EP1949468A1

EP1949468A1 - Method for grafting molecules of interest on inorganic surfaces, resulting surfaces and uses thereof

Info

Publication number: EP1949468A1
Application number: EP06831010A
Authority: EP
Inventors: Guillaume Delapierre; Florence Duclairoir; Jean-Claude Marchon
Original assignee: Commissariat a lEnergie Atomique CEA
Current assignee: Commissariat a lEnergie Atomique et aux Energies Alternatives CEA
Priority date: 2005-10-27
Filing date: 2006-10-25
Publication date: 2008-07-30
Also published as: US20090188602A1; FR2892859A1; FR2892859B1; US7919046B2; WO2007048924A1

Abstract

The invention concerns a method for grafting molecules of interest on a silicon substrate via a spacer compound, said grafting including at least one click chemistry reaction to the supports thus obtained as well as their uses in nanotechnologies and nanobiotechnologies, such as molecular electronics, the manufacture of biochips or of sensors.

Description

METHOD OF GRAFTING MOLECULES OF INTEREST ON INORGANIC SURFACES, SURFACES OBTAINED AND APPLICATIONS

The present invention relates to the grafting of molecules of interest on an inorganic substrate, in particular of silicon, by means of a spacer, said grafting implementing at least one click chemistry reaction, better known by the term "click chemistry", grafted surfaces and their applications in the fields of nano and nanobio technologies, such as molecular electronics, the manufacture of biochips or sensors.

The processes of miniaturization of electronic components are reaching their limits today and a great effort is being made to find one or more techniques to reduce the size of the current components, which seem to be becoming obsolete. Therefore, completely different systems must be proposed.

The work done in the field of molecular electronics goes in this direction, and many researches are launched in order to develop new "all-molecular" or "hybrid" microelectronics devices (for example: silicon / organic molecules). Moreover, following these numerous parallel studies, these new miniaturization approaches also find other fields of application such as biotechnologies. Indeed, in addition to answering the question of the miniaturization of electronic components, these new systems could allow, or would be adaptable to, the manufacture of medical imagers, receivers, or molecular transporters of nanoscopic sizes that can be ingested by the patient.

These new devices very often require the anchoring of a molecule or nanoscale element on a surface in order to use the system in a completely solid form. Silicon surfaces are widely used in current research because the properties of this material are known in particular through the use that is made in the field of electronics.

For about ten years, this field is in full expansion and one can list several techniques of grafting. Some are direct, others indirect, that is to say that after the preparation phase of the surface, the grafting of the desired molecule still requires one or more steps. Surfaces studied are numerous: gold, silica (SiO ₂ ), silicon, graphite, platinum. The specificity of each grafting technique is based on the material on which the experiment is attempted, as well as on the number of steps performed and on the chemistry used.

In the field of electronics, silicon surfaces are often coated with an oxide layer having insulating properties. Few examples involve a grafting on silicon directly. Gold graft directly on silicon can have advantages, such as avoiding the presence of an oxide layer that widens the dimensions of the device, or that may screen the semiconductor silicon molecule. The surfaces of native silicon are also of major interest in the field of electronics and methods other than those previously exposed have been developed to modify them.

Thus, some authors, such as Menzel H. et al., Langmuir, 1997, 13 (4), 723-728, propose a method for modifying a silicon surface with polyglutamate using a spacer. The surface must first be cleaned by different treatments that generate superficial Si-OH bonds, and then the spacer, a trichlorosilyl bromoalkane, is anchored via Si-O-Si bonds to the surface. Treatment of the surface thus obtained with a solution of NaN ₃ makes it possible to substitute azides for the bromine atoms, said azides then being reduced to LiAlH ₄ . This last reaction makes it possible to obtain a hydrosilylated surface covered by aminoalkanes. The N-carboxyanhydride benzylglutamate is then grafted by reaction with the NH ₂ terminal groups on the surface. The recovery rate of the surface has been estimated at 20-40%. This method, however, remains relatively limited by the laborious preparation of the surface and by the number of steps necessary to achieve the anchoring of the polyglutamate.

The native silicon surfaces, in addition to cleaning, may undergo preparation steps prior to grafting the compounds of interest. Thus, Effenberger F. et al., Langmuir, 2004, (24), 10375-10378 discloses a grafting method of modifying a silicon surface to provide free hydroxyl groups which react with trichlorosilanes; the method is illustrated by the grafting of long chain esters which are stable under the reaction conditions. Functions The esters are then reduced to alcohol and then, after reaction with phosgene, the corresponding chloroformates are obtained. A substitution reaction of chlorine in the presence of NaN ₃ is then carried out to yield the corresponding azidoformate. Exposure to UV radiation from this surface results in the removal of nitrogen to form a carbamate-terminated surface. Another modification that can be envisaged is a nucleophilic substitution on the electrophilic carbamate by an amino molecule. According to this method, an activated surface is obtained, which can then be reacted with molecules of interest having functions that can be added to the amines or carbamates. This protocol, similar to that proposed by Menzel H. et al. (cited above) is difficult to implement because it requires the use of phosgene and represents a significant number of steps. Moreover, the proposed functionalization, although it can lead to two different results, does not allow to graft very varied molecules outside the amines. The recovery rate is estimated at 73-78%. Other authors, such as Sudhölter EJR et al., (Langmuir, 1999,

15 (23), 8288-8291), propose a thermal method of surface functionalization using hexadecene. The operating conditions, particularly the high temperature, involve the use of particular solvents. These conditions, relatively drastic, do not seem compatible with labile or unstable groups and do not offer perspective to this method. The authors mention a second process that requires a modification of the native silicon surface by substitution of the hydrogen atoms present by chlorine or bromine atoms. The desired modified surface is then obtained by reaction of an organomagnesium or an organolithium. Recovery rates are not shown. In order to immobilize DNA molecules, Wayner D. D. M. and ah,

Langmuir, 2004, (26) 11713-11720, have shown that it is possible to graft undecenoic acid onto a silicon surface under UV irradiation. The surface, thus terminated by a carboxylic acid function, is then activated using N-hydroxysuccinimide (NHS) in the presence of N-ethyl-N '- (3-dimethylaminopropyl) carbodiimide, in order to react with a Single stranded DNA carrying a 5'-dodecylamine group. The recovery rate is not specified by the authors. He J., et al., Chem. Phys. Lett, 1998, 286, 508-514 describe a functionalization method according to which thiophene molecules are grafted on a surface of Si (111). According to this method, a bromination reaction is first carried out with N-bromosuccinimide or bromochloroform, using benzoylperoxide as a radical initiator. This gives a reactive surface having surface Si-Br bonds. Still according to this method, an alkylation reaction is then carried out between the superficial bonds and an organolithium, the lithiated thiophene. Thus, the thiophene is immobilized on Si via an Si-C bond. This type of surface chemistry can also be done by chlorinating the surface, and using an organomagnesium instead of an organolithium for the alkylation reaction.

Linford et al., In Langmuir, 2005, 2093-2097, have explored the long-chain acid chloride reaction on particular activated silicon surfaces ("scribed silicon"). The authors show that acid chlorides react directly with these silicon surfaces to lead to their alkylation. Nevertheless, it appears that side reactions also take place and so secondary products of the Si-C (O) -R or Si-OC (O) -R type are formed. The operating conditions are decisive in this procedure and the authors estimate that the recovery rate is between 20 and 60%. The molecules of interest are grafted onto the surface randomly here. According to the grafting methods described above, the surfaces are either functionalized directly by the molecules of interest or indirectly functionalized. In the latter case, after the anchoring of a first molecule, a secondary reaction takes place on the surface between the terminal function of the immobilized molecule, and the terminal function of the molecule of interest. Among the types of secondary reactions used on the surface, mention may be made especially of ester hydrolysis, ester reduction and cleavage, ester formation, activation of CH terminal bonds followed by formation. amide or sulfonamide, or the polymerization. These side reactions are interesting, but sometimes have average yields. On reading the methods presented above, it appears that the grafting of molecules of interest has a certain number of disadvantages and can be further improved in particular as regards the number of steps necessary for grafting, the chemistry used in secondary reactions and the variety of molecules that can be transplanted.

The inventors therefore set themselves the goal of developing a method of grafting molecules of interest on inorganic surfaces that is effective and easy to implement, especially in the process of manufacturing modified surfaces, which can then be used according to the wishes of the user. The use of click chemistry, more widely known under the English name of "click chemistry", recently defined by Sharpless K. B. et al. (Angew Chem Int.Ed., 2001, 40, 2004-2021) allowed the inventors to develop such a method.

The inventors therefore propose in this application a covalent grafting method of at least one molecule on the surface of an inorganic substrate carrying at least one spacer, by implementing a "click chemistry" reaction. As has been amply described in the Sharpless article KB et al. (cited above) the "click chemistry" corresponds to the reactions leading to the formation of at least one covalent bond between a carbon atom and a heteroatom under operating conditions that are simple to implement and where the presence of water or oxygen usually has no influence on the course of the reaction. These reactions are sometimes carried out without solvent or in the presence of a non-polluting solvent (such as water) or easily removed. The desired product is easily isolable and obtained in good yields, without formation of troublesome byproducts. More generally, it is recognized that this type of reaction, in addition, a higher driving force kcal.mol 20 ^". In this type of reaction, the bringing together of two main substrates, the functions of" reactive " are complementary, effectively leads to the desired product.

Some of the main reactions of "click chemistry" include:

cycloadditions of unsaturated species, such as 1,3-dipolar cycloadditions or Diels-Aider type reactions (hetero Diels Aider included), nucleophilic substitutions, especially the openings of constrained electrophilic heterocycles such as epoxides, aziridines , aziridinium ions and episulfonium ions, the reactions on the carbonyls, apart from the aldol chemistry, such as in particular the formation of ureas, thioureas, aromatic heterocycles, ether oxime, hydrazones and amides,

addition reactions on multiple carbon-carbon bonds, in particular oxidation, such as epoxidation, dihydroxylation, aziridination, addition of sulfenyl halide and Michael-type additions.

"Click chemistry" is widely used to functionalize biological molecules. Indeed, the mild operating conditions (for example: reaction at room temperature using water as reaction solvent) related to the very high yield of these reactions are very well suited to treat the fragile molecules. These advantages also suggest that other molecules might also react under similar conditions.

The present invention therefore firstly relates to a process for the covalent grafting of at least one molecule of interest on the surface of a silicon substrate comprising surface hydride functions, said substrate carrying at least one spacer, characterized in that it includes at least the following steps:

(a) a first covalent anchoring step, on the surface of said substrate, of at least one spacer compound of general formula (I) below:

(X) _n -E- (Y) _n , (I) wherein:

-E represents a group chosen from alkyl and aryl radicals,

-X represents a C = C double bond,

Y represents a terminal functional group chosen from the double and triple bonds between two carbon atoms, the triple bonds between a carbon atom and a nitrogen atom, the aziridines, the acyl chlorides and their complementary azide and amine functions; , said functional group being capable of reacting with at least one complementary functional group Z carried by a molecule of interest in a reaction leading to the formation of a covalent bond between a carbon atom and a heteroatom belonging respectively and indifferently to the functional groups Y and Z (click chemistry reaction), m and n, independently of one another, are integers between 1 and 3 inclusive,

(b) a second covalent coupling step to said spacer compound of formula (I) thus anchored to the surface of said substrate, of at least one molecule of interest by formation, according to a click chemistry reaction, at least one covalent bond of the type -C-Het- in which Het is a heteroatom, by reaction of at least one functional group Z carried by said molecule of interest and selected from the double and triple bonds between two atoms of carbon, the triple bonds between a carbon atom and a nitrogen atom, aziridines, acyl chlorides and their complementary azide and amine functions, and at least one Y group of said spacer compound of formula (I), Z and Y being capable of forming together said covalent bond.

For the purposes of the present invention, "anchoring" refers to the immobilization of a spacer compound of formula (I) on a surface of silicon bearing surface hydride functions via a covalent bond. The term "coupling" describes the reaction between at least one terminal functional group Y (non-immobilized) of the spacer compound of formula (I) and at least one complementary functional group Z of the molecule of interest. The "grafting" describes all of these two actions. Thus, the molecule of interest is grafted onto the surface of the inorganic substrate following the anchoring of the spacer compound of formula (I) according to the invention, followed by the coupling of the molecule of interest and this spacer compound with a covalent bond of type -C-Het-, according to a click chemistry reaction.

According to the invention, the alkyl radical defined for E is preferably a radical containing 1 to 20 carbon atoms, unsubstituted or optionally mono- or polysubstituted, linear, branched or cyclic, saturated or unsaturated. When mono- or polysubstituted, the substituent of such alkyl radicals are preferably selected from halogen atoms, hydroxyl, amino, carboxyl, C ₁ to C _0, substituted or unsubstituted aryl, said substituents which may themselves contain one or more halogen atoms such as F or Cl and / or one or more heteroatoms such as N, O, P, Si and S, thus giving rise, for example, to C ₁ -C ₁₀ alkoxy groups such as that for example the methoxy groups and ethoxy; ether-oxide; aryloxy; C ₁ -C ₁₀ aminoalkyl; aminoaryl; C ₁ -C ₁₀ hydroxyalkyl, hydroxyaryl; C ₁ -C ₁₀ thioalkyl such as for example the mercapto group; cyano; keto; halo C ₁ -C _0; heteroalkyl, Ci-C _10; haloaryl and heteroaryl. Among such saturated and unsubstituted alkyl radicals, mention may in particular be made of methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl and pentyl radicals.

Unsaturated and unsubstituted alkyl radicals that may be mentioned in particular are ethenyls, propenyls, isopropenyls, butenyls, isobutenyls, tert-butenyls, pentenyls and acetylenyls.

According to the invention, the aryl radical defined for E is preferably an aromatic or heteroaromatic carbon structure, optionally mono- or polysubstituted, consisting of one or more aromatic or heteroaromatic rings, the heteroatom (s) possibly being chosen from N, O, P, Si and S. According to a particular embodiment of the invention, and when the alkyl or aryl radicals are polysubstituted, the substituents may be different from each other. Among the substituents of the alkyl and aryl radicals, mention may in particular be made of halogen atoms, alkyl, haloalkyl, substituted or unsubstituted aryl groups, substituted or unsubstituted heteroaryl, amino, cyano, azido, hydroxy, mercapto, keto, carboxy, ether -oxide and alkoxy such as for example methoxy.

According to the invention, E is preferably chosen from alkyl and aryl radicals, more preferentially from alkyl radicals as defined above, still more particularly from C ₁ to C ₂₀ alkyl radicals, preferably unbranched in order not to have of steric gene when anchoring with already anchored structures and obtain an acceptable recovery rate.

The grafting method according to the invention is applicable to silicon semiconductor substrates having surface hydride (Si-H) functions. Silicon wafers (known as "wafers") are readily available. The silicon surfaces are advantageously Si (100) or Si (111) surfaces.

According to a preferred embodiment of the invention, the grafting method further comprises a step of preparing the surface, prior to anchoring the spacer compounds of formula (I), in order to maximize the amount of surface Si-H bonds and to reduce the possibility of the appearance of an oxide layer. In this case, the preparation step advantageously consists in etching the surface with a solution of a weak acid such as, for example, a solution of hydrofluoric acid. This step can be optimized by observing the infrared vibrations of the Si-H and Si-O bonds after stripping of several surfaces with solutions of hydrofluoric acid of different concentrations in order to choose the concentration leading to the best result. The surfaces are then preferably rinsed with demineralised water. The spacer compounds of general formula (I) are anchored on the surface of the substrate by at least one functional group X. Depending on the value of n, the spacer compounds of formula (I) can comprise several functional groups X. However, according to a particularly preferred embodiment of the invention, use is made of spacer compounds of formula (I) in which n = 1, that is to say having only one functional group X to reduce the steric hindrance.

According to the invention, the anchoring of the spacer compounds of formula (I) on the surface of a silicon substrate during step (a) is via an Si-C bond, this one being particularly strong and stable. The anchoring step (a) is therefore a hydrosilylation reaction between the surface of a silicon substrate carrying hydride functions (-Si-H) and at least one spacer compound of formula (I) and the CC function. of the X functional group or groups. This reaction is of radical type and may be initiated thermally, photochemically, electrochemically or catalytically. According to the invention, the photochemical and thermal initiations are preferred; advantageously, we will use photochemical initiation since it involves the use of mild conditions and adapted to the manipulation of thermo sensitive molecules. In addition, this form of initiation allows the use of smaller amounts of product.

Preferably, the functional group Y of the spacer compounds of formula (I) represents an azide, alkyne or nitrile function.

According to the invention, the grafting method described above makes it possible to graft various molecules of interest. Preferably, Z is a group functional device carrying at least one sp hybrid carbon, preferably a C≡C group (obtained by Sonogashira coupling for example) or C≡N; the functional group Z may also be an azide group which is the preferred complementary partner in a "click chemistry" reaction with an alkyne or a nitrile. Advantageously, the molecule of interest carries from one to four functional groups Z, and preferably only one.

Among the complementary pairs between the functional group (s) Y of the spacer compounds of formula (I) and the functional group (s) Z of the molecules of interest, mention may be made of: - the alkyne or nitrile (group Y) / azide pairs ( group Z);

the azide (group Y) / alkyne or nitrile pairs (group Z);

A particularly preferred pair according to the invention is the pair azide (group Y) / alkyne (group Z).

Among the spacer compounds of formula (I) above, mention may in particular be made of those in which:

- n ^≈ l;

- E represents an alkyl radical C ₆ -C _2;

Y represents an azide and m = 1.

Such compounds include, inter alia, hexenyl azide, heptenyl azide, octenyl azide, nonadecenyl azide, decenyl azide, undecenyl azide, and azide azide. dodecenyl.

The spacer compounds of formula (I) used according to the method according to the invention can be easily prepared according to conventional synthetic methods that can be used by those skilled in the art, for example by using the synthetic methods described by Scriven E et al. (Chemical Reviews, 1998, 88 (2),

297-368) or by Alvarez S. G. et al (Synthesis, 1997, 413-414).

According to the invention, the step (b) of covalent coupling between the spacer compound of formula (I) and the molecule of interest (reaction between groups Y and Z) is preferably a 1,3-cycloaddition reaction between a dipole, which may be for example an azide group N ₃ , and a dipolarophil, which may be for example:

- an alkyne group; the reaction leading in this case to a triazole, - a nitrile group; the reaction leading in this case to a tetrazole, the group Y being, in this case, preferably the dipole. When the dipolarophile is an alkyne, it is preferable that it is terminal to be accessible. The covalent coupling to the spacer, obtained in (a), of at least one molecule bearing at least one Z group may be carried out in the presence of a catalyst.

In the case of 1,3-cycloaddition coupling, and in particular 1,3-cycloaddition of Huisgen, it is recommended to use a metal catalyst, advantageously a transition metal and particularly Cu ¹ . It is possible to distinguish two kinds of alkynes and nitriles that can be used as functional groups Y or Z.

The first type consists of dipolarophils having an electron-attracting group which are said to be "activated" and which react easily with the dipole. These "activated" alkynes or nitriles can sometimes react with the azide in a quasi-quantitative manner in the absence of a catalyst.

The second kind consists of so-called "non-activated" alkynes and nitriles for which the cycloaddition reactions, and in particular of Huisgen 1,3-cycloaddition, using them require prior activation by the action of a catalyst. preferably in addition to a base. By way of example, it is possible in particular to use a cupric catalyst Cu ¹ generated in situ, by reaction of a source of Cu ¹¹ , such as (CuSO ₄ , 5H ₂ O), and a base such as sodium ascorbate, which will reduce Cu ¹¹ to Cu ¹ . If the solvent is aqueous, this approach is favored. In the case of unactivated alkynes, the addition of a base is recommended to facilitate the loss of the proton of the alkyne and thus promote the start of the reaction. Indeed, the mechanism envisaged for this reaction passes through a cycle where the deprotormed base alkyne and copper acetylide is then formed. Acetylide then reacts with azide. The formation of the new triazole derivative ligand is thus obtained. This new complex then loses Cu ¹ . The catalyst is regenerated and the final product is obtained. Although the reactions of "click chemistry", and in particular of

1,3-cycloadditions, are relatively insensitive to the solvent of the reaction, the use of polar solvents can facilitate them. Thus, the step (b) of covalent coupling is preferably carried out in the presence of at least one polar solvent which may be chosen in particular from water, alcohols, acetone, acetonitrile, dimethylformamide (DMF), and mixtures thereof.

The grafting method according to the present invention makes it possible to graft any type of molecule of interest comprising, naturally or after modification, at least one functional group Z as described above, and capable of reacting with at least one functional group Y of the compounds spacers of formula (I) according to the conditions specified above.

For the purposes of the present invention, the term "molecule of interest" is understood to mean any element of nanometric size, that is to say having at least one nanoscale dimension (advantageously between 1 and 100 nanometers). This element can in particular be an organic or inorganic molecule, an aggregate or a cluster, a supramolecular assembly, a nanowire, a nanocrystal, etc. Advantageously, the molecules of interest that can be grafted according to the method of the invention are multistable molecules, the. possessing several energy minima separated by activation barriers. Thus, the molecules of interest can pass from one state to another reversibly or irreversibly. Among the multistable molecules of interest, mention may in particular be made of electroactive molecules that exist in several redox states, photosensitive spiropyrranine derivatives, photo-isomerizable compounds of the azobenzene type, and the like.

The molecules of interest can then be grafted by the process according to the present invention are preferably selected among the organic molecules biomolecules such as nucleic acid molecules such as I ¹ DNA and RNA; nucleotides; oligonucleotides; the proteins ; peptides; sugars or polysaccharides; porphyrins such as tetrakis [meso (4-ethynylphenyl) porphyrin, 1,10- (4-ethynylphenyl) 5,15-

(4-mesityl) porphyrin and 1- (4-ethynylphenyl) 5,10,15- (4-mesityl) porphyrin) whose presence on a surface is particularly useful in the field of molecular electronics; compounds having cis-trans isomerism such as diarylethylenes, spiropyrans, spiroxazines, fulgides or azobenzenes whose presence on a surface is particularly useful for the manufacture of photocontrolled molecular switches.

The grafting method according to the invention has multiple advantages in view of the prior art. In addition to the surface preparation phase, current grafting techniques include a number of consequent and often laborious steps, in contrast to the grafting method according to the present invention.

Invention which involves only a limited number of steps.

In general, the overall recovery rate is related to the yields of the first step (a) of anchoring and the second step (b) of coupling. Given the particular nature of the support (silicon), the grafting method according to the present invention allows the implementation of reactions of high yields, such as the hydrosilylation of an alkene on a silicon surface, and as the cycloaddition of Huisgen which is a quasi- quantitative "click chemistry" reaction. The overall recovery obtained is therefore very important. The method, main object of the invention, allows a grafting on a silicon substrate, in just two steps through the click chemistry. It is adaptable to different molecules carrying the appropriate terminal functions, and makes it easy to graft the studied molecules onto a silicon surface. The advantages of the present invention may be exploited by manufacturers in different fields, such as microelectronics, biotechnology or the environmental industry. The properties of the grafted molecules will differentiate the domains concerned since they are the ones which define the field of application conferred on the surface.

The present invention therefore also relates to silicon supports comprising at least one surface functionalized by at least one molecule of interest, said supports being capable of being obtained by implementing the grafting method as defined above.

These supports are characterized in that they comprise at least one surface of silicon having surface hydride functions, on which is covalently grafted at least one molecule of interest via at least one compound spacer of formula (I) below:

(X) _n -E- (Y) ₁₁₁ (I) in which X, Y, E, n and m have the same meaning as previously indicated and in which the functional group (s) Y are engaged in a covalent -C-Het bond with at least one complementary functional group Z carried by said molecule of 'interest. The set of molecules of interest thus grafted onto the surface of the support by means of the spacer compounds of formula (I) forms a particularly stable and robust film, in particular because the anchoring of the spacer compounds of formula (I) on the surface of the support is provided by a Si-C bond.

These films are in the form of a monomolecular layer. Indeed, certain current grafting techniques, involving radical reactions, lead to the formation of multi-layer deposition of molecules on the surface. The control of these reactions, in order to limit this deposition to a monolayer, is not yet well known. In the context of the invention, even if the grafting of the spacer compounds of formula (I) is by a radical reaction, it can not lead to the formation of polymer. Thus, the reaction is done only on the available surface sites thus leading to the formation of a monomolecular layer.

Moreover, in the field of silicon surfaces, the Si-O bond is usually widely used. The Si-O and Si-C bond energies are equivalent and therefore have identical thermodynamic stability but there is a difference in kinetics. The Si-O bond is highly polarized and thus much more sensitive to acidic or basic hydrolysis than the Si-C bond. Surfaces involving these Si-O bonds may therefore degrade more rapidly. Moreover, the surfaces obtained via Si-O bonds are often less organized because they result from the reaction of a multifunctional siloxane (RSi (OR ') ₃ for example) that is capable of reacting with the surface but also with a other molecule in solution. The layers obtained by this method are difficult to monomolecular and the recovery rate can vary from one sample to another. In contrast, according to the invention, the support comprises a silicon surface on which the spacer compounds of formula (I) are covalently anchored via a -Si-C bond and form a monomolecular film. This embodiment provides a very good stability. Thus, the surfaces obtained can be stored longer, without risk of decomposition of the layer of molecules, and will not be affected by the conditions outside the device. We can easily understand that this advantage is very important if we want to transfer this kind of surfaces into the electronic compounds of the future.

Depending on the nature of the molecules of interest coupled to the spacer compounds of formula (I) (nucleic acids, nucleotides, oligonucleotides, proteins, peptides, sugars and polysaccharides, porphyrins, diarylethylenes, spiropyrans , the spiroxazines, fulgides and azobenzenes), the support according to the invention respectively constitutes a nucleic acid chip, a nucleotide chip, an oligonucleotide chip, a protein chip, a peptide chip, a sugar chip , a polysaccharide chip, a porphyrin chip, a diarylethylene chip, a spiropyran chip, a spiroxazine chip, a fuzzy chip or an azobenzene chip. The supports according to the invention can advantageously be used in the fields of nano and nano biotechnologies such as electronics, in particular molecular electronics, biotechnologies and the environment.

The use of the supports according to the invention in the field of molecular electronics is very interesting since the method of the invention makes it possible to graft bistable molecules (multistable at two minima) directly onto silicon. In the majority of examples cited, the molecules are anchored on an insulating silica layer. This oxide layer will always have an important role in the behavior of the device. It is therefore difficult to decipher the direct relationship between the change of state of the molecule and the changes in the semiconducting properties of silicon. Moreover, if this layer is too thick the properties of the material will not be affected at all by the change of state of the molecules and the interest of grafting them on this surface will be lost. The multistable grafted molecules are often electroactive molecules that exist in several redox states or compounds having cis-trans photoisomerization, such as diarylethylenes derivatives, spiropyrans, spiroxazines, fulgides or azobenzene. Thus modified, the surface should reproduce this multistability which will most often advantageously be converted into an electrical signal. The manufacture of this type of surfaces is interesting for the microelectronics industry in their quest for the miniaturization of existing devices. In addition, in this area, the molecules are directly grafted onto silicon, which will make it possible to study the effect of state switching of the multistable molecules on the semiconductor properties of Si. The manufacture of hybrid electronic devices, such as hybrid memories or molecular transistors will be facilitated by these surfaces.

In the field of bio-industry and in particular in the manufacture of nucleic acid chips (DNA, RNA), the ease of grafting of the process object of the invention will be exploited. In fact, the intermolecular recognition between a grafted molecule and a molecule in solution can be characterized by fluorescence when one of the two molecules has this property, or by a change in the semiconductive properties of the substrate when the properties of the grafted molecule are modified. to the specific supramolecular interactions (hydrogen bonds, electrostatic bonds, π-π ...) between this molecule and that which is in solution. Thus the invention allows the production of biochips with molecules of interest, which may be used, for example, to detect antibodies or to determine where lesions are located on an injured DNA strand ...

The supports according to the present invention can also be used as sensors in the field of the environment, particularly for the search and / or removal of pollutants. Indeed, receiving surfaces may be prepared according to the method according to the invention so that in a certain medium a specific recognition takes place between a grafted molecule of interest and one of the different pollutants in solution. It will thus be possible to detect and / or selectively eliminate certain pollutants. In addition to the foregoing, the invention also comprises other arrangements which will emerge from the description which follows, which refers to examples of synthesis of a spacer compound of formula (I) and of molecules of interest, an example describing test cycloaddition reactions in solution between spacer compounds of formula (I) and molecules of interest and to an example of preparation of a support functionalized with a spacer compound of formula (I) coupled to a molecule of interest, and to the appended Figures 1 and 2, in which: FIG. 1 represents the multi-reflection surface infrared spectra (MIR) of a native silicon surface (fine line) and the same surface after regeneration with hydrofluoric acid (HF) (thick line). As a result of this treatment the surface has reactive -Si-H groups; FIG. 2 represents the MIR spectra of a hydrogenated silicon surface (thin line) and of the same surface after functionalization with undecenyl azide.

It should be understood, however, that these examples are given solely by way of illustration of the subject of the invention, of which they in no way constitute a limitation.

EXAMPLES

The following examples are made on silicon surfaces. It has been chosen to immobilize in particular an aromatic electroactive molecule derived from heterocycles. It is a metalloporphyrin having four terminal C liaisonsC triple bonds. This is grafted via a spacer having an aliphatic chain of eleven carbon atoms. The "click chemistry" reaction carried out during the process is a cycloaddition between an alkyne group on the metalloporphyrin and the azide group of the spacer.

EXAMPLE 1 SYNTHESIS OF A SPACER COMPOUND OF FORMULA (I): undecenyl azide

In this example, a spacer compound of formula (I) has been prepared: the undecenyl azide of formula (Ij) below:

To do this, sodium azide (2.781 g, 43 mmol) was dissolved in 170 ml of dimethylformamide (DMF). 5 ml of undecenyl bromide

(36 mmol) were added, with stirring, to the reaction medium, which was then heated at 80 ^° C. for 24 hours. The mixture was allowed to cool to room temperature and then 100 ml of water was added in portions. Then extraction was carried out with ethanol (3 x 100 ml) then the organic phase was washed with water (3 x 100 ml) and dried over sodium sulphate (Na ₂ SO ₄ ). Finally, The solvent was evaporated and 2.58 g of undecenyl azide (20.3 mmol, 60% yield) was isolated as a pale yellow transparent oil. EXAMPLE 2: SYNTHESIS OF MOLECULES OF INTEREST

In this example, several molecules of interest have been synthesized. They derive from a central core of porphyrin type. These molecules have been chosen because they are of interest in several fields of application of the invention. The porphyrinic core, allowing the complexation of metals, is of major interest in applications to biology or the field of the environment. In addition, the metalloporphyrins having several redox states, these have a great potential in electronics and photoelectronics.

1) Synthesis of Tetrakis (4-trimethylsilylethynylphenyl) porphyrin (Protected TEPP) (1):

This molecule can be represented by the following formula (1):

5.02 g of 4-trimethylsilylethynylbenzaldehyde (25 mmol) were dissolved in 210 ml of propionic acid and then heated at 80 ^° C. Then 1.72 ml of pyrrole (25 mmol) were added portionwise over a period of 'one o'clock. The temperature was then raised and the mixture was kept under reflux for 4 hours. After returning to ambient temperature, 75 ml of methanol was added to the reaction medium, which was then allowed to stand for 14 hours so that the porphyrin crystallized. The medium was filtered through sintered glass and the product was. rinsed with methanol. 2.87 g (3 mmol, 12% yield) of the expected porphyrin (1) were obtained in the form of a violet powder. 2) Synthesis of tetrakis (4-ethynylphenyl) porphyrin (TEPP) (2)

A solution of 700 mg (0.7 mmol) of Protected TEPP (1) obtained above in 500 ml of a mixture of chloroform / tetrahydrofuran (THF) (1/1 v / v) was cooled in a bath of liquid nitrogen and acetone at a temperature between -30 and -50 ° C and then a 1 mol / l solution of tetrabutylammonium fluoride (TBAF) in THF (259 mg, 0.99 mmol in 1 ml) was added to the reaction mixture which was then stirred for 20 hours. The rise in temperature has not been controlled. The solvent was then evaporated and the deprotected porphyrin (2) was obtained as a violet powder (747 mg, 1.05 mmol). 3) Synthesis of iron tetrakis (4-ethynyl-phenyl) -porphyrin (FeCl-TEPP) (3):

A solution of FeCl ₃ , 4H ₂ O (4.56 g, 28.13 mmol) in DMF (100 mL) was added in portions to a solution of 1 g (1.4 mmol) of TEPP porphyrin (2). ), as obtained above in the previous step, in 400 ml of DMF and the whole was refluxed for 24 hours. 10 ml of 2,6-lutidine were then added, and the reflux was resumed for 48 hours. Heating was stopped and the solvent evaporated under reduced pressure. The residue was taken up in 50 ml of water and stirred to remove a maximum of salts and TBAF, then filtered on paper. The residue was dissolved in 100 ml of chloroform and the organic phase washed with saturated aqueous sodium chloride solution (3 x 100 ml) to ensure that the axial ligand of the iron is a chlorine atom. The organic phase was then dried over sodium sulfate and the solvent was evaporated. Iron tetrakis (4-ethynylphenyl) porphyrin (FeCl-TEPP) (3) (813 mg, 1.02 mmol, yield: 73%) was obtained as a purple / black powder, and brown in solution in chloroform. 4) Synthesis of cobalt tetrakis (4-ethynylhephenyl) porphyrin (Co-TEPP) (4):

A solution of 8.71 g (35 mmol) of cobalt acetate tetrahydrate (Co (CH ₃ COO) ₂ , 4 H ₂ O) in 100 ml of DMF was added in portions to a solution of 1.7 g ( 2.39 mmol) of porphyrin TEPP (2) as obtained above in step 2) in 500 ml of DMF. The resulting mixture was refluxed for 20 hours. The heating was stopped and 150 ml of water was added to the reaction medium. The mixture was then extracted with ethanol (4 x 200 ml) and the organic phase was evaporated. The residue was dissolved in 300 ml of ethanol, and the organic phase was washed with 3 x 200 ml of water and dried over sodium sulfate. The solvent was evaporated and cobalt tetrakis (4-ethynylphenyl) porphyrin (Co-TEPP) (4) (1.19 g; 1.55 mmol; yield: 65%) was obtained in the form of a powder of purple / black color, and red in solution in chloroform.

5) Synthesis of manganese tetrakis (4-ethynylphenyl) porphyrin (MnCl-TEPP) (5):

A solution of 5.5 g (27.8 mmol) of manganese chloride tetrahydrate (MnCl ₂ , 4 H ₂ O) in 160 ml of DMF was added in portions to a solution of 1 g (1.4 mmol) of porphyrin TEPP (2) as obtained in step 2) above and dissolved in 200 ml of DMF. The mixture was refluxed for 72 hours. After cooling to room temperature, the solvent was evaporated under reduced pressure. The residue was taken up in 50 ml of chloroform and the manganese salts were removed by filtration on celite. The liquid filtrate was evaporated and the residue was then dissolved in 100 ml of ethanol and washed with 3 x 100 ml of water to remove TBAF. After evaporation, the residue was dissolved in chloroform and this organic phase was in turn washed with a saturated aqueous solution of sodium chloride (3 x 100 ml) to ensure that the axial ligand of the manganese is a chlorine, and finally washed with 2 x 100 ml of water to remove traces of sodium chloride. The organic phase was then dried over sodium sulfate and the solvent evaporated. During the first extraction with ethanol and water as solvents, part of the porphyrin precipitated. This precipitate was filtered and kept. It was then redissolved in chloroform to be added to the organic phase when washing with sodium chloride. 700 mg (0.88 mmol, 63% yield) of manganese tetrakis (4-ethynylphenyl) porphyrin (MnCl-TEPP) (5) were obtained under the form of a purple / black powder that is green in solution in chloroform.

EXAMPLE 3: TEST REACTIONS OF CYCLOADDITION SOLUTION

These reactions were carried out in the absence of any support to study the materiality of the formation of a -C-Het type bond between a spacer compound of formula (I) and a molecule of interest comprising a functional group Z. 1) Synthesis of Manganese Tetrakis (Meso [4- (1-hexyl- [1,2,31triazol-4-yl] -phenphen) Porphyrin (6)

To a solution of hexyl azide (0.157 g, 1.23 mmol) in 35 ml of water was added successively with stirring a solution of copper iodide (6.7 mg, 0.035 mmol) in 60 ml. acetonitrile, then 0.09 ml of triethylamine and finally a solution of MnCl-TEPP (5) as obtained in Example 2 (5) (88.7 mg, 0.11 mmol) in 120 ml of acetone. The mixture was heated at 80 ° C for about 3 days and then the solvent was evaporated. The product was obtained as a black powder. 2) Synthesis of iron tetrakis (mesof4- (1-hexyl-ri, 2,3-triazol-4-yl) -dispendyl) porphyrin (7)

To a solution of hexyl azide (91.1 mg, 0.72 mmol) in 18 ml of water was added successively with stirring a solution of copper iodide (6.7 mg, 0.035 mmol) in 25 ml of acetonitrile, then 0.09 ml of triethylamine and finally a solution of FeCl-TEPP (3) as obtained above in example 2, step 3) (54.2 mg, 0.07 mmol) in 60 ml of acetone. The mixture was heated at 80 ° C for about 6 days and then the solvent was evaporated. The product was obtained as a black powder.

3) Synthesis of Tetrakis (Manganese Meso [4- (1-dodecanyl- [1,2,31triazol-4-yl] D-phenyl) Porphyrin (8)

(8) To a solution of dodecanyl azide (0.13 g, 0.63 mmol) in 35 ml of water was added successively with stirring a solution of copper iodide (6 mg, 0.03 mmol) in 50 ml. acetonitrile, then 0.09 ml of triethylamine and finally a solution of MnCl-TEPP (5) as obtained above in example 2, step 5) (48.9 mg, 0.063 mmol) in 60 ml of 'acetone. The mixture was heated at 80 ^° C. for about 4 days and then the solvent was evaporated. The product was obtained as a black powder.

For these 3 reactions, the mass spectra (not shown) verify that the reactions were total, and that there was no production of by-products. In order to improve the reaction times, a hindered base such as N, N-diisopropylethylenediamine (DIPEA) can be added or a larger amount of catalyst can be used.

We will also notice that in the case of a porphyrin having only one reactive function, the reaction time is considerably reduced. Moreover, it suffices that only one of these functions reacts with the spacer compound of formula (I) anchored on the silicon so that the molecule is itself grafted onto the surface. Thus, it is not necessary to react the modified surface and porphyrin for as long as in solution. EXAMPLE 4 PREPARATION OF A SUPPORT FUNCTIONALIZED BY A SPACER COMPOUND OF FORMULA (I) ON WHICH A MOLECULE OF INTEREST IS COUPLED

1) Preparation of the silicon surface

The silicon surfaces that were used in this example are pieces of silicon wafers Si (100) of dimensions 2 × 10 cm ² p-doped and resistivity 14 to 22 Ω.cm. Their preparation and the following steps were carried out in a clean room to avoid any contamination. The unoxidized silicon surface was regenerated by soaking the substrate in dilute hydrofluoric acid (5%) for a short time (a few tens of seconds). After rinsing with deionized water, the substrate was dried under a stream of nitrogen and then kept under an inert atmosphere. The surface obtained has superficial Si-H bonds. 2) Anchoring a spacer compound of formula (I)

This step is the first step of grafting a molecule of interest on the silicon, it corresponds to the anchoring of the spacer compound of formula (I) on the surface of the support. This is a radical-type hydrosilylation reaction between the undecenyl azide (1) as prepared above in Example 1 and the regenerated silicon surface as indicated above in 1). The substrate prepared as above in 1) was immersed in a solution of 6% undecenyl azide in mesitylene. The reaction was conducted under argon at 150 ° C for 12 hours.

This reaction can be initiated catalytically, thermally, photochemically or electrochemically. In this example, the reaction was thermally initiated.

The reactions leading to the grafting of the spacer compounds of formula (I) can be represented according to scheme A below:

SCHEME A

In this scheme, R corresponds to the E- (Y) _m portion of the spacer compound of formula (I). After the initiation step, an Si-H bond loses an electron (step a)) and thus an Si ^' radical is obtained, which then reacts (step b) with the terminal double or triple terminal of the electron molecule. interest in grafting. The Si-C bond is thus formed and the radical C on the β-carbon of the Si will then react, during step c), with the Si-H bond of the neighboring anchoring site and produce a -CH ₂ and a radical Si ^' . The propagation of the radical may initiate these Si-C bond formation reactions at the other sites.

The substrate was then rinsed successively with 200 ml of various solvents (toluene, acetone, ethanol, water). After reaction of the spacer compound of formula (I) on the surface, a surface is obtained that no longer has a surface Si-H bond, but very reactive azide functions.

Characterization :

The contact angle of the surface thus prepared is 68-70 °. MIR spectra (infrared surface multireflection) of the native surface (native oxide - Figure 1: fine line), the activated surface (HF 5%) (Figure 1: thick line and figure

2: fine line) and the functionalized surface (N ₃₎ (Figure 2: thick line) were obtained and compared.

These spectra are shown in FIGS. 1 and 2 respectively.

FIG. 1 represents the MIR spectra of the native silicon surface (thin line) and of the same surface after regeneration by hydrofluoric acid

(HF) (thick line); FIG. 2 represents the MIR spectra of the hydrogenated surface

(thin line) and the same surface after functionalization with undecenyl azide

(thick line);

Figure 1 clearly shows the disappearance of the native oxide (OH bonds, strip centered at 1200 cm ^-1 ). FIG. 2 shows the modification of the shape of the band at 2100 cm ^-1 , interpreted as the disappearance of the elongation vibration of the Si-H bonds and the appearance of the elongation vibration of the terminal azide group. R2006 / 002,395

27

3) Coupling of a molecule of interest on the support functionalized by "click chemistry"

The azide having been grafted to the support surface according to step 2) above, it was reacted with tetrakis (4-ethynylphenyl) porphyrin (2) as prepared above in step 2 ) of Example 2.

Since the metalloporphyrins are not soluble in water, a mixture of organic solvents: acetone / acetonitrile / water (10/5/3 v / v) was used. Following the test reactions carried out completely in solution in a homogeneous medium and as described above in Example 4, the reaction was carried out in the presence of 0.1 equivalent of catalyst (Cu ¹ ) and 2 equivalents of base (the triethylamine or diethylisopropylamine), at 80 ^° C. The reaction was transposed to a silicon surface and the molecules of interest were grafted onto the modified surface which can be controlled by MIR, ATR, XPS (Spectroscopy of photoelectrons with radiation). X)

STM / AFM (AFM - Electron force microscope, STM - Surface Tunneling Microscopy) or electrochemistry.

Porphyrin chips are thus obtained which may be used, for example, as molecular memories or as detectors for gases or biological substances.

Claims

9528 CLAIMS

1. Process for the covalent grafting of at least one molecule of interest on the surface of a silicon substrate comprising surface hydride functions, said substrate bearing at least one spacer, characterized in that it comprises at least the steps following:

(X) _n -E- (Y) _n , (I) wherein:

E represents a group chosen from alkyl and aryl radicals,

-X represents a C = C double bond,

Y represents a terminal functional group chosen from the double and triple bonds between two carbon atoms, the triple bonds between a carbon atom and a nitrogen atom, the aziridines, the acyl chlorides and their complementary azide and amine functions; , said functional group being capable of reacting with at least one complementary functional group Z carried by a molecule of interest in a reaction leading to the formation of a covalent bond between a carbon atom and a heteroatom belonging respectively and indifferently to the functional groups Y and Z;

m and n, independently of one another, are integers between 1 and 3 inclusive,

(b) a second covalent coupling step to said spacer compound of formula (I) thus anchored to the surface of said substrate, of at least one molecule of interest by formation, according to a click chemistry reaction, of at least one bond covalent of -C-Het- type, wherein Het is a heteroatom, by reaction of at least one functional group Z carried by said molecule of interest and selected from the double and triple bonds between two carbon atoms, the triple bonds between a carbon atom and a nitrogen atom, aziridines, acyl chlorides and their complementary azide and amine functions, and at least one Y group of said spacer compound of formula (I), Z and Y being capable of forming together said covalent bond.

2. Method according to claim 1, characterized in that the alkyl radical defined for E is a radical containing from 1 to 20 carbon atoms, unsubstituted or mono- or polysubstituted, linear, branched or cyclic, saturated or unsaturated.

3. Process according to claim 2, characterized in that the alkyl radical defined for E is mono- or polysubstituted and the substituent or substituents are chosen from halogen atoms, hydroxyl, amino, carboxyl and C-alkyl groups. ₁ to ₁₀ , substituted or unsubstituted aryl, said substituents may themselves contain one or more halogen atoms and / or one or more heteroatoms.

4. Method according to any one of the preceding claims, characterized in that E is a non-branched alkyl radical Q to C ₂₀ .

5. Method according to any one of the preceding claims, characterized in that it further comprises a step of preparing the surface of etching the surface with a solution of a weak acid.

6. Process according to any one of the preceding claims, characterized in that the compounds of formula (I) are chosen from those in which n = 1.

7. Process according to any one of the preceding claims, characterized in that the functional group Y of the spacer compounds of formula (I) represents an azide, alkyne or nitrile function.

8. Process according to any one of the preceding claims, characterized in that the functional group Z of the spacer compounds of formula (I) represents an azide, alkyne or nitrile function.

9. Process according to any one of the preceding claims, characterized in that the complementary pairs between the functional group (s) Y of the spacer compounds of formula (I) and the functional group (s) Z of the molecules of interest are chosen from :

the alkyne or nitrile pairs (group Y) / azide (group Z); and

the azide (group Y) / alkyne or nitrile (group Z) pairs.

10. Process according to any one of the preceding claims, characterized in that the spacer compounds of formula (I) are chosen from those in which:

- n = 1; E represents a C ₆ to C ₁₈ alkyl radical;

Y represents an azide and m = 1.

11. Process according to claim 10, characterized in that the spacer compounds of formula (I) are chosen from hexenyl azide, heptenyl azide, octenyl azide, nonadecenyl azide, Decenyl azide, undecenyl azide and dodecenyl azide.

12. Process according to any one of the preceding claims, characterized in that the step (b) of covalent coupling between the spacer compound of formula (I) and the molecule of interest is a 1,3-cycloaddition reaction. between a dipole and a dipolarophile in the presence of a metal catalyst.

13. Method according to any one of the preceding claims, characterized in that the step (b) of covalent coupling is carried out in the presence of at least one polar solvent selected from water, alcohols, acetone, acetonitrile, dimethylformamide and mixtures thereof.

14. Process according to any one of the preceding claims, characterized in that the molecules of interest are chosen from multistable molecules.

15. The method of claim 14, characterized in that the molecules of interest are organic molecules selected from nucleic acid molecules, nucleotides, oligonucleotides, proteins, peptides, sugars or polysaccharides, porphyrins and compounds with cis-trans photoisomerization.

16. The method of claim 15, characterized in that the compounds having a cis-trans photoisomerization are selected from diaryléthylènes derivatives, spiropyrannes, spiroxazines, fulgides and azobenzene.

17. Support functionalized with at least one molecule of interest and obtainable according to the grafting method as defined in any one according to one of the preceding claims, characterized in that said support comprises at least one silicon surface, having surface hydride functions, on which at least one molecule of interest is covalently grafted via at least one molecule of interest. a spacer compound of formula (I): (X) _n -E- (Y) _n, (I) wherein X, Y, E, n and m have the same significance as defined in any one of claims and in which the functional group (s) Y are engaged in a covalent -C-Het bond with at least one complementary functional group Z carried by said molecule of interest.

18. Support according to claim 17, characterized in that all the molecules of interest grafted to the surface of the support via the spacer compounds of formula (I) form a film in the form of a monomolecular layer.

19. Support according to claim 18 or 19, characterized in that the molecules of interest are chosen from nucleic acids, nucleotides, oligonucleotides, proteins, peptides, sugars and polysaccharides, porphyrins, diarylethylenes , spiropyrans, spiroxazines, fulgides and azobenzenes and that it constitutes respectively a nucleic acid chip, a nucleotide chip, an oligonucleotide chip, a protein chip, a peptide chip, a sugar chip, a polysaccharide chip, porphyrin chip, diarylethylene chip, spiropyran chip, spiroxazine chip, fulgid chip, or azobenzene chip.

20. Use of a support as defined in any one of claims 17 to 19 in the fields of electronics, biotechnology or the environment.