WO2008040769A1

WO2008040769A1 - Method for forming a polymer film on predetermined locations of a substrate

Info

Publication number: WO2008040769A1
Application number: PCT/EP2007/060519
Authority: WO
Inventors: Cyril Delattre; Arthur Soucemarianadin; Damien Vadillo
Original assignee: Commissariat A L'energie Atomique; Universite Joseph Fourier
Priority date: 2006-10-04
Filing date: 2007-10-03
Publication date: 2008-04-10
Also published as: FR2906810B1; FR2906810A1

Abstract

The invention relates to a method for forming a polymer film on predetermined locations of a substrate, that successively comprises: a) the step of depositing on said predetermined locations a droplet including an organic solvent and the polymer(s), said locations being wettable by said solvent while the areas surrounding said locations are not wettable by said solvent; b) the step of heating said substrate at a efficient temperature for evaporating said solvent. Application in the production of matrices of oligonucleotide, oligosaccharide probes.

Description

METHOD OF FORMING POLYMER FILM (S) ON PREDETERMINAL SITES OF A SUBSTRATE

DESCRIPTION

TECHNICAL AREA

The present invention relates to a method of forming a polymer film (s) at predetermined sites of a substrate.

In particular, this method applies to the protection of predetermined sites in the manufacture of a matrix of oligonucleotide probes (also called microarrays) having different sequences, for use in the analysis, diagnosis or research in genetics (mutations, polymorphisms, transcriptome), oligosaccharides or any other molecule of interest. This method can also be applied to the production of flat screens.

STATE OF THE PRIOR ART

Many chemical or biological analysis systems or devices are used for sequencing and studying gene expression. They consist of a set of molecular probes (oligonucleotides) attached to the miniaturized surface of a substrate to make a DNA microarray.

During the analysis of a sample, the extracted target nucleic acids are labeled and deposited on the probe array. Hybridization (pairing between the complementary strands of the DNA double helix) between the probes and the targets labeled allows to identify and identify the sequences of the DNA sample.

Various grafting and immobilization techniques of the probes (by covalent or electrostatic bonding) on different substrates (silicon, glass, polymer, gold electrodes) have been the subject of research and industrial developments.

The probe matrices are thus produced: either by immobilizing enzymatically or chemically presynthesized oligonucleotides by grafting them by mechanical, electrochemical or ink-jet means on a functionalized substrate or on a conductive polymer (commonly called "synthesis off chip");

or by in situ synthesis of the probe oligonucleotides on the structured substrate (commonly called "synthesis on chip").

In situ synthesis processes typically take place as follows:

1) a step of attachment to all the graft sites of a first nucleotide protected at the 5 'position by a protecting group;

2) a step of masking predetermined fixing sites;

3) a step of attachment to the non-masked sites of a second nucleotide on the first nucleotide; the masking 2) and fixing 3) steps being repeated until olinucleotides of desired sequence are obtained on each of the grafting sites. The fixation steps on the non-masked sites of a nucleotide (n + 1) on a nucleotide n take place according to a cycle generally comprising 4 phases: a phase of deprotection which activates the

5 'OH of nucleotide n chemically (e.g., when the protecting group is dimethoxytrityl), photochemically (e.g., when the protecting group is a photolabile group, such as ((α-methyl-2) (nitropiperonyl) oxy) carbonyl)) or alternatively electrochemically (for example, when the protecting group is a reducing electrolabile group, such as p-nitrobenzoyl); an addition coupling phase of the nucleotide (n + 1) protected at 5 'and activated at 3' with a phosphoramidite, phosphite or phosphonate group, with the 5 'hydroxyl of nucleotide n; a "capping" phase intended to eliminate unreacted reagents; an oxidation phase of the trivalent phosphorus in its pentavalent homolog of the 3 'position before starting the next cycle.

The masking step 2) defined above is carried out in order to protect the sites that are not intended to undergo the fixation of a nucleotide during the cycle defined above. This masking must be effective to finally obtain oligonucleotides of different predetermined sequences for given sites. In the prior art, in particular in document WO 0053310, the attachment of oligonucleotides is performed on functionalized sites in the form of cuvettes formed in the substrate.

The masking step is therefore carried out by depositing a protective cap in the appropriate cuvettes. The protective cap described in WO 0053310 is made with polyhydroxystyrene deposited in cuvettes by ejecting a solution comprising dimethylsulfoxide (solvent) and said polymer. The substrate is then heated in its entirety to evaporate the solvent and leave the polymer in the form of a film of inhomogeneous thickness in the cuvettes.

However, this technique is limited because it is technically difficult to deposit enough polymer to perfectly protect the cuvettes which must not undergo the nucleotide grafting step. In particular, the polymer accumulates unnecessarily on the walls of the cuvette without completely covering the bottom, regardless of the amount of polymer injected.

The inventors have therefore set themselves the objective of developing a method for protecting predetermined sites of a substrate from a substantially planar substrate, which would make it possible to overcome the drawbacks associated with the use of substrates having cuvettes, and which allows an exclusive localization of the polymer on the predetermined sites.

SUMMARY OF THE INVENTION Thus, according to a first object, the invention relates to a method of forming a film of polymer (s) at predetermined sites of a substrate comprising successively: a) a step of depositing on said predetermined sites a drop comprising an organic solvent and the polymer (s), said sites being wettable vis-à-vis solvents while the zones surrounding said sites are not wettable by said solvent; b) a step of heating said substrate to a temperature effective to obtain an evaporation of said solvent.

Through the judicious choice of a substrate comprising sites having wettability vis-à-vis the solvent in which the polymer (s) is (are) dissolved, while the areas surrounding the sites are non-wettable vis-à-vis With this solvent, it is thus possible to concentrate the drop on the selected sites and obtain after heating a film of said polymer covering only the selected sites.

The wettability is defined by the contact angle (or connection angle) that the drop forms with the substrate at the sites of deposition of the drop.

Thus, when it is specified that the deposition sites are wettable with respect to the solvent, this generally means that the deposited drop will form, with respect to the deposition site, a contact angle generally having a value of less than 40. ° whereas, for the non-wettable zones surrounding said site, it means that the contact angle formed between the droplet and these zones generally has a greater than 60 °, preferably greater than 80 °. From a practical point of view, this means that the drops deposited will spread over the entire surface of the wettable sites while they do not spread out or more difficult on the areas surrounding these sites. The drop thus remains localized on these sites and the polymer film obtained after evaporation of the solvent thus remains concentrated exclusively on the wettable sites. Concretely, in the context of the invention, the ejected drop comes to spread essentially on the wettable site and possibly slightly on the non-wettable areas surrounding this site. This eventual overflow is not detrimental since the evaporation step allows the polymer to refocus exclusively on the wettable site without remaining on the non-wettable areas surrounding said site.

From a chemical point of view, a liquid will wet a solid substrate if it has a chemical affinity to it. Thus a hydrophobic solid substrate will be wettable vis-à-vis the hydrophobic liquids and vice versa.

The method of the invention may comprise a step of preparing the substrate so as to create predetermined wettable sites with respect to the drops comprising the solvent and the polymer (s) intended to constitute a film of protection adhering to said sites and areas surrounding these non-wettable sites vis-à-vis said drops. Thus, the preparation of the substrate may consist in preparing on said substrate predetermined wettable sites vis-à-vis the solvent by chemical functionalization and non-wettable areas by said solvent surrounding said sites. These non-wettable areas may inherently exist because of the nature of the substrate, in which case only the wettable sites will have to be prepared or vice versa. Non-wettable areas and wettable sites may be created by appropriate treatments, if none of these properties pre-exist in the natural state on the substrate.

We explain below the preparation of a substrate comprising hydrophilic sites and hydrophobic zones, so as to be able to wet these sites with an aqueous solvent (comprising water) or DMSO.

Starting from a silicon substrate, the preparation thereof to delineate hydrophobic zones and hydrophilic sites may consist of:

subjecting said substrate to thermal oxidation so as to create a layer of silicon oxide; depositing on said silicon oxide layer a photoresist layer;

photolithographically removing the resin at predetermined areas; generating silanol functions on said predetermined areas by subjecting said predetermined areas to an oxygen plasma; - Grafting on said silanol functional groups a hydrophobic silane compound, such as perfluorooctyltrichlorosilane. removing the remaining photosensitive resin, thereby generating predetermined hydrophilic sites and hydrophobic areas surrounding said sites.

At the end of this preparation, a substrate having hydrophobic zones (zones grafted with the hydrophobic silane compound) and hydrophilic sites (sites surrounded by said zones and protected during grafting by a photoresist) are thus obtained.

These sites are wettable vis-à-vis a solvent selected by its wetting character, such as dimethylsulfoxide (DMSO). DMSO has the ability to dissolve polymers such as polyhydroxystyrenes. It will therefore be possible to protect, by a polyhydroxystyrene protective film, the aforementioned hydrophilic sites by depositing thereon a drop comprising dimethylsulfoxide (DMSO) and a polyhydroxystyrene.

The step of depositing the drop comprising the solvent and the polymer (s) at the predetermined sites can be done by mechanical addressing, for example, using selective microdeposition techniques of the piezoelectric actuator type, "pin and ring », Inkjet printing, Archimedean screw and micropipettage. Addressing the polymer by microdéposition makes it possible to distribute locally at the predetermined sites calibrated droplets of a polymer in solution. Moreover, because of the wetting of the predetermined sites relative to the surrounding areas, the drop automatically centers on the predetermined site in a few hundred milliseconds for drops having a diameter of a few millimeters.

Once the deposition step has been performed, the method of the invention comprises a step of drying the substrate at a temperature that is effective to obtain evaporation of the solvent. This drying is automatically accompanied by a centering of the polymer at the predetermined site (even if the initial drop has a larger size than the active site). The drying step is generally carried out by heating the substrate at a temperature ranging from 60 to 80 ^° C. for a duration ranging, for example, from 1 minute to 10 minutes and, preferably, from 2 minutes to 5 minutes. After drying, the polymers form at the predetermined sites tight covers fitted on these sites and can thus provide a protective role vis-à-vis these sites. Depending on the heating strategy adopted (heating power more or less important, heating directly after deposition), it is possible to control the size and the final geometry of the deposit spots, in order to obtain homogeneous protection caps centered and of controlled thickness.

Different heating means can be envisaged, such as direct contact heating substrate or via a hot air stream or by application of infra - red radiation.

This method can find application in a wide variety of fields, including all areas requiring the deposition of a polymer film exclusively at selected sites from solutions comprising it.

This may be the case for processes for producing a matrix of sequences of chemical or biological molecules formed by different sequences of molecules, such as DNA chips, oligosaccharides, proteins, etc.

Given that these matrices have different sequences on different sites, it is necessary during the in situ synthesis of these sequences to protect certain sites to avoid a grafting of molecules thereon, the protection consisting in implementing the method of Polymer film formation described above. Thus, the invention also relates, according to a second object, to a process for producing a matrix of sequences of chemical or biological molecules formed by different sequences of molecules M1, M2,. located on a substrate as defined above, comprising successive steps of grafting on predetermined sites of a molecule Ml, M2, ... or Mn constitutive of said sequences, said predetermined sites comprising functional groups able to form a bond covalent with said molecule M1, M2, ... or Mn, these grafting steps being preceded, when necessary, a site protection step not requiring this grafting, this protection step being performed by the implementation of the method of forming a polymer film as defined above, this film of polymer being removed when the site concerned must undergo a grafting step again.

By virtue of the protection of certain sites, during the production of the sequences, the sequences of chemical or biological molecules formed from different sequences of molecules M ₁ , M ₂ , .sub.m are obtained at the end of the process. _n .

The functional groups capable of forming a covalent bond with the molecules M ₁ , M ₂ ... or M _n may be derived from a molecule M ₁ , M ₂ ,... Or M _n previously grafted or, when is the first grafting cycle, a functional group directly attached to selected sites of the substrate, which group generally conferring wettability at this site vis-à-vis the solvent of the solution comprising the polymer intended to form a protective film.

The functional group resulting from a previously grafted molecule may be, when the molecules constituting the sequence are nucleotides, a 5 'OH group capable of creating a covalent bond with the phosphoramidite of the 3' position of the nucleotide to be grafted.

The functional group bonded directly to the substrate may be a silane group comprising, for example, Si-OH bonds, which group is therefore: adapted by its wetting character to allow the deposition of a localized polymer film according to the method of the invention; and

- To react, when not protected by a polymer film (s), with reactive functions facing the silane group of a molecule Mi, M ₂ , M _n to form a covalent bond.

As an example of such a group, there may be mentioned a silane group carrying a function of the diol or epoxide type.

The molecules M ₁ , M ₂ , ... M _n can be of different types. By way of example, it may be natural or synthetic amino acids L- or D-, nucleotides (RNA or DNA), pentose or hexose. In the case of amino acids, the number of molecules Ml, M2, ... M _n can be important. In the case of nucleotides, the number of molecules is more limited, since it includes A, T (or U), G and C.

In the case where the molecules to be grafted onto the matrix are oligonucleotides, the in situ synthesis on predetermined sites as defined above comprises the implementation of several cycles of synthesis respectively comprising, apart from the first nucleotide of the sequence a step of coupling the 5 '-OH end of a grafted nucleotide with the 3' activated end (phosphoramidite, phosphotriester or H-phosphonate) of another nucleotide to form a covalent bond between these two nucleotides; a step of oxidation of the phosphoramidite, phosphotriester or H-phosphonate group to a phosphate group; part of these synthetic cycles being carried out only on some of the predetermined sites, the other sites then being protected by a polymer film produced by the film-forming method as defined above, which film is subsequently removed in order to to be able to undergo a synthesis cycle, whereby at the end of the process oligonucleotides grafted at the predetermined sites, having different sequences, are obtained.

It should be noted that the grafting of the first nucleotide on each of the predetermined sites will be by coupling this nucleotide with functional groups directly linked to the predetermined sites, these functional groups also conferring the wettability characteristics mentioned above at the predetermined sites.

Prior to the coupling step, it may be necessary, if necessary, to carry out a step of deprotection of the -OH end at 5 ', this end being generally protected before coupling by a trityl group, such as the dimethoxytrityl group.

Between the coupling step and the oxidation step, a reactive functional groups (usually the 3 'end group) of the nucleotides can be blocked (called the "capping" step). graft does not have reacted, so that these nucleotides do not intervene in the subsequent synthesis cycles.

Synthetic cycles (optionally comprising a deprotection step, a coupling step, optionally a blocking step, and an oxidation step) are conventionally carried out in an automatic oligonucleotide synthesizer, by soaking the substrate in the reaction chamber. of the synthesizer. The step of forming a protective film on some of the predetermined sites during certain synthesis cycles (which corresponds to a selective protection of these sites by a protective polymer) is carried out conventionally using microdeposition techniques.

The choice of the protective polymer in the matrix embodiment is very important and must advantageously meet a certain number of criteria, such as the ejectability which will be a function of the concentration and the nature of the polymer and the resistance to the chemical steps.

First of all, the polymer must advantageously be deposited by the microdeposition techniques mentioned above, by means of a solution of the polymer in a solvent.

In the case of nucleotide synthesis, and wherein the protective polymer is to be maintained throughout the synthesis cycle as defined above, it should preferably be inert, and preferably free of reactive functions capable of coupling to molecules and synthetic reagents. It must not be soluble in the solvents used in the stages of the cycle and have a impermeability vis-à-vis these solvents.

In reality, it is sufficient that the polymer plays its role of protective cover essentially in the deprotection step (usually a detritylation step) or in the coupling step. It is then possible to adapt and optimize the polymer-solvent pair for the selected step.

For example, in the case of DNA chips, it is preferable for the protective polymer to have no functions with OH, NH ₂ or COOH free hydrogens which would be capable of coupling with the M ₁ , M ₂ molecules. , ..., M _n , for example, the nucleotides, during the coupling step, to avoid unnecessarily consuming reagents which will then be removed with the protective polymer. The polymer must be removable by a solvent inert to the nucleotides.

The choice of the protective polymer will be carried out: first, depending on the step (s) during which it must protect some of the grafting sites, taking into account its solubility in solvents that can be used for the synthesis of nucleotides; and

on the other hand, depending on the nature of the grafting sites to be covered with a film, it being understood that these sites must be wetting with respect to a solvent of this protective polymer, so that a drop comprising the solvent and the polymer can concentrate exclusively on these sites, during evaporation. In other words, the polymer must be soluble in a wetting solvent vis-à-vis the grafting sites and insoluble in the solvents used in the steps against which the polymer must protect the chosen sites. In the case of the synthesis of oligonucleotides, if it is desired to ensure protection by the polymer only during the deprotection step of the 5 'ends of a nucleotide, generally a detritylation step, it is sufficient to choose an insoluble polymer. in detritylation reagents which generally consist of dicholoroacetic acid (DCA) or trichloroacetic acid (TCA) or a ZnBr ₂ type Lewis acid, at about 2-5% in a solvent such as dichloromethane, acetonitrile or toluene.

Thus, examples of such polymers include polyhydroxystyrenes which are insoluble in the solvents mentioned above (dichloromethane, acetonitrile or toluene) and soluble in a solvent different from those listed above, which solvent is wetting with respect to grafting sites to protect.

Examples of polymer / solvent pairs are presented in the following table.

For example, starting from a substrate having predetermined sites grafted with a silane compound carrying an epoxide or diol function (serving as a spacer arm between the support and the sequence of nucleotides and conferring wettability characteristics at these sites) and areas surrounding these sites consisting of a silicon oxide layer on which is grafted a perfluorinated silane, the predetermined sites can be protected by depositing thereon a drop of a solution comprising dimethylsulfoxide (DMSO) and a polyhydroxystyrene (soluble in DMSO), this drop concentrating exclusively on the wettable sites by the nature of the silane compound grafted to DMSO and after evaporation constituting a protective polymer film on these sites. The unprotected sites may undergo the synthesis cycle steps to graft the desired nucleotides while the protected sites do not undergo this grafting. In this case, the polymer film is then removed for example, by washing with THF, thus releasing the site for subsequent nucleotide grafting.

The method of forming polymer films as well as the method of making matrix of different sequences have many advantages.

Indeed, compared with the method described in reference WO 0053310 which uses a substrate having on the surface microcuvettes, to constitute the grafting sites, the method of the invention is implemented with essentially planar substrates (that is, that is, with no surface relief), the difference in wettability between predetermined sites and the zones surrounding these sites making it possible to position the polymer films exclusively on these sites, without having to use cuvettes capable of accommodating the films.

Because the deposited drop focuses on predetermined sites because of its wettability characteristics, it is possible to increase the density of the substrates at these predetermined sites. Given that these predetermined sites are functionalized so as to give them adequate wettability properties and also to allow the grafting of molecules, it is thus possible to obtain, for example, matrices having a very high number of oligonucleotide probes. different sequences. Since the deposited drop is automatically positioned at the predetermined sites, the film forming method is a fast method of implementation.

Also, because following the evaporation of the solvent, the polymer concentrates only on the desired site, it is possible to employ solutions prior to more diluted ejection, which facilitates the ejection in the process.

It should be noted that, according to the invention, the ejection does not have to be as precise as, according to the prior art, because the difference in wettability between the zones and the sites allows easy repositioning of the polymer film on the surface. site to hide. In particular, it is entirely acceptable during the ejection of the drop comprising a solvent and the polymer, to possibly overflow from the wettable site to the non-wettable zones, the subsequent evaporation step making it possible to concentrate the polymer exclusively on said wettable site.

The film forming method of the invention has excellent reliability because it allows better protection by the polymer of the predetermined sites.

It is also possible thanks to this process to optimize the size and shape of the film of polymer, and the polymer concentration depending on the size of the site.

In addition to allowing the production of protective films in the context of the synthesis of molecules of interest, such as oligonucleotides, the method of the invention can be implemented also for the production of flat screen, which requires in their design, the deposition on a substrate at well-defined locations of droplets comprising polymeric inks, these polymeric inks may be, for example, polyimide inks.

The invention will now be described with respect to the examples below given for illustrative and not limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a view from above of the matrix produced according to the example below, this matrix presenting several pairs of lines (pairs of lines 18, T, g, C, A, 14 and 18) presenting a set of sites, each of said sites corresponding to a given oligonucleotide sequence grafted.

FIG. 2 schematically illustrates an oligonucleotide sequence grafted on a site of the matrix represented in FIG. 1. DETAILED DESCRIPTION OF PARTICULAR EMBODIMENTS

EXAMPLE 1

On a new silicon plate is carried out thermal oxidation at 1100 ⁰ C to create a silicon oxide layer of 5000 Å. On this layer is deposited a layer of photoresist followed by a photolithography step performed to create photoresist discs to define circular sites. The plate is then subjected to an oxygen plasma which has the effect of activating the surface of the zones not protected by the resin by generation of silanol sites. The entire plate is then silanized with a hydrophobic silane (perfluorooctyl-trichlorosilane) for 10 minutes at room temperature in anhydrous toluene.

(silane concentration of 9 mM). The hydrophobic silane molecules will graft only on the areas not protected by the resin. The plate is then washed with acetone, ethanol and sonicated for 5 minutes in ethanol. The chip is then baked for 1 hour at 110 ^° C. The contact angle measured with water is 110 °. The photoresist is removed during passage through the acetone and ethanol baths. The contact angle measured with water on the areas protected by the resin is close to 50 °.

Ejection experiments are carried out with a piezoelectric ejection head (robot from MicroDrop GmbH) and a solution of dimethylsulfoxide (DMSO) containing 5% by weight of poly (4-hydroxystyrene) (PHS) with a molar mass of around 20,000 g. mol ^'1 . Three drops of 90 μm diameter in flight are ejected on an active site of 85 microns in diameter manufactured by the technique described above. Then the substrate is placed on a hot plate at 80 ° C for 5 minutes to evaporate the DMSO. An image in scanning electron microscopy is taken after this heating step which shows that the polymer is centered only on the active site without overflowing. It should also be emphasized that the thickness of the polymer film (15 μm) is much greater than that which can be obtained with the previous method using cuvettes (always less than 1 μm).

EXAMPLE 2

On a new silicon plate is carried out thermal oxidation at 1100 ⁰ C to create a silicon oxide layer of 5000 Å. On this layer is deposited a photosensitive resin layer followed by a photolithography step performed so as to define patterns in the photoresist. The plate is then subjected to an oxygen plasma which has the effect of activating the surface of the areas not protected by the resin by generation of silanol sites. The whole plate is then silanized with a hydrophobic silane (perfluorooctyl trichlorosilane) for 10 minutes at room temperature in anhydrous toluene (silane concentration of 9 mM). The hydrophobic silane molecules will be grafted only on the areas not protected by resin. The plate is then washed with acetone, ethanol and sonicated for 5 minutes in ethanol. The chip is then baked for 1 hour at 110 ^° C. The contact angle measured with water is 110 °. The photoresist is removed during passage through the acetone and ethanol baths.

A photolithography step is again performed so as to define patterns in a photosensitive resin of negative polarity. The resin is left on all previously hydrophobized areas by silanization. The plate is subjected to an oxygen plasma which has the effect of activating the surface of the zones not protected by the resin (generation of silanol sites). The entire plate is then silanized with 5mM silane-5, 6-epoxyhexyltriethoxysilane silane in anhydrous toluene with 3% by volume of triethylamine for 16 hours at 80 ^° C. The plate is then washed with acetone, ethanol and sonicated for 5 minutes in ethanol. This procedure also removes the photosensitive resin that protected the hydrophobic areas. The plate is then placed in a bath of 0.2N aqueous HCl solution for 3 hours. The plate is finally cut in the form of square substrates 1 cm apart.

The substrate thus obtained is then used to produce different oligonucleotides, using an automatic synthesizer of oligonucleotides EXPEDITE 8909. Each oligonucleotide is synthesized by grafting nucleotides, the first nucleotide being grafted onto the grafted silane compound (reference 3 in FIG. 2) on predetermined locations (reference 1 in FIG. 2) of the silicon wafer.

Each oligonucleotide comprises the following sequence:

5T spacer (reference 5 in FIG. 2) - Known sequence of 7 nucleotides (reference 7 in FIG. 2) - Variable base (reference 9 in FIG. 2) - known sequence of 7 nucleotides (reference 11 in FIG. 2); the spacer 5T corresponding to a sequence of 5 bases T, linked to the silane compound grafted

(Reference 3 in Figure 2) at predetermined locations (reference 1 in Figure 2) of the silicon wafer, the variable base being A, C, G, T or a single bond. The synthesis protocol comprises a first step of deposition of a first base common to all the pads: the modified thymine, which reacts via its 3 'phosphoramidite function with the OH group of the silane compound grafted to form a bond covalent between the silane compound and the thymine.

Then, the 4 bases T as well as the known sequence of 7 nucleotides are grafted base by base according to a conventional protocol using phosphoramidite chemistry comprising a cycle of four steps: a step of detritylating the grafted base to release the 5'-reactive OH function; a coupling step of the base to be grafted with the grafted base, by reaction of the -OH function in 5 'of the grafted base and the phosphoramidite function of the base to be grafted; a capping step for the reactive -OH functions 5 'unreacted graft nucleotides, so that these nucleotides do not intervene in the subsequent synthesis cycles;

an oxidation step for oxidizing the trivalent phosphorus to its pentavalent homologue before starting the next cycle.

This cycle is repeated 11 times so as to obtain the sequence of 5 bases T and the first known sequence of 7 nucleotides.

Then, the coupling of the variable base A is carried out according to the following synthesis cycle: after removal of the synthesizer substrate, deposit in a polymer deposition robot (robot of MicroDrop GmbH) on the sites not to undergo grafting base A, 2 drops of a solution of 3% PHS / DMSO which is then evaporated at 80 ^° C. for 2 minutes; detritylation of unmasked grafted bases by subjecting them to 1 ml of DMT-Removal solution (3% trichloroacetic acid / dichloromethane) for 1 minute with shaking;

rinsing in a dichloromethane bath; - coupling of the base A;

- Removal of the polymer in a tetrahydrofuran (THF) bath.

Given the difference in wettability of the DMSO between the silanized sites and the non-wetting silanized zones, the film is localized only and uniformly at the selected sites and thus prevents the base A from grafting onto the unwanted sites. This synthesis cycle is repeated to obtain the coupling of the base C, G and T.

Once the 4 synthesis cycles have been carried out, the second known sequence of 7 nucleotides is carried out by a protocol identical to that described above for the synthesis of the first known sequence of 7 nucleotides.

Finally, the substrate is taken out of the synthesizer and is incubated in NH ₄ OH (Sigma-Aldrich 32014-5) for 45 minutes at 60 ^° C., in order to eliminate the protective groups of the nucleotide bases, and then it is rinsed with water and dried under a stream of nitrogen.

At the end of this process, a matrix comprising a set of pairs of lines is thus obtained, each of the lines having a predetermined number of sites grafted by oligonucleotide probes of known sequence (see FIG. 1). For a given pair of lines, all the sites are grafted by the same nucleotide probes (28 probes in total for each pair of lines). The characteristics of these lines are given in the following table.

The hybridization is carried out with a solution of oligonucleotide targets of 5 'CAT AgA sequence gTg ggT TTA TCC 3' (complementary to the oligonucleotide of the "C" lines) at 10 nM. These targets carry a fluorescent group Cy3 on the 5 'end. After hybridization, the substrate is rinsed and dried.

The fluorescence reading is performed with the Genetac scanner (Genomic Solutions) with a gain of 34.

The image obtained in fluorescence corresponds to the result that was expected: the "C" line corresponding to the hybridization between two complementary oligonucleotide sequences produces the highest fluorescence signal; - The lines "T", "g", "A" and "14" give a weaker signal because the hybridization is not perfect because of the difference of a base in the sequence; the "18" lines have a very weak, almost negligible signal because the oligonucleotide sequences differ by 3 bases.

Claims

1. A method of forming a polymer film (s) at predetermined sites of a substrate comprising successively: a) a step of depositing on said predetermined sites a drop comprising a solvent and the polymer (s) ), said sites being wettable by said solvent, while the areas surrounding said sites are not wettable by said solvent; b) a step of heating said substrate to a temperature effective to obtain an evaporation of said solvent.

2. The process for forming a film according to claim 1, comprising, before step a), a step of preparing the substrate so as to create predetermined wettable sites with respect to the drops comprising the solvent and the polymer. and areas surrounding these non-wettable sites vis-à-vis said drops.

3. A method of forming a film according to claim 1 or 2, wherein the predetermined sites are hydrophilic sites and the areas surrounding said sites are hydrophobic areas.

The method of forming a film according to claim 3, wherein, when the substrate before preparation is a silicon substrate, the step of preparing said substrate comprises the steps of: subjecting said substrate to thermal oxidation so as to create a layer of silicon oxide; depositing on said silicon oxide layer a photoresist layer;

photolithographically removing the resin at predetermined areas; generating silanol functions on said predetermined areas by subjecting said predetermined areas to an oxygen plasma; grafting on said silanol functional groups a hydrophobic silane compound, such as perfluorooctyltrichlorosilane. removing the remaining photosensitive resin, thereby generating predetermined hydrophilic sites and hydrophobic areas surrounding said sites.

5. Formation method according to any one of the preceding claims, wherein the deposition step is carried out by mechanical addressing.

The forming method according to claim 5 dependent solely on claim 4, wherein the drop comprising a solvent and the polymer is a drop comprising dimethyl sulfoxide and polyhydroxystyrene.

7. Process for producing a matrix of sequences of chemical or biological molecules formed by different sequences of molecules Ml, M2, ..., Mn by in situ synthesis on a substrate as defined in claim 1, said method comprising successive steps of grafting on predetermined sites of a molecule Ml, M2, ... or Mn constituting said sequence, said predetermined sites comprising groups functional groups able to form a covalent bond with said molecule M1, M2,... or Mn, these grafting steps being preceded, where necessary, by a site protection step that does not require this grafting, this protective step being carried out by implementing the process for forming a polymer film as defined in any one of claims 1 to 6, this polymer film being removed when the site concerned must undergo a grafting step again.

The method of claim 7, wherein the molecule sequences are oligonucleotides.

9. The method of claim 8, wherein a step of grafting a nucleotide, except the first nucleotide of the sequence, comprises successively the following substeps: - coupling the -OH end 5 'of a nucleotide grafted with the 3'-activated end (phosphoramidite, phosphotriester or H-phosphonate) of another nucleotide to form a covalent bond between these two nucleotides; oxidation of the phosphoramidite, phosphotriester or H-phosphonate group in phosphate group.

The method of claim 7, wherein the molecule sequences are oligosaccharides.

11. A method of producing a flat screen comprising a step of implementing the method as defined in claim 1.