium alloys, cobalt chromium alloys, molybdenum, manganese, vanadium and nitinol. By way of nonlimiting examples of semiconductor substrates, mention may be made of doped or non-doped silicon, mono- or polycrystalline silicon, hydrogenated silicon, tantalum nitride, titanium nitride, tantalum nitride and silicon carbide. , indium phosphide or gallium arsenide. Finally, as non-limiting examples of substrates made conductive, mention may be made of graphitized polymers on the surface. As non-limiting examples of non-conductive substrates but activated by ultrasound, photons or even by microwaves, mention may be made of oxides, clays and polymers.

In an advantageous embodiment, said "surface of a support" may contain different zones, each independently of the others being conductive, semiconductive, made conductive or non-conductive but activated by ultrasound, photons or by micro -ondes.

By the expression "able to initiate a photopolymerization" is meant according to the present invention functional groups capable of forming the first species of a polymerase chain reaction, by reaction under UV excitation with a functional monomer, where appropriate in presence of a polymerization co-initiator.

The expression "able to play this role after chemical transformation" means that it may be necessary to chemically transform said graft functional groups to make them able to initiate a photopolymerization according to the above definition. Said chemical route may comprise one or more stages, chosen by those skilled in the art.

By "diazonium salts" is meant according to the present invention the salts having the formula of the following general formula (1):

X ^" N ₂ ⁺ -A

(1)

in which :

A represents a functional group initiator of polymerization or adapted to play this role after chemical transformation,

- X ^" represents an anion advantageously chosen from halides (Γ, Br ^" , Cl ^" ), nitrates, sulphates, phosphates such as hexafluorophosphate, perchlorates, carboxylates and haloboranes such as tetrafluoroborate, tetrafluoroborate, which is deemed to lead to stable and non-explosive diazonium salts, will preferably be chosen.

In an advantageous embodiment of the invention, the at least one functional monomer is chosen from compounds comprising at least one terminal double bond, for example vinyl compounds (acrylic, methacrylic, styrene) and in particular styrene, which is optionally substituted. , alkyl acrylates, substituted alkyl acrylates, alkyl methacrylate, substituted alkyl methacrylate, acrylonitrile, acrylamide, methacrylamide, N-alkylacrylamide, N-alkylmethacrylamide, N -dialkylacrylamide, the N-dialkylmethacrylamide, isoprene, butadiene, ethylene, vinyl acetate and its derivatives, vinyl ethers, N-vinylpyrrolidone, 4-vinylpyridine, 3-vinylpyridine, 2-vinylpyridine , and in particular methyl methacrylate, ethyl methacrylate, propyl methacrylate and isomers thereof, butyl methacrylate and isomers thereof, 2-ethylhexyl methacrylate, isobornyl methacrylate, methacrylic acid, methacrylate, benzyl, phenyl methacrylate, methacrylonitrile, methylstyrene, methyl acrylate, ethyl acrylate, propyl acrylate and isomers thereof, butyl acrylate and its isomers, 2-ethylhexyl acrylate, isobornyl acrylate, acrylic acid, benzyl acrylate, phenyl acrylate, glycidyl methacrylate, 2-hydroxyethyl methacrylate, hydroxypropyl methacrylate and isomers thereof, hydroxybutyl methacrylate and the like. its isomers, N-di methacrylate methylaminoethyl, 2- (diethylamino) ethyl methacrylate, triethylene glycol methacrylate, itaconic anhydride, itaconic acid, benzyl methacrylate, 2- (dimethylamino) ethyl methacrylate, 2- (dimethylamino) ethyl acrylate, glycidyl acrylate, 2-hydroxyethyl acrylate, hydroxypropyl isomers of acrylates, hydroxybutyl acrylate and isomers thereof, N-diethylaminoethyl acrylate, triethylene glycol acrylate, N-methylacrylamide, N, N-dimethylacrylamide, N-tert butylmethacrylamide, N-butylmethacrylamide, N-methylolmethacrylamide, N-isopropylacrylamide, N-ethylolmethacrylamide, N-tert-butylacrylamide, Nn-butylacrylamide, N-methylolacrylamide, N-ethylolacrylamide, vinylbenzoic acid and its isomers, diethylaminostyrene and its isomers, methylvinylbenzoic acid and its isomers, diethylamino methylstyrene and its isomers, p-vinylbenzoic acid and its sodi salt trimethoxysilylpropyl methacrylate, triethoxysilypropyl methacrylate, diisopropoxymethylsilylpropyl methacrylate, dimethoxysilylpropyl methacrylate, diethoxysilylpropyl methacrylate, dibutoxysilylpropyl methacrylate, disopropoxysilylpropyl methacrylate, trimethoxysilylpropyl acrylate, triethoxysilylpropyl acrylate, triethoxysilylpropyl methacrylate and the like. tributoxysilpropyl acrylate, dimethoxymethylsilypropyl acrylate, diethoxymethylsilypropyl acrylate, dibutoxymethylsilypropyl acrylate, diisopropoxymethylsilypropyl acrylate, dimethoxysilylpropyl acrylate, diethoxysilylpropyl acrylate, dibutoxysilypropyl acrylate, acrylate of diisopropoxysilylpropyl, maleic anhydride, N-phenylmaleimide, N-butylmaleimide, butadiene, isoprene, chloroprene, 2- (2-oxo-1-imidazolidinyl) ethyl, 2-methyl-2-propanoate N-vinyl pyrrolidone, N-vinyl imidazole, l- [2 - [[2-hydroxyl) y-3- (2- propyl) propyl]] amino] ethyl] -2-imidazolidin-one, crotonic acid, vinylsulfonic acid and its derivatives, 2-methacryloyloxyethylphosphorylcholine, phosphorylcholine type monomers, oligo (ethylene glycol) methacrylates ), the ammonium salt of sulfatoethyl methacrylate, glyceromethacrylate, 2- (N-morpholino) ethyl methacrylate and 2- (N-3-sulfopropyl-N, N-dimethylammonium) ethyl methacrylate, the compounds comprising at least a vinyl function such as telechelic and α-tertbutoxy-ro-vinylbenzyl polyglycidol (PGL).

Similarly, in an advantageous embodiment of the invention, the at least one crosslinking monomer is chosen from divinylbenzene, ethyleneglycol dimethacrylate and tetramethyleneglycol.

dimethacrylate, poly (ethylene glycol) dimethacrylate, 2,2-bis (hydroxymethyl) butanol trimethacrylate, Ν, Ν'-dimethylacrylamide, Ν, Ν'-ethylenebisacrylamide, 1,3-diisopropenylbenzene,

2,6-bisacryloylamidopyridine, N, N'-methylenediacrylamide,

N, N'-1,4-phenylenediarcylamine, 2,6-bisacryloylamidopyridine,

N, O-bis (acryloyl) phenylalaninol, 1,4-di (acryloyl) piperazine and pentaerythritol tetraacrylate.

By the expression "model molecule" ("template", T) is meant an ionic, molecular, macromolecular species, organic or otherwise, around which the molecular imprinted polymer will be structured during the polymerization step.

In a preferred embodiment of the invention, the step of removing the at least one model molecule is carried out by extraction in a suitable solvent.

In a particularly advantageous embodiment of the process according to the invention, the diazonium salts are represented by the general formula (1), in which -A represents a type I or II photoinitiator group.

The method according to the invention can therefore be illustrated according to the following scheme:

By the term "type I photoinitiators" is meant compounds well known to those skilled in the art, capable of generating free radical initiators of polymerization under UV excitation by intramolecular homolytic fragmentation. Similarly, the term "free-radical type II photoinitiators" means compounds that are well known to those skilled in the art, capable of generating free radicals initiating polymerization by bimolecular reaction with another compound called a co-initiator, said reaction causing hydrogen transfer from the co-initiator to said type II photoinitiator.

The polymerization composition used in c) may therefore comprise, if appropriate, a co-initiator necessary for starting the photopolymerization, said co-initiator being preferably chosen from: ethyl-p-dimethylaminobenzoate, Ν, Ν-dimethylaniline ( DMA), triethylamine (TEA), 4-dimethylaminobenzoate (EDAB), p-hydroxymethyl-N, N-dimethylaniline, Ν, Ν-dimethyltoluidine, p-methyl-N, N-dihydroxyethylaniline, dimethylaminoethanol, N-Methyldiethanolamine (NDEA), N, N-dimethylethanolamine (DMEA), P-methyl-N, N-dihydroxyethylaniline, ethanolamine (EA), triethanolamine and THF. Polyethylene glycol (PEG) and polyethyleneimine (PEI) can also be mentioned.

In yet another embodiment of the invention, the diazonium salts are represented by the general formula (2), which corresponds to the general formula (1) in which -A is a -phenyl-R group, as represented below :

wherein X ^" is as defined above and R represents a substituent selected from the group consisting of:

a hydrogen atom,

aliphatic radicals, linear or branched from 1 to 20 carbon atoms, optionally comprising one or more double (s) or triple (s) link (s), optionally substituted by carboxyl radicals, N0 ₂ , OH, protected amino disubstituted protected monosubstituted amino, cyano, diazonium, alkoxy of 1 to 20 carbon atoms,

alkoxycarbonyl of 1 to 20 carbon atoms, alkylcarbonyloxy of 1 to 20 carbon atoms, optionally fluorinated vinyl or allyl, the halogen atoms, ammonium groups comprising from 1 to 20 carbon atoms, acid anhydrides comprising from 1 to 20 carbon atoms, at 20 carbon atoms, derivatives of urea or thiourea comprising from 1 to 20 carbon atoms, oximes functions and phosphonic acids,

aryl radicals optionally substituted by carboxyl, acyl, especially benzoyl, N0 ₂ , OH, disubstituted protected amino, protected monosubstituted amino, cyano, diazonium, alkoxy radicals of 1 to 20 carbon atoms, alkoxycarbonyl radicals of 1 to 20 carbon atoms, , alkylcarbonyloxy of 1 to 20 carbon atoms, optionally fluorinated vinyl or allyl, halogen atoms, ammonium groups comprising from 1 to 20 carbon atoms, acid anhydrides comprising from 1 to 20 carbon atoms, derivatives thereof. urea or thiourea comprising from 1 to 20 carbon atoms, oximes functions and phosphonic acids,

the carboxyl radicals, N0 ₂ , OH, disubstituted protected amino, protected monosubstituted amino, cyano, diazonium, alkoxy of 1 to 20 carbon atoms, alkoxycarbonyl of 1 to 20 carbon atoms, alkylcarbonyloxy of 1 to 20 carbon atoms, vinyl optionally fluorinated, halogen atoms, ammonium groups comprising from 1 to 20 carbon atoms, acid anhydrides comprising from 1 to 20 carbon atoms, derivatives of urea or thiourea comprising from 1 to 20 carbon atoms, oximes functions and phosphonic acids.

By way of non-limiting example of an aliphatic initiator photopolymerization radical, there may be mentioned a radical chosen from: -C _n H 2 _n Br in which n varies from 1 to 4, and is preferably equal to 1 or 2, a radical - CH (CH ₃ ) Br, a radical - (CH ₂ ) nCH ₃ a -C ₆ H ₄ - (CH ₂ ) n CH ₃ (DDP) radical used in the presence of benzophenone (S. Gam-Derouich et al. The present invention relates to a surface modification of carbon, metals and semi-conductors by diazonium salt-initiated photopolymerization, Surface Science 605 (201 1) 1889-1899) or a benzophenone derivative in solution (L.-L. Cheng, Y. Zhang, W. Shi, Photoinitiating characteristics of benzophenone derivatives as type II macromolecular photoinitiators used for curable UV resins, Chemical Research of Chinese Universities 2011, 27 (1), 145-149). As a non-limiting example of a non-aliphatic initiator photopolymerization radical, there may be mentioned a radical selected from -NO ₂ and -SH. In an advantageous embodiment of the invention, the group R of the formula

(II) is located in the para position of the -N ₂₊ group, so that the orientation of the growing polymer chains is close to the local vertical of said surface, as opposed to growing polymer chains whose orientation would be close to the surface itself. In a preferred embodiment of the invention, the diazonium salts used are selected from the group consisting of BF ₄ ^~ ₂ N ⁺ -C ₆ H ₄ -CO-C ₆ H _5, BF ₄ ^"+ ₆ N2-C H ₄ -N (CH ₃ ) ₂ and Cr ⁺ N ₂ -C ₆ H ₄ -n (CH ₃ ) ₂ The subject of the present invention is also a process according to the preceding definition, characterized in that the grafts mentioned in d) are selected from the group consisting of nitrophenyl, bromoalkylphenyl and chloralkylphenyl groups, and photo initiators of type I or II comprising benzil, benzyl diketal and its derivatives, thioxanthone and its derivatives, 2,2-azobisisobutyronitrile, benzophenone and its derivatives, thioester, dithioester and dithiocarbamate type photoiniferters, quinones, irgacides, benzoin ethers, peroxides, 4-nitrobiphenyl, acetophenone derivatives, aniline, hydroxyalkylphenone and its derivatives, Γα-aminoketone and its derivatives and acylphosphine oxide , whose formulas are given in Table 1 below.

Table 1 - Favorite PhotoStars

Names of

Types Formulas

photoinitiators

Nitrophenyl / -C ₆ H ₄ -NO ₂

-C ₆ H ₄ -CH (CH ₃ ) Br

Bromoalkyl phenyl

or -C ₆ H ₄ -CH ₂ CH ₂ Br

-C ₆ H ₄ -CH (CH ₃ ) C1

Chloroalkyl phenyl /

or -C ₆ H ₄ -CH ₂ CH ₂ C1

Benzile Type II

Names of

Types Formulas

photoinitiators

Benzyl diketal

Type I

and its derivatives in which:

R and R 'represent, independently of one another, a linear or branched alkyl chain of 1 to 6 carbon atoms.

Benzyl diketal: R = R '= H. o

Derivatives from

Type II

benzophenone in which:

R represents H, Cl, a linear or branched alkyl chain of 1 to 12 carbon atoms or a phenyl group.

For anthraquinonyl, the grafting from the corresponding diazonium salt is described by Li Q. et al. (The synthesis and characterization of controlled thin sub-monolayer films of 2-anthraquinone groups on graphite surfaces, New Journal of Chemistry, in press, DOI: 10.1039 / clnj20461k). Certain anthraquinone derivatives which can be used according to the present invention have been described by (Allen NS et al., Photochemistry and photoinitiator properties of 2- substituted anthraquinones: 2. Photopolymerization and flash photolysis, Polymer, 36 (1995) Pages 4665-4674).

The term "photoiniferters" (abbreviation for "Photo-Initiator-Transfer-Terminators") refers to compounds well known to those skilled in the art, capable of acting as radical polymerization initiators, transfer agents and reaction terminators. radical. The mechanism of action of these compounds is given in patent application EP0844256.

In a particularly advantageous embodiment of the invention, a diazonium salt of a thioxanthone derivative (TX) corresponding to the following formula is used:

in which X ^" represents an anion advantageously chosen from halides (Γ, Br ^" , Cl ^" ), nitrates, sulphates, phosphates such as hexafluorophosphate, perchlorates, carboxylates and haloboranes such as tetrafluoroborate,

R 1 represents a hydrogen atom or a chlorine atom,

P ₂ represents either a hydrogen atom or a chlorine atom or a straight or branched (Ci-C ₄ ) alkyl group, for example an ethyl or isopropyl group, or an -SH group, or a disilyleacetylene group, and

P 3 represents either a hydrogen atom or a straight or branched (Ci-C ₄ ) alkoxy group.

Advantageously, a diazonium salt of 2-isopropylthioxanthone (ITX), or 2-ethylthioxanthone, or 2-chloro-thioxanthone, or 1-chloro-4-propoxythioxanthone, or 2- mercapto-thioxanthone.

According to the invention, the diazonium salt of the TX derivative can be grafted to the surface. Certain thioxanthone derivatives, for example those described by Yagci et al (Yagci Y, Jockush S., NJ Turro, Photoinitiated polymerization: advances, challenges, and opportunities, Macromolecules 2010, 43, 6245-6260) do not require the presence of co-initiators. On the other hand, with certain thioxanthone derivatives, for example with 2-isopropylthioxanthone, a co-initiator is necessary. In this case, the co-initiator is chosen from hydrogen donors such as ethyl-p-dimethylaminobenzoate, Ν, Ν-dimethylaniline (DMA), triethylamine (TEA), 4-dimethylaminobenzoate (EDAB), p-hydroxymethyl-N, N-dimethylaniline, Ν, Ν-dimethyltoluidine, p-methyl-N, N-dihydroxyethylaniline, dimethylaminoethanol, N-methyldiethanolamine (NDEA), Ν, Ν-dimethylethanolamine (DMEA) , P-methyl-N, N-dihydroxyethylaniline, ethanolamine (EA), triethanolamine, THF, polyethylene glycol (PEG) and polyethyleneimine (PEI) and is in the reaction medium. The use of this thioxanthone derivative via the surface chemistry of diazonium salts makes it possible to prepare ultrathin polymer films by surface-initiated photopolymerization (surface-initiated photopolymerization, SIPP), even in the presence, in the reaction medium, of scavengers. radicals (radical scavenger) such as polyphenols, especially in the presence of flavonoids, such as quercetin.

In another embodiment, instead of grafting the thioxanthone derivative at the surface, another diazonium salt is synthesized, for example dimethylaminobenzene diazonium tetraborofluorate (BF ₄ ^{~ +} N ₂ -C ₆ H ₄ -N (CH ₃ )). ₂ ) which by reduction leads to the grafting of dimethylaminophenyl groups (-C6H ₄ -N (CH ₃ ) ₂ ) which act as hydrogen donors for the thioxanthone derivative employed in solution. The radicals generated on the surface make it possible to prime the polymerization surface even in the presence of quercetin or any other polyphenol or molecule known to be a radical scavenger.

These embodiments are based on the results described by Fouassier's team for polymerization in solution (F. Mauguière-Guyonnet, D. Burget, JP Fouassier Journal of Applied Polymer Science, Vol 92, 1 154-1164 (2004). F. Mauguière-Guyonnet, D. Burget, JP Fouassier Progress in Organic Coatings, (2006), Vol 57, 23-32) which has shown that the presence of 2-isopropylthioxanthone in solution allows the acrylates to be light cured. using a co-initiator, even in the presence of scavengers such as quercetin.

In an advantageous embodiment of the invention, the initiator is a diazonium salt of 2-isopropylthioxanthone grafted on the surface and the co-initiator is dimethylaniline in solution.

In another advantageous embodiment of the invention, the initiator is the

2-isopropylthioxanthone free in solution and the co-initiator (hydrogen donor) is the dimethylaminophenyl group grafted on the surface by (electro) chemical reduction of 4-dimethylaminobenzenediazonium chloride (Cl ^"+ N ₂ -C ₆ H ₄ -N (CH ₃ ) ₂ ). Of course other combinations are possible, such as, for example, a ia diazonium salt for the greff -based grafting and PEG (hydrogen donor) as a co-initiator in solution. It is also possible to envisage the surface grafting of another thioxanthone derivative by means of one of its diazonium salts with in solution any hydrogen donor as defined above, in particular DMA. in solution or conversely the grafting on the surface, by means of one of their diazonium salt, of any hydrogen donor as defined above, in particular DMA and ΓΙΤΧ or other free thioxanthone in solution . All these combinations are also part of the invention.

The diazonium salts employed in the context of the present invention may be prepared extemporaneously (Example 1) or in situ, in particular, from their amine precursors, and then reduced on a surface immersed directly in the medium used for their synthesis. The reduction of the diazonium salts generated in situ can, for example, be carried out without the addition of a solvent, by the addition of isoamyl nitrite and heating at 60 ° C. for 3 hours, according to the technique described by Li et al. (Functionalization of Single-Walled Carbon Nanotubes with Well-Defmed Polystyrene by "Click" Coupling, J. Am., Chem Soc., 2005, 127 (41): 14518-14524). It can also be made in an acidic medium according to the technique described by Baranton et al. (In situ generation of diazonium cations in organic electrolyte for electrochemical modification of electrode surface Electrochimica Acta 2008, 53: 6961-6967), or as described by Corgier et al. (Diazonium-protein adducts for graphite electrode microarrays modification: direct and addressed electrochemical immobilization, J. Am Chem Soc 2005, 127: 18328).

In addition to the grafting technique mentioned in Example 1, the extemporaneously prepared diazonium salts can be grafted onto the surface by electrochemical reduction according to one of the following reactions:

spontaneously

or spontaneously in the presence of surfactant:

or in the presence of acetonitrile:

Yet another object of the present invention is a process according to one of the preceding definitions, characterized in that the functional monomer is selected from the group consisting of vinyl monomers, acrylic monomers, methacrylic monomers and mixtures thereof .

By the term "mixtures thereof" is meant any mixture of at least two different monomers selected from the group of compounds mentioned above. Preferred functional monomers are given in Table 2 below.

Table 2 - Preferred Functional Monomers

Names of

monomers Formulas

preferred functional

Methacrylate

methyl

Names of

monomers Preferred functional formulas

CH ₂

Acid

methacrylic H ₃ cA ^H

0

methacrylamide

acrylamide

Acrylic acid

styrene

Boronic acid

4-ethylstyrene

Itaconic acid Names of

monomers Formulas

preferred functional

P-vinylbenzoic acid

4-vinylpyridine

1 -Vvinylimidazol

α-tert-butoxy-ω-vinylbenzyl-polyglycidol (PGL) 1 (OCHCH ₂ ) _n OC (CH ₃ ) ₃

CH ₂ OH with n varying from 1 to 35.

Yet another object of the present invention is a process according to any one of the preceding definitions, characterized in that the crosslinking monomer is a one selected from the group comprising divinylbenzene, ethylene glycol dimethacrylate, tetramethylene glycol dimethacrylate, polyethylene glycol) dimethacrylate, 2,2 bis (hydroxymethyl) butanol trimethacrylate, Ν, Ν'-dimethyl acrylamide, Ν, Ν'-ethylenebisacrylamide, 1,3 diisopropenyl benzene, 2,6-bisacryloylamidopyridine, Ν, Ν ' methylenediacrylamide, 1,4-phenylenediarnylamine, 2,6-bisacryloylamidopyridine, N, O-bis (acryloyl) phenylalaninol, 1,4-di (acryloyl) piperazine and pentaerythritol tetraacrylate.

The preferred crosslinking monomers are given in Table 3 below. Table 3 - Preferred Crosslinking Monomer

The present invention also relates to a process according to any one of the preceding definitions, wherein the carrier is selected from the group consisting of porous and non-porous, planar and non-planar, inorganic and organic carriers. In another embodiment of the method of the invention, the support is in the form of plates or particles.

In yet another embodiment of the process of the invention, the plates are selected from the group consisting of gold, silicon, indium-tin oxide, iron, palladium, copper, nickel, zinc, doped and undoped diamond, vitreous carbon, carbon felt, carbon fiber, highly oriented pyrolytic carbon, mono- and multi-carbon nanotubes walls and gallium arsenide.

In a particular embodiment of the process of the invention, the particles are chosen from the group comprising carbon black, mono- and multi-wall carbon nanotubes, graphene and undoped or doped diamond nanoparticles. nitrogen or boron, metal nanoparticles, in particular Au, Pt, Ru, Co, Al, Ni, Zn, Cu, bimetallic nanoparticles, in particular Pt-Ru, nanoparticles of oxides, in particular of silicon, of aluminum, of iron and magnesium, quantum dots based on cadmium sulphide or InAs / GaAs.

In an advantageous embodiment of the method of the invention, the target molecule is chosen from the group comprising ions, antigens, antibodies, amino acids, peptides, proteins, nucleotides, DNA bases, drugs, pesticides, food products, heavy metals, pollutants, volatile and non-volatile organic compounds, and derivatives thereof.

By the term "derivatives thereof" is meant derivatives of all the compounds mentioned above.

The present invention further relates to a molecular fingerprint polymer having an ultrafine thickness confined to a support obtainable by the method according to any one of the preceding definitions.

The present invention also relates to the use of at least one diazonium salt carrying photopolymerization initiator functional groups, as a photopolymerization initiator in a process for the preparation of molecular imprinted polymers, in ultrathin layers on a support, which are useful for the recognition of target molecules, said method comprising a step of photopolymerizing a composition comprising at least one crosslinking monomer, at least one functional monomer and at least one target molecule, the photopolymerization being confined to the surface of the support.

The present invention also relates to chips comprising one or more molecular imprinted polymers having an ultra-thin thickness confined to a support, characterized in that the polymer (s) is (are) capable (s) to be obtained (s) by the method according to one of the preceding definitions. These chips can also understand an array of electrodes on each of which a different potential is sequentially applied in ascending or descending order.

The subject of the present invention is also a kit for analyzing at least one target molecule comprising at least one molecular-fingerprint polymer obtainable by the method according to any one of Claims 1 to 9, said polymer being suitable interacting with the at least one target molecule, and at least one marker, said marker being able to interact with all or part of the recognition sites of the at least one target molecule.

The present invention also relates to the use of a molecular fingerprint polymer obtainable by the process according to any one of claims 1 to 9 for the recognition and / or extraction of target molecules.

Figures 1 to 21 and the following Examples 1 to 5 serve to illustrate the invention.

FIG. 1 represents the cyclic voltammetric (or voltammetry) diagrams of 4-benzoylphenylbenzene diazonium tetrafluoroborate in 0.1 M TACN + of Bu ₄ BF ₄ on an Au plate (FIG. 1a) or ITO (FIG. ECS v = 0.2 Vs ^"1 ), obtained in the first scan cycle (curve a), and in the second scan cycle (curve b).

Figures 2 represent the chronoamperometry chart on Au plates (Figure 2a) and ITO (Figure 2b) in ACN + 0.1M Bu ₄ BF ₄ + 2mM DZ. The reference is the saturated calomel electrode (SCE).

Figure 3 shows the Polarization Modified Polarization Absorption Reflection (PM-IRRAS) spectrum of a gold surface grafted with a 4-benzoylphenyl or BP (Au-BP) group and the IR spectrum of a KBr pellet. diazonium salt (DZ (BP)).

Figure 4 shows broad-scan XPS spectra; FIG. 4a scanning of the BP-grafted glassy carbon surface (CV-BP) and FIG. 4b scanning of the BP-grafted gold surface (Au-BP).

FIG. 5 represents the Cls spectra of a vitreous carbon (FIG. 5a) or gold (FIG. 5b) surface, grafted with BP (CV-BP and Au-BP).

Figure 6 shows the general XPS spectrum of the Au, BP and Au samples.

MIP.

Figure 7 shows the XPS Cls spectrum of Au-BP and Au-MIP samples.

Figure 8 shows the general XPS spectrum of a naked ITO and ITO-MIP sample (Figure 8a) and the XPS Cls spectrum of an ITO-MIP sample (Figure 8b).

Figure 9 shows the PM-IRRAS spectra of Au-BP and Au-MIP. Figure 10 shows AFM (Atomic Force Microscopy) images for Au, Au-BP and Au-BP-MIP.

Figure 11 schematizes the steps of formation of MIPs with dopamine as a model molecule and the step of analysis by cyclic voltammetry.

Figure 12a shows the electrochemical response of dopamine immobilized on the Au-MIP electrodes compared to non-printed Au-NIP electrodes. Figure 12b is a histogram showing the influence of the model (T) / functional monomer (MF) / crosslinking monomer (MR) ratio on the anodic response.

Figure 13 shows the electrochemical responses obtained during the study of the specificity of the ITO-MIPs system.

Figure 14 shows the electrochemical responses (cyclic voltammetry) of the ΜΙΡ-dopamine (MIP-DOP), non-printed polymer (NIP) and MIP-L-3,4-dihydroxyphenylalanine (MIP-L-dopa) systems.

Figure 15 shows the electrochemical response of dopamine on the gold plates and 15b the semi-logarithmic linearization of this response according to the logarithmic dopamine concentration of 10 ^-4 to 10 ^-7 M.

FIG. 16 shows the electrochemical response obtained for the ΓΓΌ-MIP system with a T / MF / MR formulation = 2/8/30 and 16b the semi-logarithmic linearization of this response. Figures 16c and 16d show the same variables for a T / MF / MR formulation = 2/8/25.

Figure 17 shows the Square Wave Stripping Voltammetry (SWSV) voltammetry diagram for the detection of dopamine by ultrathin layers of MIPs.

FIG. 18 represents the chronoamperometry diagram of the gold plates according to example 5. Diazonium salt: 4-dimethylaminobenzenediazonium chloride (Cl N ₂ -C ₆ H ₄ --N (CH ₃ ) ₂ ) 0.1 M, solvent: acetonitrile, bottom salt: (C ₄ H ₉ ) ₄ NBF ₄ .

Figure 19 shows the XPS spectrum before electrografting (Au-nu), after electrografting of DMA (Au-DMA) and Au-MIP under the conditions of Example 5.

FIG. 20 represents the Cls A spectra after electrografting of DMA (Au-DMA) and B) after grafting of a polymethacrylate-type MIP under the conditions of Example 5.

FIG. 21 represents the Square Wave Stripping Voltammetry (SWSV) voltammetry diagram for the detection of quercetin by ultrathin layers of MIPs according to the invention compared with polymer layers. not printed (Au-NIP) under the conditions of Example 5. Conditions: Pulse amplitude = 50 mV, speed: 100 mV / s. solvent: PBS, pH 7.4.

EXAMPLE 1 EXTEMPORANEOUS SYNTHESIS OF DIAZONIUM SALT ⁺ N ₂ -

The tests were carried out with salts having as counterion X ^" = BF4 ^" .

The starting diazonium salt ^(BF 4 N 2 -C ₆ H ₄ -CO-C ₆ H ₅₎ was synthesized from 4-amino-benzophenone according to the method described by Finch et al. (Attachment of Polymers to Organic Moieties Covalently Bonded to Iran Surfaces, Chem Mater 2002, 14 (11): 4576-4585).

This salt (DZ) is suitable for surface photopolymerization since free benzophenone initiates photopolymerization in solution, as described by Dyer and Kato et al. (Dyer, D. J. Adv., Polym Sci 2006, 47: 197, Kato, E. et al, Polym Sci, 2003, 28: 209-259). This salt is then reduced electrochemically on the carbon plates in order to determine the optimal grafting conditions which were then transposed to the substrates of gold, silicon, 316L steel, ITO, etc. Plates modified with the 4-benzoyl phenyl group (BP) served as macro -photo surface-induced polymerization initiator (surface-initiated photopolymerization, SIPP).

In a flask placed in an ice bath, 17 mmol of 4-aminobenzophenone dissolved in 11.5 ml of tetrafluoroboric acid (HBF4, 48%) was added. The mixture was kept stirring. The temperature having reached -3 ° C., 34 mmol of sodium nitrite (NaN0 ₂ dissolved in a minimum of water) were added dropwise. An orange-brown precipitate appears. Stirring is maintained for 30 min at -3 ° C and then the solution was placed in the refrigerator overnight to precipitate the diazonium salt. After filtration of the sintered glass solution, the product was washed with pure water and cold diethyl ether and dried under vacuum. The powder obtained is brown in color; it was characterized by 1 H NMR (DMSO): δ ppm: 8.87 and 8.25 (d, 4H), aromatic group of the diazonium salt, 7.5 to 7.8 (m, 5 aromatic H). EXAMPLE 2 Preparation and Crafting of the Surfaces to be Polymerized

1. Electrochemical grafting of the aryl groups of DZ (+ ₂ -C ₆ H ₄ -CO-C ₆ H ₅ ) on the surface The gold plates and ITO (Aldrich® 1 cm × 2 cm) obtained in Example 1 were washed with distilled water and ethanol and then cleaned with UV / O3 (3 cycles of 10 min). This step makes it possible to eliminate the superficial organic matter.

The reactions were carried out on glassy carbon electrodes and then transposed on a gold plate and ITO (FIGS. 1a and 1b). Two irreversible, broad, monoelectronic waves are observed at Ep _c = -0.366 V / SCE and -0.180 V / SCE which respectively correspond to the concerted reduction of the diazonium salt to aryl radical which reacts respectively with the gold surface and ITO. During successive scans, the height of this wave becomes very low as is usual with the diazonium salts, the surface being progressively blocked by the grafting of aryl groups.

The photoinitiator derived from the diazonium salt was then grafted by chronoamperometry at -0.700 V / SCE (or at -0.400 V / SCE for ITO). At the beginning, a large current is observed for a few seconds, which tends very rapidly towards zero (Figures 2a and 2b). This drop explains the cessation of the reduction process due to an organic film deposited on the surface and which reflects a blockage of the electrode, resulting in a weak current, a sign of passivation. Chromoamperometry will be used for systematic grafting studies of MIPs and their use for analyte detection.

After grafting, the plates are carefully rinsed in acetonitrile (ACN) under ultrasound to remove molecules that are only physically adsorbed on the surface. The electrodes were then stored under an inert atmosphere to protect them from external contamination.

2. Characterization of the grafted surface by IR spectroscopy

The IR spectrum obtained is given in FIG. 3. The most striking difference between the diazonium spectrum and the spectrum of the grafted organic layer is the absence of the band at 2228 cm ^-1 indicating that the diazonium has been well reduced and is not only adsorbed on the surface, PM-IRRAS analysis of 4-benzoylphenyl-grafted plaques clearly shows the appearance of the aromatic group signature with 1600-cycle vibration cm ^-1 and bands in the 3060 cm ^{-1 area} characteristically CH elongation of aromatic groups. The appearance of a peak located at 1650 cm ^-1 characteristic of the vibration mode of v (C = 0) of the ketone function and the band located at 1270 cm ^-1 corresponding to the vibration of v (CC) conjugate confirms the presence of the initiator (-C ₆ F L -CO-C ₆ H ₆ ) on the surface.

3. Characterization of the grafted surface by XPS

The results are shown in Figures 4a and 4b. The spectrum contains the characteristic lines Au4f, Cls, Nls and Ois, centered at 84, 285, 400 and 532 eV, respectively. As can be seen previously, the gold spectrum has very attenuated characteristic lines, but an intense continuous background which indicates a good coverage of the plates by a layer of photopolymerization initiating aryl groups whose thickness is 1 μm. order of 3 ± 1 nm in accordance with the literature. With regard to carbon, the high intensity ratio of 01 s / Cl s is a good indication of the success of electrografting on vitreous carbon.

The corresponding Cls spectra are given in Figures 5a and 5b. The gold plate has a high resolution Cls peak of the initiator (Figure 5b) which is reconstructed with four components. The assignment of these components to chemical functionalities according to the binding energy is as follows:

a component centered at 285.0 eV corresponding to the carbon of type C-C / C-H, characteristic of the carbons of diazonium salts;

- a component centered at 286.5 which groups the C-0 bond (due to the aryl layer surface oxidation) and CN due to the formation of azo bridges between the aryl groups (aryl-N = N-aryl) . Indeed, it is well known that the diazonium salts very generally lead to grafted oligomers and not to monomolecular monolayers;

a component centered at 287.9 corresponding to the carbon atom of the ketone function (C = O) of the initiator;

a high energy link component centered at 291.9 ± 0.2. The latter, the satellite line of the main peak centered at 285 eV and called "skake up π → π *", is specific to aromatic compounds. In the case of the CV-BP macroinitiator, the line C1s is decomposed into 5 contributions centered at 284.8; 285.3; 286.7; 287.9 and 291.5 eV respectively attributed to the C- links C / CH of the aromatic carbon atoms, carbons of Csp3, CO, C = O and "shakeup" type.

It can be concluded from these two spectroscopic techniques that the 4-benzoylphenyl (BP) group was grafted onto the gold and CV plates.

The contact angle measurements of the drops of water gave a value of 72 °. Photoinitiator aryl groups from other diazonium salts, postmodification polymerization initiator photo groups, as well as aryl groups which do not function to photoinitiate the polymerization, but are part of the general process of photopolymerization when a free photoinitiator in solution is used, have also been grafted. The following characteristics have been obtained.

For the diazonium salts of general formula (II) where X = BF ₄ ^" : For R = - (CH ₂ ) n-CH ₃ XPS has obtained an intense peak Cls centered at 285 eV and a strong attenuation of the signal The presence of nitrogen reflects the presence of azo bridges Infrared spectroscopy shows the disappearance of the N ₂ signature (expected at around 2200 cm ^-1 ), demonstrating that covalent grafting location. A strong signal detected around 2900 cm ^-1 is attributed to the vibrations of the CH ₂ groups.The drops of water make an angle of 92 ° with the surface, which indicates a hydrophobic character that the long alkyl chain gives to the surface.

For R = -CH (CH ₃ ) Br, a peak Br3d centered at 70eV due to the C-Br bond was obtained by XPS, a Cls line at 285 eV with a shoulder at approximately 286 eV (C-Br bond) as well as a strong attenuation of the signal of the gold. The presence of nitrogen reflects the presence of azo bridges. Infrared spectroscopy shows the disappearance of the N ₂ signature (expected at about 2200 cm ^-1 ), demonstrating that the covalent grafting took place, a strong signal around 2900 cm ^-1 due to the vibrations of the CH groups. ₂ . The drops of water make an angle of 57 ° with the surface.

The -CH (CH ₃ ) Br group was then modified with sodium Ν, Ν-diethyldithiocarbamate to obtain on the surface the C ₆ H ₄ -CH (CH ₃ ) -SC (= S) -N (C) ₂ H ₅ ) ₂ . The presence of sulfur by XPS (S2p at about 162-163 eV) can be noted. Infrared spectroscopy shows bands assigned to SC = S and N-SCS around 1005 and 1560 cm ^{-1, respectively} . The functional group -CH (CH ₃ ) Br can be replaced by the functional group -CH ₂ Br or -CH ₂ CH ₂ Br and proceed in the same way by using sodium N, Ndiethyldithiocarbamate or a derivative product to obtain at the surface a photoinitiator for the controlled growth (in thickness) of layers of MIPs.

For R = -N0 ₂ a peak NIs was obtained by XPS centered at about 406 eV from the nitro group and another at 400 eV due to the azo bridges. We notice a strong attenuation of the signal of the gold. Infrared spectroscopy shows the disappearance of the N ₂ signature (expected at about 2200 cm -1), demonstrating that covalent grafting has taken place. The symmetrical and anti-symmetric bands of NO ₂ appear at about 1350 and 1520 cm ^{-1, respectively} .

The group -NO ₂ was then electrochemically reduced to -NH ₂ (a single peak NI s in

XPS centered at 400 eV). The amino group was attacked by CH3-CH (C≡N) ₂ (malonodinitrile) (Steenackers et al., Morphology control of structured polymer brushes, Small 2007, 3 (10): 1764-1773) to obtain the group -N = NC (C≡N) ₂ CH3 bonded to the phenyl from the starting diazonium salt ^(BF 4 N 2 ⁺ C ₆ H4-N0 _2). The -C ₆ H 4 -N = NC (C≡N) ₂ CH 3 group bonded to the substrate is equivalent to ΓΑΙΒΝ and makes it possible to photoinitiate the surface polymerization.

It is also possible to limit oneself to obtaining -C _Ô FL-NLL and to carry out photopolymerization leading to MIP by adapting the method of Steenackers et al. (Structured and Gradient Polymer Brushes from Biphenylthiol Self-Assembled Monolayers by Self-Initiated Photografting and Photopolymerization (SIPGP), Langmuir, 2009, 25: 2225).

More simply, it has been verified that the grafting of -C ₆ H ₄ -NO ₂ aryl groups makes it possible to directly initiate surface photopolymerization.

For R = -SH, this diazonium salt was used to immobilize on the surface, gold nanoparticles (AuNP) as described by Porter et al. (Attachment of Gold Nanoparticles to Glassy Carbon Electrodes via a Mercaptobenzene Film J. Am Chem Soc 2001, 123: 5829-5830). A new substrate-aryl-AuNP interface is thus created on which it is possible to graft a new layer of photo-initiator aryl groups described above to initiate the formation of MIPs. This approach to the preparation of MIPs in the presence of AuNPs is similar to that described by Matsui et al. (SPR Sensor Chip for Detection of Small Molecules Using Molecularly Imprinted Polymer with Embedded Gold Nanoparticles, Anal Chem 2005, 77: 4282-4285) to obtain MIP layers useful in SPR detection of small molecules. For electrochemical detection, Xu et al. (A novel molecularly imprinted sensor for selectively probing imipramine created on ITO electrodes modified by nanoparticles., Talanta, 2009, 78:26) immobilized gold NPs on mercaptosilanes prior to photopolymerization of a prepolymer containing the model molecule. However, we can note in this work the absence of covalent bond between the MIPs and the surface, so unlike our method which has precisely the advantage of robustness of the anchoring layers of MIPs to surfaces. EXAMPLE 3: GROWTH OF IMPRINTED POLYMERS

MOLECULARS FROM ARY GRAFTS AND CHARACTERIZATION OF SURFACES CARRYING SAID POLYMERS

MIPs were grown on the surface by photopolymerization from the aryl grafts obtained in Example 2 and used as a photoinitiator. 1. Light curing

In different cells, a solution containing the template molecule ("template"), a functional monomer (eg methacrylic acid or 4-vinylpyridine (VP)), a crosslinking monomer such as dimethacrylate has been introduced. ethylene glycol or EGDMA, methanol as a pore-forming solvent and Ν, Ν-dimethylaniline (DMA) as a hydrogen donor. The solution is purged by means of a stream of nitrogen (N ₂ ) to expel the oxygen for 10 to 15 min. Gold plates (or ITO, Si (III), 316L stainless steel, vitreous carbon, micro and nanoparticles modifiable by diazonium salts or amino precursors, etc.) grafted by the initiator are covered by this solution, under an atmosphere of nitrogen, then placed inside a reactor (spectro-LINKER ™, 17.6 mW / cm ² at 365 nm), then exposed to the irradiation source for 1000 seconds at room temperature. After irradiation, the plates are recovered and then immersed in a Erlenmeyer flask containing methanol to extract unreacted monomer. The plates are then dried and stored under nitrogen for analysis.

The non-reactive reference plates were prepared in the same way, but this time in the absence of the target molecule, which leads to the non-imprinted polymer (NIP).

The model molecules were extracted using the "Soxhlet" to obtain MIPs. The body of the Soxhlet containing the plate is fixed on a solvent tank (acetone in which the molecule is soluble) and is surmounted by a coolant. The solvent is vaporized and then condensed and remains in contact with the plate. The solution is drawn off periodically by priming a siphon. The solution of the balloon is gradually enriched in solute and the solid is always put in contact with freshly distilled solvent. The extraction time is 24 hours.

The model molecule can also be extracted by cyclic voltammetry, which is an alternative method to solvent extraction. In this case, after about 30 redox cycles between -200 and 1000 mV, dopamine oxidation peak is no longer observed, which indicates that it has been completely extracted from the polymer. This extraction naturally leaves the molecular fingerprints of dopamine and the film can then be used to detect it. With a scanning speed of 100 mV / s, about thirty cycles require only 10-12 min to extract the model molecule, which represents a considerable time saving compared to the "Soxhlet" method (24h).

2. Characterization of molecular imprinted polymer coated surfaces by XPS

Wide-scan XPS spectra of naked and grafted gold plates are shown in Figure 6. The main lines Au4f, Cls and Ois are centered at, respectively, 84, 285 and 532 eV. It is also possible to see the Nls line of nitrogen at 400 eV (this however requires several tens of accumulations of spectra) which testifies to the incorporation of 4-vinylpyridine into the crosslinked copolymer. The modification of gold by electrografting is very clear: there is a significant increase in the intensity of the lines Cls and Ois compared to those of the peaks of gold. For the Au-MIP sample, in situ grafting of the polymer completely obscures the gold substrate. However, the atomic% of the surface nitrogen is low (generally around 1%) because in the synthesis medium the 4-vinylpyridine generally represents only 20% at most of the "model molecule" mixture. MF / MR. In addition, the N / C ratio in this functional monomer (4-VP) is only 1 / 7th, which ultimately leads to a very low proportion of nitrogen in the MIP film.

The high-resolution Cls spectrum of Au-MIP (Figure 7) has components centered at 289, 286.5 and 285 eV which respectively characterize the carbon atoms 0-C = 0 and C-0 and CC: the carbon signal is in agreement with the chemical structure of MIP which is here dominated by the EGDMA motifs, with in particular a line centered at 289 eV corresponding to the ester groups. As has been shown previously, the line Nls is very weakly intense compared to the lines Cls and Ois. For Au-BP: the C = 0 component (288 eV) of the Cls line accounts for the electrografting of the aryl groups derived from the diazonium salt. The 291-292 eV satellite is a proof that the electrografted species is aromatic.

Figures 8a and 8b respectively represent the general XPS spectrum of a naked ITO and ITO-MIP sample (ITO-BP-MIP), and the XPS Cls spectrum of a ΙΤΌ-MIP sample. It can be observed a screening of TITO signals and obtaining intense lines Cls and Ois reflecting a transition from an inorganic substrate to an organic coating. The polymer is characterized by a Cls line characteristic of a polymethacrylate as observed on a gold substrate.

This approach has also been extended to Si (111), 316L stainless steel and glassy carbon substrates. Assays were also performed on carbon microparticles and on carbon nanotubes modified by the aryl groups BP grafted by in-situ generation of diazonium salts from aminobenzophenone in the presence of isoamyl nitrite. The set of results indicates attenuation of the substrate signals and the appearance of Cls and Ois peaks characteristic of the EGDMA ester function.

3. Characterization of surfaces coated with molecular imprinted polymers by IR

Figure 9 shows the Au-BP and Au-MIP spectra. The most remarkable difference between the two spectra is the appearance, after the polymerization step, of a very intense band at 1735 cm ^-1, which signifies the presence of the polymer by its carbonyl group. 1261 and 1167 cm ^-1, which corresponds respectively to v (C = 0) and v (COC), reflects the successful growth of poly (EGDMA) at the surface.

The small peak located at 1632 cm ^-1 characteristic of the vibration v (C = C) is weak compared to that of the v (C = 0) of grafted EGDMA, which shows that the EGDMA has polymerized via these two doubles C = C bonds.

The distinction of the characteristic peaks of the MAA units from those of the EGDMA units is difficult for two reasons: on the one hand the copolymer was prepared with a molar ratio EGDMA / MAA is 30/8, hence the dominance of the EGDMA motifs at the area.

On the other hand, the spectral signatures of the MAA and EGDMA patterns are very similar.

In contrast, the small broad band at about 3300 cm- ¹ reflects the presence of the MAA functional monomer in the MIP. 4. Characterization of molecular imprinted polymer coated surfaces by AFM

Figure 10 shows the images obtained by AFM on plaques of Au, Au-BP, Au-

MIP.

The image shows that the morphology of the gold surface is significantly modified by the grafting of the initiator and the consequent polymer coating. The maximum height increases in the Au <Au-BP <Au-MIP direction. It may be noted that the Au-MIP surface has a spongy appearance that could allow better diffusion of the analytes to the metal-polymer interface where they will be detected electrochemically. EXAMPLE 4: ELECTROCHEMICAL DETECTION OF AMINE DOP AND

STUDY OF THE SPECIFICITY OF THIS DETECTION

1. Electrochemical detection of the model molecule (dopamine)

Cyclic voltammetry makes it possible to study the detection of the model molecule by MIPs and to establish whether they are specific and selective. For this purpose, the electrodes obtained in Example 3 are immersed in the solution of dopamine (or L-dopa, an interferon) for 20 minutes, then rinsed with a PBS solution (phosphate buffer saline solution, pH 7.4). to eliminate all physisorbed molecules. Dopamine trapped by artificial receptors is characterized by an irreversible wave when a potential is applied between -0.3 and 0.9 v in a PBS solution (0.1 M, pH = 7.4) as shown in Figure 11.

2. Study of specificity on Au and ITO

Figure 12a shows the electrochemical response of dopamine immobilized on the Au-MIP electrodes compared to non-printed Au-NIP electrodes. The difference is very significant and proves the specificity of these electrodes vis-à-vis the target molecule.

Modification of the ratio of model molecule or template (T), functional monomer (MF); Crosslinking monomer (MR): T / MR / MF acts on the electrochemical response; therefore, the more artificial receptor sites, the better the anodic response (Figure 12b).

The same experiment was transposed on ITO (inexpensive surface compared to gold plates). Figure 13 confirms that the concept works on various surfaces and clearly demonstrates the specificity of ITO-MIP. The electrochemical response of NIP (unprinted surface) is almost the same as that of ΓΙΤΟ nu.

3. Study of selectivity on Au

The selectivity of Au-MIP with respect to dopamine was studied by comparing the oxidation waves of dopamine and L-dopa, after incubation for 20 minutes, in the respective catechol solutions. L-dopa was chosen because of its resemblance (chemical composition and form) to dopamine.

Figure 14 shows the electrochemical response (cyclic voltammetry) of the "dopamine-templated" film after incubation in a solution of 0.1 M PBS (pH 7.4) of L-dopa or dopamine. A very low oxidation current was observed for L-dopa, as in the case of an Au-NIP surface.

- No recognition of L-dopa by the cavities of dopamine,

- Electrochemical response: anodic current (L-dopa) = anode current of PIN. These results highlight the selectivity of MIPs. 4. Detection threshold

The detection threshold was first determined on the Au plates, then on the ITO plates.

Figure 15 shows a detection limit better than 10 ^-7 M on gold plates and also a linear variation of the current with the logarithmic concentration of dopamine from 10 ^-4 to 10 ^-7 M.

The detection threshold and the effect of the formulation were also studied on ITO. Figure 16 shows the (logarithmic) relationship between the intensity of the dopamine oxidation current and its solution concentration, i.e., in the medium in which the grafted MIP plates are incubated. This relationship is linear over a wide range of concentrations, between 10 ^{~ 9} and 10 ^-4 M. The formulation T / MF / MR = 2/8/30 gives a better detection at low concentration of the analyte compared to the formulation 2/8/25 The latter gives a better detection at high concentration of the analyte (dopamine) The limit of detection of analytes by cyclic voltammetry being of the order of 10 ^-8 mol or better; this threshold was lowered to 10 ^-9 mol or better using voltammetry of square wave dissolution (SWSV) that is deemed to be more sensitive to analytical aspects of Γ electrochemistry (Gam-Derouich et al. Aryl diazonium knows Surface chemistry and ATRP for the preparation of molecularly imprinted polymer grafts on gold substrates. Surf. Analog interface. 2010, 42 (6-7): 1050-1056). Figure 17 shows that starting from a concentration of dopamine equal to 1 nanomole, the SWSV still allows to detect an electrochemical signal to the oxidation potential of dopamine.

EXAMPLE 5 PREPARATION OF THIN LAYERS OF MIP ON GOLD VIA DMA GRAFT IN SURFACE AND ITX IN SOLUTION - APPLICATION TO THE DETECTION OF QUERCETIN

1. Preparation of thin layers

The grafting of 4-dimethylaminobenzenediazonium chloride (Cl ^"+ N ₂ -C ₆ H ₄ -N (CH ₃ ) ₂ ) to gold is carried out according to the following scheme in 0.1 M acetonitrile in the presence of: C ₄ H ₉ ) ₄ NBF ₄ as background salt.

DMA Au -DMA

2. Characterization of thin layers

The grafting is followed by chronoampérommétrie the gold plates (Figure 18).

The grafted plaque is placed in free isopropyl thioxanthone (ITX) in solution (0.088 g ITX in 5 ml of a methanol / chloroform mixture (70/30 v / v), ie a molar concentration of ITX of 0.083 mol / L). and the photopolymerization is induced according to Example 3 (UV irradiation at 365 nm). The surface-grafted DMA groups provide hydrogen radicals under the effect of light and thus the polymerization is initiated.

The XPS surface analysis spectra of the gold plates before and after DMA grafting followed by MIP grafting by photopolymerization are given in FIG. 19. A C / N ratio = 7.5 close to 8 theoretical value is obtained. Grafted DMA and a C / O ratio close to 2.5 for the MIP layer, which proves that the product obtained corresponds to the expected product. The structure is confirmed by the Cls line of the graft surface of DMA (Figure 20A) and that of the grafted surface of the MIP (Figure 20B). 3. Electrochemical detection of quercetin

The plates prepared previously are immersed in a solution containing quercetin (template T) at 0.132 mol L (0.2 g / 5 ml of a methanol / chloroform mixture (70/30 v / v)), an acid copolymer methacrylic acid (MAA) as a functional monomer (MF) at a concentration of 0.532 mol / L (0.23 g / 5 ml of a methanol / chloroform mixture (70/30 v / v)) and ethylene glycol dimethacrylate as the crosslinking monomer ( MR) at a molar concentration of 2.01 mol / L (2 g / 5 ml of a methanol / chloroform mixture (70/30 v / v)). The molar ratio T / MF / MR is therefore about 1/4/15. The detection is carried out under the following conditions: Pulse amplitude = 50 mV, speed: 100 mV / s. solvent: PBS, pH 7.4.

Figure 21 shows that even starting from a quercetin concentration equal to 10 nanomoles, the SWSV can detect an electrochemical signal at the oxidation potential of quercetin. On the other hand, the non-printed polymer (Au-NIP) does not recognize quercetin.

Claims

A process for preparing a molecular fingerprint polymer confined to the surface of a support, said method comprising the steps of:

a) a step of preparing said grafting surface on said surface, either of polymerization initiator functional groups or capable of performing this role after chemical transformation, or of co-initiating functional groups, in particular hydrogen donors, grafting of said groups; functional by chemical or electrochemical reduction of diazonium salts by which said functional groups were initially carried,

b) if necessary a step of transforming said graft groups on said surface to make them able to initiate a photopolymerization,

c) a step of bringing the resulting surface into contact with a polymerization composition comprising at least one functional monomer, at least one crosslinking monomer, at least one model molecule, optionally a co-initiator, in particular a hydrogen donor in a medium of polymerization,

e) a step of obtaining an ultra-thin layer molecular imprinted polymer having a thickness less than or equal to 1 μιη, preferably less than or equal to

100 nm, more preferably less than or equal to 5 nm,

f) a step of removing the at least one model molecule from the matrix of the molecular imprinted polymer,

g) optionally repeating steps a) to d) using a composition comprising at least one identical or different functional monomer, at least one identical or different crosslinking monomer and at least one identical or different model molecule.

2. Process according to claim 1, characterized in that the grafts mentioned in d) are chosen from the group comprising nitrophenyl, bromoalkyl phenyl and chloralkyl phenyl groups, and type I or II photoinitiators comprising benzyl, benzyl diketal and its derivatives, thioxanthone and its derivatives, 2,2-azobisisobutyronitrile, benzophenone and its derivatives, thioester type photoiniferters, dithioester and dithiocarbamate, quinones, irgacides, benzoin ethers, peroxides, 4-nitrobiphenyl, acetophenone derivatives, aniline, hydroxyalkylphenone and its derivatives, omega-aminoketone and its derivatives and the 'acylphosphine.

3. Method according to claim 1 characterized in that in step b) is implemented a co-initiator selected from hydrogen donors such as ethyl-p-dimethylaminobenzoate, Ν, Ν-dimethylaniline (DMA) triethylamine (TEA), 4-dimethylaminobenzoate (EDAB), p-hydroxymethyl-N, N-dimethylaniline, N, N-dimethyltoluidine, p-methyl-N, N-dihydroxyethylaniline, dimethylaminoethanol, N- methyldiethanamine (NDEA), Ν, Ν-dimethylethanolamine (DMEA), P-methyl-N, N-dihydroxyethylaniline, ethanolamine (EA), triethanolamine, THF, polyethylene glycol (PEG) and polyethyleneimine (PEI).

4. Process according to any one of the preceding claims, characterized in that the initiator is a diazonium salt of a thioxanthone derivative corresponding to the following formula:

in which

X ^" represents an anion advantageously chosen from halides (T, Br ^" , Cl ^" ), nitrates, sulphates, phosphates such as hexafluorophosphate, perchlorates, carboxylates and haloboranes such as tetrafluoroborate,

R 1 represents a hydrogen atom or a chlorine atom,

P ₂ represents either a hydrogen atom or a chlorine atom, or a straight or branched (Ci-C 4) alkyl group such as for example an ethyl or isopropyl group, or a -SH group, or a disilyleacetylene group and

P 3 represents either a hydrogen atom or a straight or branched (Ci-C 4) alkoxy group.

5. Process according to claim 4, characterized in that the initiator is a diazonium salt of 2-isopropylthioxanthone, or 2-isopropylthioxanthone, 2-ethylthioxanthone, or 2-chloro-thioxanthone, or 1-chloro-thioxanthone. 4-propoxy-thioxanthone, or = 2-mercapto-thioxanthone.

6. Method according to claim 4 characterized in that the initiator is a diazonium salt of 2-isopropylthioxanthone grafted on the surface and the co-initiator is dimethylaniline in solution.

7. Method according to claim 1 characterized in that the initiator is the

2-isopropylthioxanthone free in solution and the co-initiator is diméthyaminophényle group grafted reduction surface (electro) chemical chloride diméthylaminobenzènediazonium 4- (N2 Cl-C ₆ H 4 N (CH ₃₎ 2).

8. Process according to any one of the preceding claims, characterized in that the functional monomer is a member selected from the group consisting of vinyl monomers, acrylic monomers and methacrylic monomers and mixtures thereof.

9. Process according to any one of the preceding claims, characterized in that the crosslinking monomer is chosen from the group comprising divinylbenzene, ethylene glycol dimethacrylate and tetramethylene glycol.

dimethacrylate, poly (ethylene glycol) dimethacrylate, 2,2-bis (hydroxymethyl) butanol trimethacrylate, Ν, Ν'-dimethyl acrylamide,

Ν, Ν'-ethylenebisacrylamide, 1,3-diisopropenylbenzene, 2,6-bisacryloylamidopyridine, Ν, Ν'-methylenediacrylamide, N, N'-1,4-phenylenediarcylamine,

2,6-bisacryloylamidopyridine, N, O-bis (acryloyl) phenylalaninol, the

1,4-di (acryloyl) piperazine and pentaerythritol tetraacrylate.

The method of any one of the preceding claims, wherein the support is selected from the group consisting of porous and non-porous, planar and non-planar, inorganic and organic carriers.

11. The method of claim 9, characterized in that the carrier is in the form of plates or particles.

12. The method of claim 10, characterized in that the plates are selected from the group consisting of gold plates, silicon, indium tin oxide, iron, palladium, copper, nickel, zinc, doped and undoped diamond, vitreous carbon, carbon felt, carbon fiber, highly oriented pyrolytic carbon, mono- and multiwall carbon nanotubes and gallium arsenide.

13. Process according to claim 10, characterized in that the particles are chosen from the group comprising carbon black, mono- and multi-wall carbon nanotubes, graphene and undoped or doped diamond nanoparticles. nitrogen or boron, metal nanoparticles, in particular Au, Pt, Ru, Co, Al, Ni, Zn, Cu, bimetallic nanoparticles, in particular Pt-Ru, oxide nanoparticles, in particular silicon, aluminum, iron and magnesium, quantum dots based on cadmium sulphide or InAs / GaAs.

14. Process according to any one of the preceding claims, characterized in that the target molecule is chosen from the group comprising ions, antigens, antibodies, amino acids, peptides, proteins, nucleotides, bases of DNA, drugs, pesticides, food products, heavy metals, pollutants, volatile and non-volatile organic compounds, and derivatives thereof.

15. A molecular fingerprint polymer having an ultrafine thickness confined to a support obtainable by the method according to any one of claims 1 to 12 characterized in that it allows a detection limit of 0.1 nmol / L .

16. Use of at least one diazonium salt carrying photopolymerization initiator functional groups, as a photopolymerization initiator in a process for the preparation of molecular imprinted polymers, in ultrathin layers on a support, useful for the recognition of target molecules, said method comprising a step of photopolymerizing a composition comprising at least one crosslinking monomer, at least one functional monomer and at least one target molecule, the photopolymerization being confined to the surface of the support.

17. Fleas comprising one or more molecular imprinted polymers according to claim 14 having an ultra-thin thickness and confined to a support.

18. Kit for analyzing at least one target molecule comprising at least one molecular imprinted polymer according to claim 13, said polymer being able to interact with the at least one target molecule, and at least one marker, said marker being suitable interacting with all or part of the recognition sites of the at least one target molecule.

19. Use of a molecular imprinted polymer according to claim 14 for the recognition and / or extraction of target molecules.