WO2002026373A1 - Method and device for making a support bearing a plurality of different polynucleotide and/or peptide sequences - Google Patents

Abstract

The invention concerns the manufacture of electronic micro-sensors (chips) for analysing nucleotide and/or peptide sequences. The object is to fix at the surface of said chips a very large number of various probe sequences easily and economically. The method aims at making arrays of sequence probes on a support by anchoring and/or local chemical synthesis in the presence of volatile solvents. Said method is characterised in that it consists in using a support whereon is produced a grid of reaction wells made of light-cured (epoxy) resin; in placing the support in a reaction chamber which is saturated with vapours of the synthesis solvent (acetonitrile); in finally producing the anchorage and optionally the localised chemical synthesis in situ of the probe sequences in the wells using a localised supply of liquids/reagents in each well corresponding to a specific probe-sequence, and in supplying collectively the wells with common liquids/reagents and in ensuring that prior to each localised supply operation the liquid contained in each well is eliminated. The invention also concerns a device for implementing said method.

Description

METHOD AND DEVICE FOR MANUFACTURING A SUPPORT CARRIING A PLURALITY OF DD7FERENT POLYNUCLEOTTDE AND / OR PEPTIDE SEQUENCES

TECHNICAL AREA

The field of the invention is that of the manufacture of microsensors useful in the analysis of nucleotide and / or peptide sequences. This type of analysis opens access to the detection and identification of all or part of genes or peptides which may be the tracers of higher biological entities (cells, tissues, bacteria, viruses, fungi, yeasts,. ..).

The production in question in this presentation is an anchoring and / or a localized chemical synthesis of polymer probes (oligonucleotides and / or peptides) on a support intended to form a network of probes, some of which are likely to pair with target sequences contained in the analysis medium. The revelation of these pairings can be carried out using fluorescent, colored, radioactive markers or by electronic transductance.

More specifically, the invention relates to a method of manufacturing a support carrying on at least one of its faces a plurality of sequences - poly - preferably oligo - nucleotide and / or peptide (OGN sequences) advantageously different from each other and apparent from their complementary (target sequences) by affine interaction, these OGN sequences being obtained by localized chemical synthesis, in the presence of volatile solvents. The present invention also relates to the device for implementing this method, as well as the support included in this device and used in the method.

The invention also relates to a method for detecting and / or identifying target OGN sequences using biosensors manufactured by the above-mentioned method. Within the meaning of the present description, the OGN sequences correspond to poly- preferably oligo-nucleotide and / or peptide sequences. In other words, it can be: - oligonucleotides each carrying an ATCG nitrogen base,

- oligopeptides whose recurring unit is an amino acid,

- or peptide-nucleic acids - APN - ("PNA = Peptide Nucleic Acid") which are polymers with petptide backbone comprising for example N- (2-aminoethyl) glycine repeating units linked to each other by amide bonds , each unit carrying a pyrimidine or purine nitrogen base,

- or even chimeras with a mixed structure of nucleic acids and peptide-nucleic acids. The explosion of genetics, genomics and biotechnologies in the medical field and in the whole industry, creates a need for a tool for analysis of nucleic or amino acid sequences, this tool having to be high resolution, extremely fast and reliable. With regard to nucleic acid sequences, this need for an efficient analysis tool is expressed for numerous applications such as for example the study of gene mutation, the detection of pathogens, or even the sequencing of genomes.

The principle of the analysis of nucleotide and / or peptide sequences is relatively simple since it involves reading the arrangement of the 4 nucleotide comonomers A, C, T and G in the case of polynucleotides and PNAs and the arrangement about twenty amino acids with regard to the polypeptides. This simplicity of principle is only matched by the difficulty arising from the considerable amount of information to be processed. The genome of the bacterium Escherichia Coli, which is one of the shortest, contains 4,000 genes with 4.7 million nucleotides while the human genome consists of approximately 100,000 genes, which represents 3 billion nucleotides. . Added to this is the fact that the decryption of a gene or a protein is nothing compared to the understanding of the complex action of these nucleic or peptide polymers in normal or abnormal living mechanisms.

This further underlines, if need be, the fact that it would therefore be particularly useful to have tools or sensors enabling rapid and reliable analysis of a large number of nucleic or amino acid sequences. . PRIOR ART

To meet this need, it has therefore been proposed in the prior art various analysis tools and different techniques for producing these tools. PCT international application WO 89/10 977 (inventor: Southern), describes a method and a device for the analysis of a polynucleotide sequence. According to this invention, a support is used, for example a glass plate, comprising a network or a complete set of oligonucleotide sequences (OGN probes) capable of hybridizing with complementary target OGN sequences contained in the analysis medium. . The revealing of the hybridizations likely to occur on the network of OGN is carried out using labeling agents which can be carried by the probes, and / or by the targets. This concept of biosensors carrying a network of OGN probes makes it possible to obtain a fingerprint of the genetic content of the analysis medium as well as a partial or complete sequencing of said genetic content, and this in a single analysis. In theory, this concept is all the more attractive since it should be possible with a network of OGN probes (which one can also call "pixels" by analogy with photography), different between them and numerous (from a few hundred several million), to perform analyzes in a medium comprising multiple populations of polynucleotide sequences, without the need for cloning. But beyond the obvious theoretical interest, we are faced with a difficult practical problem which is that of the production of supports carrying networks of OGN probe sequences of length equal to one, two or three tens of seas each. It is indeed a question of fixing OGN probes of macromolecular size on surface area units of extremely small size and according to a precise and determined localization of each probe.

The concrete solution to this practical problem adopted in the examples of PCT application WO 89/10 977 is as follows. Glass microscope slides are used as a support. These plates are subjected to silanization, that is to say to a treatment using 3-glycidoxypropyltriethoxysilane intended to be fixed to the surface of the glass to form an anchor point for the OGN sequences. The silane units are subjected to an acid treatment to offer hydroxyl groups capable of reacting with the overhead monomers (rank 1) of the OGN sequences. This synthesis of the OGN oligonucleotides is carried out according to known chemistry known as phosphoramidites. The 5 'ends of the nucleotides are protected by DMT (dimethoxytrityl) and the 3' ends by β-cyanoethylphosphoramidite. The couplings of the nucleotides of rank 1 and on the silanes and of the nucleotides of rank 2 to n between them are carried out using tetrazole. The solvent is acetonitrile. This reaction is carried out under anhydrous conditions in a sealed container. This container includes the glass plate covered with a silicone rubber ribbon which is hollowed out to leave only the edges which define a single reaction site. A Teflon plate of the same size and thickness as the glass support plate covers the reaction site and the silicone border. A short plastic tube connects the reaction volume with the outside and allows the injection and draining of the reaction liquids as well as the filling of the reaction volume with argon, these operations being carried out by means of a syringe. After each nucleotide coupling, this assembly is. disassembled to continue the reaction by immersing the support of the reagent baths or reaction liquids, to then be reassembled in order to carry out the following coupling.

It goes without saying that this technique for manufacturing sensors with OGN probe arrays is absolutely not industrial, in particular because of its heaviness and its complexity. In addition, it makes it possible to produce, only in the examples of this PCT, networks comprising very few oligonucleotides, namely 24 and 72 units. These networks represented in Tables 1a and 1b of the PCT application. The composition of the OGN sequences varies very little and, moreover, a good number of bases are missing. It therefore appears that the technique for manufacturing networks of OGNs practiced according to the PCT WO 89 / 10,977 can be greatly improved.

The authors of PCT application WO 89/10 977 hypothesize that it should be possible to manufacture the matrix of OGNs by synthesizing oligonucleotides in the cells of a network, using automatic equipment comprising a printing device controlled by a computer and comprising an ink jet printing head. But this is only an idea that has not been reduced in practice. And in reality, this reduction in practice turns out to be most delicate. The complexity and the number of reactions to be implemented are such that the manufacture of an OGN network cannot be a simple adaptation of known techniques of anchoring and synthesis on a support of oligonucleotides or oligopeptides. The techniques mentioned in PCT application WO 89/10 977 fall under a manufacturing method that can be called "in situ" consisting in synthesizing the probes on the substrate by incrementation, base by base, simultaneously for all the PIXELS of support.

There is another group of manufacturing techniques which can be described as "ex-situ" based on the prior production of the set of OGN probes independently of the treatment of the support, this production being followed by the fixing of said probes on the support. .

These techniques prove to be difficult to implement when the set of OGN probes exceeds a few tens of units and prove impractical beyond a few hundred units. PCT international patent application WO 92/10 092 (inventors:

FODOR et al) corresponding to the article Science vol 251, 767 - 773, 1991; discloses an in situ synthesis localized on the support, photochemically. To this end, the nucleotide bases are protected at their 5 ′ end by a photolabile group such as nitrotératryloxycarbonyl (NVOC) which replaces the protective group (DMT), used in the synthesis according to the chemistry of phosphoramidites. The support is a glass plate carrying amine or hydroxyl groups also protected by N OC. The action of localized light radiation on the substrate leads to the photochemical cleavage of the photolabile group and to the deprotection of the irradiated surface. It is then possible to locally add an oligonucleotide, also protected by an NVOC group. By illuminating different areas of the support using a combinatorial synthesis strategy, FODOR et al synthesize a network of OGN probes. The major drawback of this FODOR et al technique is linked to the harmful influence of the NNOC group on the synthesis yield. This leads to sequencing errors and therefore to a reduction in the useful area of the sensors. In addition, this FODOR process is expensive and rigid in its implementation (mask, reagents not commercially available). Another drawback of this manufacturing method is that the combinatorial strategy implemented leads to networks the composition of which varies little from one pixel to another, most often the composition of the probe varies only by a base d pixel to pixel. Differentiation of hybridizations of neighboring pixels can therefore be difficult.

This technique for synthesizing OGNs using photolabile protective groups of the NVOC type, is also mentioned in the international PCT application WO 93 / 09,668 which describes combinatorial strategies for the synthesis of networks of nucleotide or peptide polymers on a substrate. Reference is made in this PCT application to a localized in situ synthesis of the various OGN monomers in predefined regions of the substrate. The techniques recommended in this PCT application for chemical syntheses are of two types:

1 - circulation of a flow of reaction liquids inside channels formed in a block and one of the chips of which is formed by the surface of the substrate in defined or predefined regions;

2 - deposition or projection of drops of reaction liquid onto predefined regions of the substrate,

3 - combination of techniques 1 and 2. In technique 1, a block of channels is used comprising a set of parallel grooves which are brought into contact with the surface of the substrate so as to thus define the channels in which the reaction liquids are intended to circulate. The method then consists in fixing one or more different monomers of rank 1, in dismantling the substrate assembly, channel block, for the steps of washing the protections, in reassembling the assembly to anchor the monomer (s) of rank 2 and so on until the monomer of rank n. The combinatorial strategy consists of moving the substrate relative to the block of channels in rotation (90 °) or even in translation. According to a variant of technique 1, there is provided as shown in FIG. 12, a support comprising a plurality of reaction wells and a reagent dispenser comprising a set of tubes for dispensing reagents or reaction liquids into the wells of the support. According to this variant, it is possible to interpose a mask between the distributor tubes and the wells of the support so as to control the supply of the reaction liquids in given regions of the support. Concerning technique 2, this PCT application generally discloses supports comprising at least 10,000 reaction regions per cm ² . No concrete embodiment of this type of support is exemplified. This PCT application also describes means for distributing reaction liquids in these reaction regions of the support. The inkjet printer heads are cited as examples of means for distributing reaction liquids in the reaction regions of the support. One of the techniques proposed for producing reaction regions on the support is that of obtaining surface tension wells on the surface of the support by creating hydrophobic zones delimiting hydrophilic zones, for example by applying a layer of hydrophilic resin carrying protected hydrophobic groups and by deprotecting these hydrophobic groups in the zones intended to form the contours of the surface tension wells. According to an alternative, the reaction regions can be formed by three-dimensional wells delimited by walls as shown in FIG. 12.

The deprotection of the hydrophobic groups is advantageously carried out according to this PCT request by photolysis. This PCT application WO 93/09 668 also alludes to the problems of evaporation of solvents which can affect the syntheses of the OGN sequences. To overcome this difficulty, it is proposed to use sealed reaction chambers, to reduce the vapor pressure (low temperature) and / or to saturate the reaction chamber with volatile solvents. It should be noted that there is a significant step between these speculative generalities and concrete practical realizations which work. This is confirmed by the fact that the examples given in this PCT application relate only to illustrations of technique 1 which uses reaction fluid circulation channels. This technique makes it possible, in the examples, to produce a network comprising 2,500 reaction regions. OGN sequences of oligonucleotides are synthesized in all the reaction regions. For the attachment of the 7 ^th and 8 ^th monomers, the block of reaction liquid circulation channels is used which it is attached to the surface of the substrate already comprising OGN sequences of 6 seas each. The 7 ^th nucleotide is distributed by a set of parallel channels of given direction and the 8 ^th nucleotide is distributed by flows circulating in channels whose direction is perpendicular to that of the channels intended to receive the 7 ^emβ nucleotide. In this example, we therefore create octamer OGN sequences only at the intersections between the channels.

It does not appear that the method according to this PCT application WO 93 09 668 allows rapid, reliable and easy obtaining of networks of oligonucleotides of different compositon. At each rotation of the substrate with respect to the block of channels, the device should be disassembled, which considerably burdens the in situ synthesis process and localizes OGN.

Patent No. 5,658,802 of the United States of America (DJ HAYES et al) relates to a process of ex and in situ synthesis based on the localized projection of drops of reagents on a substrate, using piezo systems. electric. Since in practice the most widely used solvent for diluting nucleotide bases is acetonitrile and this solvent has a low wetting angle, this results in problems of spreading the drops of solvent on the substrate on several hundred microns. This makes it difficult to locate reactions on an appropriate scale. In addition, the acetonitrile thus used is volatile and therefore subject to massive evaporation.

The device according to US Pat. No. 5,658,802 comprises a substrate of the type of those used for the production of integrated circuits optionally having patterns. There is no question of delimiting reaction sites of very small area according to this patent. Each piezoelectric ejection means is associated with a liquid reservoir. All these piezoelectric ejection means and its reservoirs form a distribution assembly. Also provided in this device are positioning means, means for ejecting liquid and / or the substrate.

It appears from the above that the method and the device according to this US Pat. No. 5,658,802 are not operational for the production of networks of OGN sequences by localized chemical synthesis, using volatile solvents.

Concerning the problem of controlling the spreading of the drops of reaction liquid on the substrate and their confinement in multiple reaction sites distinct and isolated from each other, reference is made in PCT application WO 93/09 668 studied above, to the technique of surface tension wells which consist in creating hydrophobic zones surrounding hydrophilic zones of approximately 100 μm in diameter so as to delimit a plurality of reaction sites by means of surface tension differentials. The document AP BLANCHARD et al (biosensors - bioelectronics 11, 687 - 690 - 1996) also deals with this technique. However, it must be recognized that the latter satisfactorily solves this problem only for aqueous solvents. However, it turns out that these are not suitable solvents for reactions of synthesis of oligonucleotides or oligopeptides. In reality, the surface tension well technique is not appropriate in the case where the reaction liquids are constituted by volatile organic solvents such as acetonitrile. In fact, taking into account the surface tension characteristics of such volatile solvents, the separation of the reaction sites by the play of a surface tension differential is not very effective. In addition, the problem of the evaporation of solvents distributed in very small quantities remains unresolved in technical proposals which involve surface tension wells.

PCT application WO 94/27,719 concerning a method and a device for producing a chemical reaction network on the surface of a support. According to this PCT request, surface tension wells are formed on the surface of the support using photoresists of the phenol / formaldehyde or polymethacrylate type. The use of means for distributing reaction liquids of the piezoelectric inkjet printer head type is recommended in this PCT application.

It is therefore clear that the prior art does not propose an efficient and effective method for producing the surface of a support, of a network comprising a very large number of OGN sequences of different and varied nature. It appears that many of the methods and devices described in the literature have no concrete application and have not been reduced in practice. In addition, those which escape this rule lead to OGN sequence networks comprising a small number of pixels or having many erroneous OGN sequences. This last point reflects the weakness of these methods with regard to the synthesis yields. We know that an average efficiency of 92% reduces the useful area of each pixel to 51% for 8-sea probes and 36 for 12-sea probes. The presence of erroneous sequences on a large part of the sensors complicates the detection of hybridizations or fine pairings and obliges to design redundant sensors or to limit the length of the probes. Such a defect is particularly true with regard to photochemical processes involving NVOC protective groups. In addition, the known manufacturing methods are not very flexible, in the sense that it is difficult to easily change the composition of the OGN sequences constituting the networks.

Finally, it can be considered that the known processes are generally complex, unreliable, not fast enough, not very economical and ultimately not very industrial.

Under these circumstances, one of the essential objectives of the present invention is to provide a method and a device for manufacturing a support carrying on at least one of its faces a plurality of poly - preferably oligo - sequences. nucleotide and / or peptide, these OGN sequences different from each other being, on the one hand, apparent to their complementary by affine interaction and, on the other hand, obtained by chemical synthesis in the presence of volatile solvents;

- This process must work properly in practice with good flexibility, reliability, speed and simplicity;

- This process should also allow obtaining networks comprising from a few tens to several hundred million OGN sequences, and this with good chemical yield and subsequently with a low percentage of error in the sequences.

Another essential objective of the present invention is to provide a simple, economical and industrial device for implementing the above process.

Another essential objective of the present invention is to provide a support usable in the method and in the above-mentioned device, which is easy to obtain and which allows optimal implementation of the fabrication of OGN sequence networks.

Another essential objective of the present invention is to provide a method for detecting and / or identifying target OGN sequences, involving a biosensor comprising a support carrying a network of OGN probes obtained by the above-mentioned method, such a detection and / or identification method having to be most reliable and most specific.

STATEMENT OF THE INVENTION

These objectives, among others, are achieved by the present invention which firstly relates to a process for manufacturing a support carrying on at least one of its faces a plurality of poly-preferably oligo- nucleotide and / or peptide, these sequences (OGN), advantageously different from each other, being, on the one hand, visible to their complementary by affine interaction and, on the other hand, obtained by chemical synthesis in the presence of volatile solvents; said process essentially consisting in: 1) using a support whose (or the) face (s) intended (s) to carry the OGN present) a network of reaction wells, each of these wells being intended to serve as seat for anchoring and / or localized chemical synthesis (s) of a given OGN sequence; these wells being obtained by application of a photocrosslinkable resin, by crosslinking of this resin by exposure to actinic radiation in the zones intended to define the wells and by elimination of the non-crosslinked resin to obtain a grid of resin forming the walls which delimit the wells ;

2) placing this support in a reaction vessel and saturating this vessel using vapors of the volatile solvent (s) used in the anchoring and localized chemical synthesis (s) of OGN, preferably by making circulating through the reaction vessel a gas stream comprising the appropriate saturated vapors;

3) carrying out the anchoring and / or localized chemical synthesis (s) (by incrementation) of the OGN sequences in at least part of the wells, this operation being carried out:

(i) by supplying locally and separately each well concerned with reagents and liquid consumable products capable of allowing the anchoring and / or synthesis of the corresponding OGN sequence,

(ii) by supplying collectively (not locally) the wells concerned with reagents and / or liquid consumable products common to the reactions intended to intervene in all these wells; (iii) and by ensuring, prior to each localized supply operation of the wells (4), at least partially eliminating the liquid possibly contained in each well intended to be supplied in a localized manner.

The method according to the invention corresponds to the production of oligonucleotide networks by chemical synthesis in situ (or even ex situ) which better meets the needs of the practice than the methods of the prior art.

The advantages of this process are numerous. One can cite among others the fact that it makes it possible to manufacture networks of OGN sequences of composition, size, and very diverse density with a simple and flexible implementation. This responds, in particular, to the needs of genetic research. In addition, the process takes place on a scale such that the consumption of very expensive reagents used for the synthesis of OGN in particular, of oligonucleotides, is particularly low. This makes it possible to envisage mass production of networks at a reduced cost. In addition, it should be noted that the method according to the invention uses only conveniences which relate to the reaction liquids and the equipment used.

Furthermore, the method according to the invention makes it possible to envisage a wide diffusion of the technology of the OGN networks in particular of oligonucleotides in all the techniques of genetic analysis applicable in medicine and in bioindustries. The method according to the invention makes it possible to flexibly manufacture networks of very diverse composition, combinatorial or not, on supports of a few square millimeters at 300 cm ^2, for example silicon plates, the surface of a pixel. being between 1000 x 1000 μm ² and 10 x 10 μm ² . The number of pixels can therefore vary from a few units to several hundred million units.

The OGN sequences which are fixed and possibly which are produced in situ on the support can be likened to probes capable of reacting with complementary target sequences by affine interaction. These OGN sequences advantageously consist of several comonomers which are nucleotides, and / or repeating units of Nucleic Acid Peptide (APN) and / or amino acids. The number of nucleotide and / or APN and / or amino acid comonomers that these polymers, or more precisely these oligomers, count, can vary from a few units to a few tens of units, preferably from 15 to 25 units.

It is preferable in accordance with the invention to carry out not only the anchoring but also the localized synthesis in situ of the OGNs in the wells. However, it is also possible to synthesize the OGNs ex situ and to fix them only then in the wells of the support in a localized manner. Ideally, the material constituting the support is chosen from the group of material comprising: glass, quartz, silicon optionally coated with at least one layer of oxide or nitride.

Silicon or other semiconductor materials are preferred in particular in the case where the support is intended to be integrated in a biosensor which operates on a mode of revealing pairings of the electronic transductance type, for example with field effect.

Another characteristic of this support is to present on its surface a network of reaction wells formed by borders of photocrosslinkable resin preferably selected from the group comprising: positive or negative resins, preferably in the subgroup comprising negative resins and more preferably still in the class comprising: (meth) acrylate, epoxy, polyester and / or polystyrene resins photocrosslinkable by the radical and / or cationic route. By "positive" resin is meant in the sense of the invention a resin made very soluble by irradiation. By "negative" resin is meant in the sense of the invention a resin made insoluble by irradiation.

Negative resins, more especially used, have the advantage of making it possible to obtain thick chemically resistant layers which adhere to the substrate.

Advantageously, the removal of the non-crosslinked resin at the unmasked locations corresponding to the wells is carried out by dissolving the resin in an organic solvent. It may, for example, be acetone, γ-butyrolactone, Propylene-Glycol-Methyl-Ether-Acetate (PGMEA), acetonitrile or even other solvents known to those skilled in the art. This production of the lithographic resin microwells follows known experimental protocols for cleaning the support, depositing, baking, exposure to actinic radiation (UV), and dissolving the non-crosslinked resin.

The examples which follow and which are supplemented by the illustrations provided by the drawings, in particular FIGS. 1 and 2, provide other details useful for the non-limiting description of the support and its production.

According to an advantageous variant of the invention, the support is treated at the surface so as to give it anchoring sites capable of forming non-labile bonds with the leading comonomers of the OGN sequences, this treatment preferably being a silanization with using an epoxidized alkoxysilane or else an ester function before and / or after the preparation of the network of microwells.

Conventionally, the silanizing agent is glycidoxypropyltrimethoxy-silane. As other silanizing agents, mention may be made of glycidoxypropyldimethoxysilane, aminopropyltriethoxysilane, or alternatively carboxymethyltriethoxysilane.

The purpose of this silanization is to provide solid anchor points for all of the rank 1 monomers (e.g. nucleotides) constituting the OGN n-sequences of the network produced in situ. The bridging of silanes with the monomers (nucleotides) of rank 1 is advantageously carried out by means of hydroxyl groups, for example.

Taking into account the evaporation of volatile solvents necessary for the anchoring and synthesis reactions of the OGN sequences, is perfectly assumed by the method according to the invention. Thus it is provided according to the latter a saturation step, preferably using a gas stream comprising suitable saturating vapors and a neutral gas. In doing so, the evaporation rate of the solvents used is controlled and all the microreactions necessary for obtaining the network are controlled.

If the evaporation is controlled by its saturation technique, it is possible to be able to cause the evaporation of the solvents so as to stop the reactions localized on the support.

Naturally, it is also possible to limit evaporation by reducing the vapor pressure of all the volatile solvents used, for example by lowering the temperature, preferably the support.

Advantageously, the carrier gas used for saturation can be: argon or helium.

Preferably, this gas flow is such that it determines an overpressure in the reaction chamber relative to the surrounding atmosphere. To fix the ideas, it can be indicated without limitation that the gas flow determines a pressure of 0.01 to

0.3 bar compared to the atmosphere.

As regards the localized anchoring and / or chemical synthesis of the OGNs on the support, provision is made in accordance with the invention for implementing localized supply means and non-localized supply means for the reaction wells of the support.

Advantageously, the localized supply means and the support are movable relatively with respect to each other in the three dimensions X, Y, Z, by means of displacement. In accordance with the invention, to carry out anchoring and / or synthesis on the support of n-OGN sequences each comprising x comonomers:

± o stores in memory the composition of the network of n OGN sequences to be produced by apprehending this composition according to an organization in x successive rows each comprising a comonomer and each corresponding to a series of withdrawal, supply and liquid deposition actions reagents located in the n wells of the support, which liquids determine the nature of the comonomers in the row Ri = 1 to x in the n wells; A and on the one hand, the localized supply means and the displacement means are controlled, the execution of the various series of actions mentioned above, and on the other hand, the non-localized supply means carry out the racking, transport and supply of liquids not located in the n wells of the support at different times during the anchoring and synthesis of OGN sequences, each localized supply being followed by a step of evacuating the liquids present in the wells and more generally in the reaction vessel, these commands and the actions which they induce being repeated by incrementing i by 1 in R up to i = x. During the various stages of manufacturing a network of sequences

OGN (for example oligonucleotides), it is necessary to supply the reaction vessel with fluids (gases, solvents, etc.) and reagents (bases, deprotection agents, coupling agents, etc.). These fluids (liquid consumables) and these reagents can be classified into two categories: 1) The fluids and reagents intended to be sent pixel by pixel to obtain the network and which will be designated below by

"localized liquid";

2) The fluids and reagents intended to be sent simultaneously to all or part of the support and more precisely to all or part of the reaction wells of the support in common to all these wells. These fluids and reagents are designated below by "non-localized liquid". According to a preferred characteristic of the invention, the elimination of liquid according to step 3 (iii) of the process is carried out so that the wells concerned are free of liquid up to at least 90% of their volume, preferably at least 95% and more preferably still at least 98%.

Advantageously, the liquid is removed by drying, by injecting gas, preferably neutral, into the reaction vessel, and / or by increasing the temperature of the reaction vessel and / or of the support and / or by lowering the vapor pressure of the liquid to be eliminated in the reaction vessel.

These provisions are intended to allow the projection or deposit of localized liquid, to be precise and not to give rise to soiling in the neighboring wells of the well which is supplied with given localized liquid, in particular by splashing, as this risks being the case if the well contains liquid before the start of the feed considered.

This elimination of liquid by drying using neutral gas also makes it possible to remove any traces of water which may compromise the synthesis and / or the anchoring of OGNs. Indeed, it is well known that the bases and the solvents capable of being used as reaction liquids must be stored in an anhydrous medium. It is also conceivable to carry out a partial and controlled vacuum of the reaction vessel. Ultimately, the method according to the invention makes it possible: to provide the support with a superstructure defining microreactors corresponding to the pixels of the network and making it possible to control the spreading of localized liquids; to control the rate of evaporation of the solvents from the liquids located in the microreactors, so as to control the chemical microreactions necessary for obtaining the network of OGN sequences; and to dispose of the used chemicals at the end of these microreactions so as to continue manufacturing the network.

According to another of these aspects, the present invention relates to a device for implementing the method as defined above. This device is characterized in that it comprises: at least one support whose (or the) face (s) intended to carry the OGN, present) a network of reaction wells, each of these wells intended to serve as a seat for anchoring and localized chemical synthesis of a given OGN sequence, these wells being obtained by application of a photocrosslinkable resin, by crosslinking of this resin by exposure to actinic radiation in the zones intended to define the wells and by elimination of the non-crosslinked resin to obtain a grid of resin forming the walls which delimit the wells; at least one reaction enclosure intended to contain the support; means for localized supply of the reaction wells with specific localized liquids (reagents / consumables) suitable for allowing the anchoring and synthesis of a given OGN sequence in each of the wells of the support, means for moving the supply means localized relative to the support and / or vice versa; means for non-localized supply of the reaction wells 10 with liquids (products / consumables) not localized and common to the reactions intended to take place in all or part of the wells; possibly means for moving the non-localized supply means; Means for evacuating non-localized liquids; these evacuation means being associated with non-localized supply means; organs capable of permitting saturation of the reaction vessel with the aid of vapors of the volatile solvent (s) used in the localized anchoring and chemical synthesis of OGNs; containers of localized liquids, containers of non-localized liquids, containers of effluent drainage liquids; 25 - means for moving the support relative to the localized supply means and / or vice versa, possibly gas supply members in the enclosure, possibly at least one memory of the different OGN sequences to be anchored and synthesized on the support, 30 - possibly at least one central unit for reading the memory and for generating control signals the supply of fluids and the displacement of the localized supply means and of the support with respect to one another. The invention will be better understood in the light of the detailed description which follows of preferred, but nonlimiting, examples of embodiments of the device and implementation of the method which it relates to.

BRIEF DESCRIPTION OF THE DRAWINGS

This detailed description is made with reference to the accompanying drawings in which:

- Fig. 1 shows a perspective view of the support on which the OGN sequence network is intended to be manufactured in accordance with the invention.

- Fig. 2 is a view in vertical section along line II-II of FIG. 1.

- Fig. 3 is a schematic representation of a first embodiment of the device according to the invention.

- Fig. 3A is a schematic view of a variant of a drop projection system of localized reagents.

- Fig. 4A is an extract from FIG. 3 comprising the reaction vessel, the support and the localized supply means, this FIG. 4A illustrating the displacement of the support and of a part of the reaction vessel with respect to the supply means located along the X axis.

- Fig. 4B is a sectional view along the line IV-IV of FIG. 4A, this FIG. 4B illustrating the movement of the support and of a lower part of the reaction vessel with respect to the supply means located along the Y axis. - FIG. 4C uses the same elements as FIG. 4A but illustrates for its part the displacement of the lower part of the reaction vessel and of the support with respect to the supply means located along the axis of the Z.

- Figs. 5 and 6 are enlarged photographs of a support 1 comprising a grid 3 made of photoresist defining several tens of microwells intended to serve as seats for chemical reactions of anchoring and / or synthesis of OGNs. The difference between Figs. 5 and 6 is due to the presence of a drop of reagents located in the microwell surrounded by a circle of the support of FIG. 6. - Fig. 7 is a general schematic perspective view of a device according to a second embodiment.

- Fig. 8 is a detailed perspective view of FIG. 7 showing the support 1 and its displacement means 7 as well as the localized supply means and their displacement means 30.

- Figs. 9A, 9B, 9C and 9D represent the non-localized supply means.

- Fig. 9A is a front view of the piston 80 belonging to these non-localized supply means 8. - FIG. 9B is a diametrical section view of the piston of FIG. 9A.

- Fig. 9C is a top view of the piston 80 of FIGS. 9 A and 9B.

- Fig. 9D is a partial view in diametral section of the piston 80 applied to the support 1.

- Fig. 10 represents the network 64 oligonucleotides with a length of 3 seas each produced in the examples.

Figs. 1 and 2 show the support 1 which is an integral part of the device according to the invention.

BEST WAY TO IMPLEMENT THE INVENTION

Preferably, this support 1 comprises:

- At least one substrate plate 2 made from a material chosen from the group comprising: glass, quartz, silicon optionally coated with at least one layer of oxide or nitride;

at least one network 3, preferably a grid, of photocrosslinked resin secured to one of the faces of the substrate, this network defining a matrix of reaction wells 4, the photoresist preferably being selected from the group comprising epoxy resins or ( meth) acrylates photocrosslinkable by the cationic and / or radical route, epoxy resins being very particularly preferred. Advantageously, the grid 3 of resin is compatible with the treatments of the substrate necessary for the attachment of the monomers (nucleotides) of rank 1. This grid 3 can be manufactured before or after these treatments. This grid 3 of resin adheres to the plate 2 of substrate during the manufacture of the OGN sequence network. It can possibly be eliminated before being used in a biosensor although this grid is not in itself troublesome for the operations of reading the networks in these biosensors.

It is important that the grid produced in a resin which is never completely crosslinked does not contaminate the synthesis reagents and that, conversely, these do not degrade the grid or cause a grid / substrate separation.

The reaction wells 4 defined by this grid 3 define as many reaction sites which will give rise to the pixels of the array. These wells limit the spread of very wetting solvents such as acetonitrile. This network or this grid 3 of reaction wells 4 is obtained by conventional lithography techniques with positive or negative resins, by depositing resin on the substrate, and, for example in the case of negative resins, by exposing the corresponding zones. to the walls with actinic radiation so as to crosslink them and by dissolving selectively the noncrosslinked zones so as to form the wells. Without this being limiting, it is possible according to the invention to manufacture microwells of height varying between 0.1 to 1000 μm, preferably from 50 to 500 μm with surfaces of between 10 × 10 μm and 1000 μm × 1000 μm. According to an advantageous arrangement of the method according to the invention, the network or grid 3 of reaction microwells 4 makes it possible to localize the chemical reactions independently of the nature and / or of the preparation of the support, so that it is possible to use supports of various, non-homogeneous nature having undergone physicochemical surface treatments (oxidation, silanization, nitriding, etc.). The first embodiment of the device according to the invention shown in FIG. 3 comprises a support 1 having on its surface reaction wells 4 defined by a grid of resin 3, this support 1 being contained in a reaction enclosure designated by the general reference 5. The device of FIG. 3 also includes localized supply means 6, displacement means 7 for the support 1 relative to the localized supply means 6.

To ensure the non-localized supply of non-localized reaction liquids, means 8 are provided. These means 8 are associated with means 9 for evacuating non-localized liquids.

The device further comprises members 10 for saturation of the reaction vessel 5 using a gas flow loaded with neutral gas and solvent. References 11, 12 and 13 in FIG. 3 respectively designate the localized liquid, non-localized liquid and effluent drain containers. Also provided are organs 14 for supplying gas into the reaction vessel 5.

Finally, according to a preferred characteristic of the invention, the automated control of the process is ensured by a memory 15 and a central unit 16 for reading the memory 15 and for generating control signals (dotted arrows).

The various constituent elements of this device are, in practice, supported by chassis and / or gantries and / or adapted bases.

According to a first form of implementation of this first embodiment, the device is characterized in that the reaction chamber comprises a sealed bellows one of whose ends carries the localized supply means and whose other end has , opposite the latter, a reaction site intended to receive the support and comprising the non-localized supply means, the carrying end of the localized supply means being preferably fixed and the other carrying end of the support preferably movable in the three dimensions x, y, z, by means of displacement. According to a second embodiment of the first embodiment, the device is characterized in that the localized supply means are capable of being set in motion in the 3 dimensions x, y, z under the action of the means displacement, relative to a fixed part of the reaction vessel intended to receive the support and associated with the non-localized supply means.

Fig. 3 corresponds to an example of a device according to the first embodiment of the first embodiment, in which the locahsee supply means are fixed relative to the movable part of the enclosure reaction intended to receive the support. This movable part is provided to the displacement means 7 in the directions x, y, z.

As shown in Fig. 3, the reaction chamber 5 is composed of a lower part 5ι which is useful as a receptacle for the support 1, an upper part 5 ₂ equipped with part of the localized supply means 6, and with an intermediate part 5 ₃ constituted by a sealed bellows connecting the lower 5ι and upper 5 _{2 parts} to each other. The upper part 5 ₂ of the reaction enclosure may or may not be integral or not with the bellows 5 ₃ . The waterproof 5 ₃ bellows is advantageously made of a chemically inert material such as polytetrafluoroethylene (TEFLON ^®). In accordance with a preferred embodiment of the invention, the localized supply means 6 comprise a projection system. The projection system is advantageously chosen from the following systems:

1) mechanical ejection using devices of the type: a) drop on demand by piezoelectric effect; b) drop by jet interrupted by electrostatic effect;

2) pressure ejection using microvalve devices.

In the example shown in FIG. 3, the projection system is of type 1-a. and comprises at least one nozzle 6ι for spraying or depositing localized liquids. This piezoelectric nozzle 6ι is connected, via a conduit 6 ₂ , to one or more localized liquid tanks 11.

The l.a. drop ejection on demand by piezoelectric effect are described in the article "Techniques for printing digitized images" by J.J. ELTGEN (ENGINEERING TECHNIQUES - treatise on electronics E5670). One of the advantages of these piezoelectric systems is that they make it possible to deposit in the microwells 4 of the support 1 very small volumes of localized liquids (reagents), for example 50 picoliters, on the most reduced surfaces of microwells 4 ( eg 70 x 70 to 300 x 300 μm).

Another advantage of piezoelectric systems is the commercial availability of products with 64 to 128 6 _X nozzles controlled independently at frequencies from 1 to 10 KHZ, for example the company's P64 and P128 systems.

MODULAR INK TECHNOLOGY (Sweden). With such products, it is therefore it is possible to supply numerous wells simultaneously by spraying the reagents diluted in acetonitrile.

According to a variant, the projection system can be of the l.b. type. drop by jet interrupted by electrostatic effect as described in the article by J.J. ELTGEN mentioned above. This system achieves higher resolutions

(typically 30 x 30 μm).

Finally, the variant of the type 2 projection system as represented in FIG. 3 A, involves, in place of the piezoelectric nozzle or nozzles of the type (6ι), mechanical parts (20) produced from of materials inert towards non-localized liquid reagents. It can be stainless steel, polytetrafluoroethylene (PTFE), polyamide (NYLON ^® ). Each piece (20) has a fine through channel (21). The inlet opening (22) of this channel (21) is connected via a conduit (23) to a solenoid valve (24) which governs the admission of liquid located in the channel (21) of the piece (20). This solenoid valve (24) is supplied with localized reactive liquid (25) by a reservoir (26). A conduit

(27) connects the reservoir (26) to the solenoid valve (24). A neutral gas (28) (for example argon) in overpressure (e.g.: 0.5 bar) propels the liquid (25) towards the solenoid valve (24).

The conduits (23) (27) whose diameter is for example 1 mm are advantageously made of the same material as the part (20) e.g.: PTFE. The outlet opening (29) - that is to say the nozzle - of the part (20) is arranged opposite a substrate 1 comprising microwells 4 defined by a grid 3 made of photoresist.

The diameter of the nozzle (29) is for example 100 μm.

The solenoid valve (24) is controlled by the central electronic control unit 16 according to FIG. 3 (not shown in Fig. 3A). This unit 16 is able to command the opening (eg: 0.1 to 10 ms) for a short controlled period and then the closing of the solenoid valve (24) so as to expel a small amount (30) of localized reactive liquid ( 25) and to supply the microwell 4 of the support 1.

With duct diameters (23) (27) of 1mm, an argon overpressure of 0.5 bar, an opening time of the solenoid valve (24) of 0.1 to 10 ms, a nozzle diameter ( 29) of 100 μm, the quantity (30) delivered is adapted to a support 1 with microwells 4 with a surface area of 400 × 400 μm and a height of 400 μm. The elements (20) (21) (29) can be, for example, the constituent elements of a microsyringe with a diameter less than 100 μm.

In practice, the solenoid valve (24) can be made up of the valve INKX 0510100 from the company LEE COMPANY (USA) which also forms a system with 7 nozzles (29) under the reference INZX 0510100. To increase the compatibility of this valve with acetonitrile, it would be advantageous to replace the elastomer valve with a fluorinated elastomer valve (KALREZ ^® or CHEMRAZ ^® ).

The variant of the projection system according to FIG. 3 A is attractive because of its reduced cost, the simplicity of its operation and its reliability. In the embodiment shown by way of example in FIG. 3, the localized supply means 6 also comprise a pipe 6 ₃ for supplying pressurized gas allowing the localized liquid contained in the reservoir 11 to be conveyed to the nozzle 6j via the pipe of the pipe 6 ₂ . The latter is equipped with a solenoid valve 6 ₄ for controlling the projection. The elements 6 to 6 which form the localized supply means 6 are controlled by the central control unit 16 which advantageously governs the opening and closing of the valve 6. The central unit 16 also controls the supply of propellant gas through line 6 ₃ . In the most frequent case where there are several reservoirs 11 of localized liquids, the central unit 16 also governs the selection of the liquid to be sprayed using appropriate organs.

It should be observed that in the example of FIG. 3 only one projection nozzle 6 _X is provided, but according to variants, it is possible to envisage having as many projection nozzles as different localized liquids to be sprayed.

The non-localized supply means 8 comprise a supply conduit 8ι for liquid not located in the lower part 5ι of the reaction vessel containing the support. This pipe 8 _X connects an opening 8 ₂ formed in the lower part 5 of the reaction vessel 5 to the reservoir 12 of localized liquid. This pipe 8 is equipped with a solenoid valve 8 ₃ making it possible to control the flow of non-localized liquid intended to supply the support. These non-localized supply means 8 also comprise a conduit 8 ₄ for propellant gas supply making it possible to generate the flow of liquid not located in the conduit 8ι. It is easily understood that it is possible to include in these non-localized supply means 8 other non-localized liquid tanks 12 by providing means for selecting the non-localized liquid to be introduced into the lower part of the reaction vessel 5 , on the support 1. Just like the means 6 of localized supply, the means 8 of non-localized supply are controlled by the central unit 16 (dotted arrows) which controls the routing of the non-localized liquids via propellant gas supplied via line 8 ₄ and via solenoid valve 8 ₃ .

These means 8 are associated with means 9 for evacuating non-localized liquids which are brought in by means 8 and which temporarily stay in the lower part 5j of the reaction vessel 5, to drown the support 1. These evacuation means 9 comprise a pipe 9ι connecting an outlet orifice 9 ₂ formed in the lower part 5ι of the enclosure 5 and the effluent tank 13. The pipe 9ι is equipped with a solenoid valve 9 ₃ controlled by the central unit 16 which governs effluent discharge at the appropriate time. The engine of this evacuation can be for example an overpressure of gas and / or the action of a suction pump (not shown in Fig. 3) making it possible to draw off the liquids.

The members 10 allowing the circulation of a saturation flow in volatile solvents comprise a solvent tank 10ι, an inlet 10 ₂ and an outlet 10 ₃ of the saturation gas stream in the lower part 5 _t of the reaction vessel. The flow is generated by a neutral gas such as argon penetrating through line 10 into the reservoir 10χ of solvent and carrying with it solvent vapors in line 10 ₅ which enters the enclosure through inlet 10 ₂ and which can be evacuated from the latter via the outlet 10 ₃ which is extended by an evacuation duct 10 ₆ . The conduits 10 _s and 10β comprise solenoid valves 10 ₇ and 10 ₈ controlled by the central unit 16 which thus controls the flow of saturation gas.

Preferably, the saturation members 10 of the reaction vessel are designed so as to allow the circulation of a saturating gas flow comprising vapors of the volatile solvent (s) used and at least one neutral gas.

The optional gas supply members 14 communicate with the reaction vessel via an inlet made in the lower part. These organs 14 are equipped with a solenoid valve not referenced and also managed by the central unit. With regard to the means 7 for moving the support 1 relative to the means 6, in the context of the first form of putting into practice the first embodiment of the device according to the invention, it can be seen that these means 7 are shown in a manner symbolic in fig. 3 which also shows through the dotted arrow the control of these displacement means 7 to the central unit 16. In general, it is essential that the displacement means have sufficient chemical resistance to the reagents used for the synthesis of OGN, in particular the solvent (s) used for saturation. The solvent (s) being flammable, the design of the means of movement must make it possible to avoid any risk of fire. (hence the interest of neutral gas). These conditions are ensured for the device of FIG. 3 since the displacement means are placed outside the chemical reactor thanks to the bellows.

These displacement means 7 can be constituted by any known and appropriate means for setting in motion the lower part 5 _X of the reaction vessel 5. This lower part 5 _t has, thanks to the bellows 5 ₃ , a certain mobility relative to the upper part 5 ₂ of the enclosure 5 comprising the projection nozzle 6χ. By way of examples, mention may be made of rack / pinion systems in the three directions which can be driven mechanically, of sliding systems or of systems comprising cross-motion carriages and / or micrometric displacement plates motorized in translation and / or in rotation. . Commercial products corresponding to these systems are offered in particular by the companies MICROCONTROLE, NEWPORT, OWIS.

Figs. 4A, 4B and 4C illustrate the displacements along x, y and z of the support 1 carried by the lower part 5ι relative to the projection nozzle 6χ. Such displacement means make it possible to supply the reaction wells 4 of the support 1 with localized liquid.

This example of a first embodiment of a device according to the invention as shown in FIG. 3, allows to distribute in the microwells 4 of the support 1, drops of localized liquids of very small volume. By way of illustration, FIGS. 5 and 6 are enlarged photographs of a support (1) comprising several tens of microwells (4) each having a surface of 150 × 150 μm and a height of approximately 100 μm. These microwells are defined by a grid (3) of photocrosslinked epoxy resin. The photograph of FIG. 5 shows the microwell array 4, in particular the microwell 4 'surrounded by a circle, empty of any trace of localized liquid. The photograph of FIG. 6 shows the network of FIG. 5 in which the microwell (4 ') surrounded by a circle contains a drop G of acetonitrile of about 50 picoliters, which was sent by the projection system by 6ι piezoelectric nozzle of the drop on demand type (1-a) . If the support 1 containing the drop G was not in the conditions provided by the device of FIG. 3 (saturation) the drop would evaporate in a fraction of a second and it would not be possible to take a picture of it. On the other hand, with the device of FIG. 3, this drop is found, for example, in a saturated volume of acetonitrile in the reaction vessel. Thus, it is maintained between 5 and 30 min, sufficient time to allow chemical reactions of anchoring and / or synthesis of OGNs.

The embodiment of the method according to the invention described in Figures 3, 3 A and 4 A, 4B, 4C has the advantage of being easy to perform with a flexible PTFE bellows. The saturated volume is low, which facilitates its control (chemical composition, concentration (s) of solvent (s), pressure, temperature, etc.). The displacement means are entirely outside the saturated volume and are therefore protected from corrosion. On the other hand, the amplitude of the mean displacements of microdeposition / bellows is limited to the deflections of the bellows, ie a few centimeters.

To fabricate arrays on large supports or substrates, for example silicon substrate "wafer 4 to 8 inches", that is to say 4 to 8 inches, ie 10.16 to 20.32 cm, it is preferable to use the device according to a second embodiment described in FIGS. 7 and 8 and be explained in more detail in Figures 9A, 9B, 9C, 9D.

Figs. 7, 8 and 9 show a second embodiment of the device according to the invention. As can be seen in these figures, the essential differences between this second mode and the first mode described above are due to the fact that, in addition to the support that can be displaced using the means 7 for moving the support 1 along the X axis, the means 6 of localized feed are movable along the Y axis and the means 8 of non-localized feed are movable along the Z axis and have large deflections, at least along the axes X, Y to allow working on large substrates.

The means 7 for moving the support 1, as well as the means 30 and 40 for moving the localized 6 and non-localized 8 supply means respectively, are at least partially included in the reaction enclosure 5. The latter is advantageously of greater dimension as the device according to the first embodiment, for example 1.5 x 1.5 x lm. This enclosure 5 is designed so as to be able to be saturated, for example, with a gas flow charged with neutral gas such as argon and with vapor of volatile solvent. To this end, the saturation bodies 10 of the enclosure 5 are provided. They are identical to those described for the first embodiment of the device according to the invention. Identical elements are designated by the same references.

The device according to the second embodiment is more suited to an industrial operating mode. The important thing in this regard is to be able to constantly maintain the saturation of the reaction chamber, as it is clear that any break in saturation would entail a significant consumption of time for re-saturation, given the large volume of this chamber.

Another characteristic of this second embodiment of the device is that the parts of the displacement means 7, 30, 40 arranged outside the enclosure 5 are the motors 31, 41 and 71 as well as the electrical supplies (not shown in the drawings) of said engine while the internal mechanical parts to (5) described in more detail hereinafter moving means 7, 30, 40 are made from inert materials such as TEFLON ^® (PTFE), steel stainless, perfluorinated elastomers (KALREZ ^® , CHEMRAZ ^® ), polyamides (NYLON ^® ). In order not to break the saturation of the enclosure 5, during the loading and unloading of the support 1 carried by the displacement means 7, the enclosure 5 is equipped with a lateral airlock 50.

Fig. 8 shows in more detail the localized supply means 6 with their displacement means 30 along the Y axis, as well as the support 1 and these displacement means 7 along the X axis. The wall of the reaction vessel 5 is symbolized in FIG. 8 by mixed lines. The means 7 for moving the support or the substrate 1 comprising a grid of resin 3 defining a network of reaction microwells 4, comprise a translational carriage 72 capable of sliding on sliding guides 73 under the action of a motor 71, capable of generating a rotational movement transformable into translation by means of a worm 74 transmission. The sliding guides 73 which are for example metal rods of polygonal or circular section, and the worm gear 74 for transmission are parallel to each other. Each end of the sliding guides 73 is provided with a stop block 75. The end of the transmission worm 74 opposite to that connected to the motor 71 is also equipped with a stop block 76. The end parts of the elements 73 , 74, the stop blocks 75, 76 and the motor 71 are arranged outside the enclosure 5.

Advantageously, the sealing at the level of the passages of the sliding guides 73 and of the transmission endless screw 74 through the wall of the enclosure 5, are fitted with O-rings, for example made of KALREZ. According to an advantageous variant of the invention, the stop blocks 75 of the sliding guides 73 can be an integral part of the wall of the enclosure 5 so as to save openings which must be sealed. The materials constituting the parts located at least in part in the enclosure 5 are chosen from materials resistant to chemical reagents for anchoring and / or synthesis, in particular of OGNs. Examples of constituent materials include PTFE, stainless steel, KALREZ.

The external motor 71 is protected from chemical reagents, which prevents any risk of fire or explosion. With regard to the localized supply means 6 and their displacement means 30, it should be noted that said displacement means 30 are designed to ensure displacement in translation along the Y axis of a microphone station 60 -deposits of liquids or reagents located in the microwells 4 in the network of the support 1. Like the displacement means 7 described above, displacement means 30 comprise a motor 31 connected to one end of a screw without end 34 of transmission terminated at the other end by the stopper 36, as well as sliding guides 33, the ends of which carry the stopper block 35. As indicated above, some of these elements and the terminal part of others these elements are arranged outside the enclosure 5. Reference will be made to the description of the means 7 above for more details.

Regarding the micro-deposition station 60 capable of being driven by the endless screw 34 in translation along the sliding guides 33 (along the Y axis), it is formed, on the one hand, by a carriage provided with an element 61 carrying reagent bottles 62 (only one of which is shown in FIG. 8) and parallel to the Y axis and, on the other hand, a battery 64 which supports a series of micro-organ deposits 65 (only one of which is shown in Fig. 8). This battery 64, parallel to the Y axis, is laterally opposite the element 61 carrying reagent bottles 62.

As indicated above, the elements of the means 6 and 30 disposed inside the saturated enclosure 5 are made of materials resistant to the reagents used, for example acetonitrile, and / or are sealed from said pregnant. Examples of resistant materials are given above.

The transfer of the reagents contained in the bottles 62 to the organs 65 of corresponding microdeposits is carried out via lines 66 (only 1 is shown in FIG. 8). Propulsion of the reagent or localized liquid is carried out by means of a neutral gas flow, for example argon, supplied under pressure through line 67.

According to a variant, the reagent bottles 62 could be arranged outside the working volume defined by the reaction enclosure 5. It would then be necessary to provide the "ad hoc" fluidic connections. The difference along the axis Z between the substrate or support 1 and the micro-deposit members 65 is adjusted so as to address the drops of reagent located on the entire network of microwells 4 defined by the grid of resin 3 of the support 1, using the movements of the carriages 60 and / or 72 along the axes X and / or Y. This addressing by movement of the carriages 60, 72 and these micro-deposits using the organs 65 (with upstream the reagent bottles 62 and the propellant gas) is controlled and managed by a central control unit equipped with a memory (computer), not shown in the drawing. Figs. 9A, 9B, 9C, 9D detail the non-localized supply means 8 and more specifically a particular element thereof constituted by a piston 80. As shown in FIG. 7, the piston 80 of the non-localized supply means 8 is displaceable in translation along the axis Z. The displacement means 40 provided for this purpose comprise, analogously to the displacement means 7 and 30 described above, a motor 41 connected to an endless screw 44 for translational translation of the Z axis of the piston 80 along two sliding guides 43 parallel to said axis Z and to the endless screw 44. The lower ends of the guides 43 and of the screws 44 include stop blocks 45 and 46. The same applies to the upper ends of the sliding guides 43. Reference is made to the descriptions of the displacement means 7 and 30 above for more details. The piston 80 is integral with a carriage 81 cooperating with the displacement means 40. The vertical axis Z of the piston 80 is perpendicular to the axis X of movement of the support 1 and to the axis Y of movement of the station. micro-deposits 60, so that it is possible by suitably actuating the displacement means 7 and 40 to make the axis of the piston 80 coincide with the center of the support 1 and also to cause the piston 80 to come over the substrate support 1. As shown in Figs. 9A, 9B, 9C and 9D, the underside of the piston 80 is provided with an annular O-ring 82 produced, preferably in KALREZ. This annular O-ring is coaxial with the piston and has a diameter substantially smaller than the diameter of the underside of said piston 81.

Thus, as shown in FIG. 9D, this annular O-ring 82 defines an interstitial space 83 between the underside 84 of the piston 81 and the surface of the support 1 against which said piston 81 is applied, by means of displacement means 40.

In this situation where the piston 81 is pressed against the substrate 1, the substrate + annular O-ring 82 + lower face 84 of the piston 81 assembly forms a tight enclosure 83 of small dimension, for example from 5 to 10 cm ³ , for a substrate 4 inches in diameter (= 10.16 cm). The piston 81 consists of a wall 85 which has a lower end portion of greater thickness and which defines an internal hollow volume 86. The lower part of the wall 85 which is arranged in the diametral plane, has an external face forming the lower face 84 of the piston 81 and an internal face 87 equipped with tubular elements 88 peripheral arranged at the angular positions 0 °, 90 °, 180 ° and 270 °. These tubular elements 88 put the interior 86 of the piston 81 in communication with the exterior and in particular with the interstitial space 83 in the pressed position of the piston 80 on the support 1 as shown in FIG. 9D. These tubular elements 88 are connected by means of pipes not shown in the drawing to non-localized reagent tanks, which are also not shown in the drawing and corresponding to the tanks 12 and 13 of FIG. 3 illustrating the first embodiment. It is thus possible to supply non-localized reagents with the interstitial space 83 of very reduced volume, without breaking the saturation of the entire reaction vessel 5. In addition to the supply of non-localized reagent, the tubular elements 88 allow also ensuring the evacuation of said non-localized reagents and / or the drying of the substrate, by passing for example using a gas flow or even by suction, pumping.

Like the localized supply means 6 and their displacement means 30, as well as displacement means 7 of the substrate 1, the non-localized supply means 8 and their displacement means 40 are controlled by a central unit control capable of managing all the washing, rinsing, evacuation, drying, etc. feeding procedures useful in the context of the inking and / or synthesis process of localized OGNs specific to the invention.

It should be noted that a person skilled in the art has numerous technical variants for ensuring the tightness of the reaction vessel 5. In particular, after having recourse to sealed ball bearings and seals of various shapes, these mechanical construction elements being for example exposed in the book "MEMOTEC, PRODUCTICS, design and drawings" (C.Barlier and R. Bourgeois).

According to another of these aspects, the invention relates to a support carrying an OGN sequence network as defined above.

The present invention also relates to a method for detecting and / or identifying target OGN sequences using biosensors comprising probes formed from OGN sequences complementary to the target OGN sequences and capable of pairing with each other by affine interaction, characterized

In that a support carrying an array of OGN probes as defined above is implemented,

• and in that the probe / target pairing is revealed using a fluorescent marker of the "picogreen" type.

The "picogreen" is a molecule presenting a fluorescence signal in the presence of double strands of DNA. The picogreen does not require any particular reaction, and must simply be placed in the presence of the double strands for 5 min before the fluorescence measurement. This molecule therefore has several advantages over the products conventionally used for the detection of double strands, whether they are fluorescent markers (fluorescein, rhodamine, etc.), or intercalators (ethidium bromide, Hoechst 33258).

Picogreen does not require any labeling reaction, unlike fluorescent probes used in DNA chips. Its fluorescence signal has excellent linearity over a wide range of concentrations. It thus makes it possible to detect double strands at concentrations much lower than those measurable with conventional intercalators, even in the presence of contaminants (single strands of DNA, RNA) in the measurement solution. Thus the sensitivity of picogreen is up to 400 times better than Hoechst 33258, itself having better sensitivity than ethidium bromide.

This high sensitivity makes it possible to measure local fluorescence levels that would not be possible with conventional intercalators. The picogreen is therefore more suitable than these intercalators for the detection of hybridization on high or medium density chips, which involve hybridization units of reduced size. The fluorescence signal is independent of the base composition of the sequence tested, unlike the Hoechst product.

The "picogreen" is excited in the same way as fluorescein (excitation 480 nm, emission 520 nm), which makes it very practical to use with standard equipment. Finally, the subject of the invention is a method for detecting and / or identifying target OGN sequences using biosensors comprising probes formed from OGN sequences complementary to the target OGN sequences and capable of pairing with each other by affine interaction, characterized

In that a support carrying an array of OGN probes as defined above is implemented,

• and in that this support is conformed by an affinity sensor comprising at least one structure comprising at least one semiconductor material Se, coated on at least one of its faces with at least one layer of Is insulator, the latter having OGN probes on its surface,

In that these OGN probes are brought into contact with an LC conductive liquid medium comprising the target OGN sequences,

• and in that the following operating mode is applied: a) select unmarked OGN probes, b) ensure that the Fermi level of Se corresponds substantially to, or passes through the intrinsic level at the surface of Se, c) subjecting the Se to periodic illumination comprising photons whose energy is> the energy of the forbidden band of the Se, d) directly or indirectly measuring the variations ΔVbp of the potential of flat bands Vbp of the Se, induced by a charge effect phenomenon directly and essentially linked to the specific pairings of the target OGN sequences of the LC conducting medium with their complementary ligands of the OGN probe (s), except:

(i) variations resulting from possible charge effects and / or charge transfers caused by chemical reactions catalyzed by enzymes and in which consumption of part of the substances to be detected occurs, (ii) and variations in the photoresponse linked to the appearance in the LC medium of at least one tracer product capable of being revealed through variations in pH or Redox potential, and / or through markers, preferably of the type of those absorbing or emitting radiation (fluorescent, radioactive, colored, eg). e) and interpret the signals collected in terms of identification and / or assay of the target OGN sequences of the LC. This method combines the networks of OGN sequences prepared in accordance with the method described above and the method of identification and / or detection of biological substances as described in application PCT / WO 98 / 57,157 which is completely incorporated into this presentation by reference.

This association leads to a particularly efficient method for detecting and / or identifying target OGN sequences. The examples which follow illustrate the operation of the method and of the device according to the invention and more particularly of the device according to the first embodiment.

EXAMPLES

EXAMPLE I:

1.1. Reagents and methodology

Table 1 shows an example of classification in the case of phosphoramidite chemistry which will be used later. Many variations are possible without changing the scope of the invention.

Table 1: Example of classification of the modes of action of products and reagents (phosphoramidite chemistry) 1.2. Device

The device used is that described above with reference to FIG. 3.

This device comprises a projection system (6) formed by 6 microvalve devices of the type of those shown in FIG. 3 A, to project reagents A, C and D from Table 1 (1 valve for reagent A, 1 valve for reagent D, valves for the 4 bases of C).

The device further comprises 6 non-localized supply means (10) of the type of those shown in FIG. 3 for reagents A, B, E, F and G of Table 1.

It is expected that product A, acetonitrile can be sent both localized and non-localized. The device also includes means for drying the substrate with argon gas (ie the reagent H in Table 1).

The support consists of a silicon or glass plate comprising 256 wells in lithographic resin (epoxy).

Each microwell has a surface of 415 x 415 μm and a height of approximately 415 μm. The volume of the wells must be such that it is filled without overflowing by the projection of a drop of base and a drop of activator.

The protocol for manufacturing microwells on this size of silicon is as follows: 1. Cleaning of the substrate (acetone, alcohol, etc.) 2. Drying with dry argon

3. Deposit on the substrate with a layer of lithographic resin with a spinning wheel, for example, with an epoxy resin with cationic polymerization (ex EPON SU8-100 resin developed by IBM which is supplied diluted with γ-butyrolactone solvent in different proportions ); the speed of the spinner makes it possible to control the thickness of the resin from 1 to 1000 μm, typically 100 μm; the resin cited as an example is used in the manufacture of microsystems and makes it possible to obtain vertical wells of significant thickness with form factors up to 1/20; 400 μm high microwells are manufactured

- Either by spreading at a low speed of rotation a single layer of resin, - Or by successively spreading from 2 to 4 layers and repeating steps 4, 5, 6 below for each layer. 4. Baking at 90 ° C for 5 to 30 'depending on the thickness of the resin and the substrate;

5. Selective exposure with a UV source (UV lamp with a set of masks or directly with a UV laser). The dose of energy (mJ / m ² ) absorbed by the substrate must be adjusted so as to obtain good resin / substrate adhesion during the various stages of cooking and development. The size of the microwells varies from

5 μm x 5 μm to 1000 μm x 1000 μm, typically from 415 μm x 415 μm;

6. Second firing at 50 to 100 ° C for 5 to 30 'depending on the thickness of the resin and the substrate;

7. Dissolution of the unexposed resin with a solvent (acetone, y-butyrolactone, PGMEA ...) with optionally activation by ultrasound;

8. Final baking to increase the crosslinking of the resin: 90 to 150 ° C for 5 to 30 '. This step is decisive for the resistance of the microwells to reagents for the synthesis of oligonucleotides.

9. Cleaning the substrate with sulfochromic mixture for 5 ', then rinsing with double distilled water;

10. Silanization by the aqueous route by soaking for 45 'in a GPTS / water mixture at 1% v / v, then passage in an oven at 125 ° C for 45';

The process parameters are adjusted so that:

- the solvents (acetonitrile, dichloromethane, THF, pyridine, etc.) and the reagents (iodine, etc.) used in the synthesis of oligonucleotides do not cause the degradation of the resin superstructure or the detachment thereof;

the crosslinking of the resin is sufficiently advanced for the superstructure to be inert with respect to the synthesis of oligonucleotides.

EXAMPLE π: ANCHORING AND CHEMICAL SYNTHESIS PROTOCOL ACCORDING TO THE CHEMISTRY OF PHOSPHORAMIDITES

The control of the displacement means is adjusted so that the distance between the tip of the nozzles is approximately 1 mm from the opening of the well 4, when projecting localized liquids. The pressure of the bottles containing the localized reagents is between:

- a minimum value below which the reagent is not completely sprayed but adheres to the base.

- a maximum value corresponding to the volume of the well. To reduce the risk of contamination between wells, only 1 in 2 wells is used so that of the 256 wells in the substrate, only 64 constitute the nodes of the OGN network.

The memory 15 and the central unit 16 are loaded so as to produce the network comprising all the variants of the following OGN (written in the manufacturing direction 3 'to 5'):

3 'GAG GAT GGT X _X X ₂ X ₃ CCT GCT AGG TAT 5'

Xi, X ₂ , and X ₃ form the 64 possible combinations of A, C, G and T. This example was chosen because the special case XιX ₂ X ₃ ≈ ACG is the OGN L226 of biological interest.

In practice, for questions of steric hindrance during hybridizations, it is useful to move the OGN away from the substrate. To do this, we extend the sequence with a header of 10 C for example, which gives: 3'CCC CCC CGC CGA GGA TGG TXιX ₂ X ₃ CC TGC TAG GTA TC 5 '. The first and third part of the network are the sequences: CCC CCC CCC CGA GGA TGGT and CC TGC TAG GTA TC were manufactured with an OGN Expertise Perkin Elmer synthesizer modified so as to be able to use substrates. The central part XιX ₂ X ₃ was manufactured with the device of FIGS. 1 to 4 depending on Fig. 10. We see in this Fig. 10 that the OGN L 226 is in the central position and surrounded by very different composition probes so that the hybridization of the ACG pad is easy to detect. In fact, it was possible to detect the O22 U226 complementary to L226 with such a network.

INDUSTRIAL APPLICATION

The invention finds a preferred application for the manufacture of a support carrying on at least one of its faces a plurality of polynucleotide sequences preferably oligo nucleotide and / or peptide different from each other and corresponding to their complementary.

Claims

CLAIMS:

1 - Process for manufacturing a support carrying on at least one of its faces a plurality of poly sequences - preferably oligo-nucleotide and/or peptide sequences, these sequences (OGN), advantageously different from each other, being, on the one hand, matchable to their complements by affine interaction and, on the other hand, obtained by chemical synthesis in the presence of volatile solvents; said method essentially consisting of: a) implementing a support (1) whose face(s) intended to carry the OGN present) a network of reaction wells (4), each of these wells ( 4) intended to serve as a seat for localized anchoring and/or chemical synthesis of a given OGN sequence; these wells being obtained by application of a photocrosslinkable resin, by crosslinking of this resin by exposure to actinic radiation in the zones intended to define the wells and by elimination of the non-crosslinked resin to obtain a grid

(3) resin forming the walls which delimit the wells (4); b) to place this support (1) in a reaction enclosure (5) and to saturate this enclosure using vapors of the volatile solvent(s) used in the anchoring and the localized chemical synthesis(s) of OGN, preferably by circulating through the reaction chamber (5) a gas flow comprising the appropriate saturating vapors; c) to carry out localized anchoring and/or chemical synthesis of the OGN sequences in at least part of the wells, this operation being carried out:

(i) by locally and separately supplying each well concerned with reagents and liquid consumable products capable of allowing the anchoring and/or synthesis of the corresponding OGN sequence, (ii) by collectively (not locally) supplying the wells concerned with reagents and/or liquid consumable products common to the reactions intended to take place in all these wells;

(iii) and, by ensuring, prior to each localized well supply operation (4), to eliminate at least

^' 5 partially the liquid possibly contained in each well intended to be supplied in a localized manner.

2 - Method according to claim 1, characterized in that the support is chosen from the group of materials comprising: glass, quartz, silicon optionally coated with at least one layer of oxide or nitride.

3 - Method according to claim 1 or claim 2, characterized in that the surface support is treated so as to give it anchoring sites capable of forming non-labile bonds with the head comonomers of the OGN sequences, this treatment being, preferably, a silanization using an epoxidized alkoxysilane,

15 before and/or after the preparation of the microwell network.

4 - Method according to any one of claims 1 to 3, characterized in that the photocrosslinkable resin is selected from the group comprising: positive or negative resins, preferably from the subgroup comprising negative resins and more preferably still in class 20 comprising: (meth)acrylate, epoxy, polyester and/or polystyrene resins photocrosslinkable by radical and/or cationic means.

5 - Method according to any one of claims 1 to 4, characterized in that the gas flow for saturation of the reaction chamber comprises at least one neutral gas in addition to the vapors of the solvent(s) concerned, this flow gaseous being

25 preferably such that it determines an excess pressure in the enclosure compared to the ambient atmosphere.

6 - Method according to any one of claims 1 to 5, characterized in that to ensure the localized supply and the non-localized supply of the wells, corresponding means are implemented, the means -30 of localized supply and the support being movable relatively to each other in the three dimensions (X, Y, Z) using movement means; and in that to carry out the anchoring and synthesis on the support of n OGN sequences each comprising x comonomers:

*we store in memory the composition of the network of n OGN sequences to be produced by understanding this composition according to an organization in x successive ranks each comprising a comonomer and each corresponding to a series of actions of drawing off, supplying and depositing liquids reagents located in the n wells of the support, which liquids determine the nature of the comonomers in the rank Ri = 1 to x in the n wells;

A and we order, on the one hand, the localized supply means and the movement means to execute the different series of actions mentioned above, and on the other hand, to the non-localized supply means to carry out the withdrawal , the transport and supply of liquids not localized in the n wells of the support at different times of the procedure of anchoring and synthesis of the OGN sequences, each localized supply being followed by a step of evacuation of the liquids present in the wells and more generally, in the reaction chamber, these commands and the actions they induce being repeated by incrementing i by 1 in Ri until i = x.

7 - Method according to any one of claims 1 to 6, characterized in that the elimination according to step 3 (iii) of liquid is carried out so that the wells concerned are free of liquid to the extent of at least 90% of their volume, preferably at least 95% and even more preferably at least 98

%.

8 - Method according to claim 7, characterized in that the elimination of liquid is carried out by drying, advantageously by injecting gas, preferably neutral, into the reaction enclosure and/or by increasing the temperature of the enclosure reaction and/or the support and/or by lowering the vapor pressure of the liquid to be eliminated in the reaction chamber.

9 - Device for implementing the method according to any one of claims 1 to 8, characterized in that it comprises: - at least one support (1) whose face(s) intended to carry the OGN, present) a network of reaction wells (4), each of these wells intended to serve as a seat for the localized anchoring and chemical synthesis of a given OGN sequence, these wells being obtained by application of a photocrosslinkable resin, by crosslinking of this resin by exposure to actinic radiation in the zones intended to define the wells and by elimination non-crosslinked resin to obtain a grid (3) of resin forming the walls which delimit the wells (4); at least one reaction chamber (5) intended to contain the support;

10 - means for localized supply (6) of the reaction wells (4) with specific localized liquids (reagents/consumables) capable of allowing the anchoring and synthesis of a given OGN sequence in each of the wells (4) of the support (1),

15 - means for moving (7) (30) the localized supply means (6) relative to the support (1) and/or vice versa; means of non-localized supply (8) of the reaction wells (4) with liquids (products/consumables) that are not localized and common to the reactions intended to take place in

20 all or part of the wells (4); possibly means for moving (40) the non-localized power supply means (8); means for evacuating (9) (88) non-localized liquids; these evacuation means (9) (88) being associated with the means

25 non-localized power supply (8); organs (10) capable of allowing the saturation of the reaction chamber using vapors of the volatile solvent(s) used in the anchoring and localized chemical synthesis of OGN;

30 at least one container (11) (62) of localized liquid containers, at least one container (12) of non-localized liquids, at least one container (13) of effluent drain liquids; optionally gas supply members into the enclosure, - optionally at least one memory (15) of the different OGN sequences to be anchored and synthesized on the support, optionally at least one central unit (16) for reading the memory and for generating control signals for the supply of fluids and the movement of the localized supply means and the support relative to each other.

10 - Device according to claim 9, characterized in that the support 1 comprises:

- at least one substrate plate 2 made from a material chosen from the group comprising: glass, quartz, silicon optionally coated with at least one layer of oxide or nitride;

- at least one network 3, preferably a grid, of photocrosslinked resin secured to one of the faces of the substrate, this network defining a matrix of reaction wells 4, the photoresin being preferably selected from the group comprising: epoxy resins or (meth)acrylates photocrosslinkable by cationic and/or radical means.

11 - Device according to claim 9 or 10, characterized in that the localized supply means (6) comprise a system for projecting or depositing localized liquids, this system being advantageously chosen from the following systems:

1) mechanical ejection using devices of the type: a) drop on demand by piezoelectric effect; b) drop by jet interrupted by electrostatic effect; 2) pressure ejection using microvalve devices.

12 - Device according to one of claims 9 to 11, characterized in that the members (10) for saturation of the reaction enclosure (5) are designed so as to allow the circulation of a saturating gas flow comprising vapors of the volatile solvent(s) used and at least one neutral gas.

13 - Device according to any one of claims 9 to 12, characterized in that the reaction enclosure (5) comprises a sealed bellows (5 ₃ ) one of the ends (5 ₂ ) of which carries the localized supply means (6) and whose other end (5ι), on the one hand, presents, facing the latter, a reaction site intended to receive the support (1) and, on the other hand, comprises the supply means non-localized (8), the end (5 ₂ ) carrying the localized power supply means being preferably fixed and the other end (5χ) carrying the support (1) being, preferably, movable in the three dimensions x, y, z, thanks to the movement means (7).

14 - Device according to any one of claims 9 to 13, characterized in that the localized power supply means (6) are capable of being set in motion in the 3 dimensions x, y, z under the action of the means displacement (7), relative to a fixed part of the reaction enclosure (5) intended to receive the support (1) and associated with the non-localized supply means (8).

15 - Device according to any one of claims 9 to 12, characterized

- in that the support (1) can be moved in translation along an x axis via the movement means (7), the localized power supply means (6) can be moved in translation along an axis y via movement means (30), the non-localized power supply means (8) can be moved in translation along a z axis, via movement means (40); x, y, z being orthogonal to each other;

- in that at least part of the movement means (7), (30), (40) is located outside the sealed and saturable reaction enclosure (5), this external part for the means (7), ( 30), (40) preferably comprising at least one electric motor (7ι) (3χ), (4j) and its power supply:

- and in that it comprises at least one airlock (50) for loading and unloading the support (1). 16 - Support carrying an OGN sequence network as defined in claims 1 and 10.

17 - Method for detecting and/or identifying target OGN sequences using biosensors comprising probes formed from OGN sequences complementary to the target OGN sequences and capable of pairing with each other by affine interaction, characterized

- in that we use a support carrying an array of OGN probes according to claim 16, - and in that we reveal the probe / target pairing using a fluorescent marker of picogreen type.

18 - Method for detecting and/or identifying target OGN sequences using biosensors comprising probes formed from OGN sequences complementary to the target OGN sequences and capable of pairing with each other by affine interaction, characterized

- in that a support carrying a network of OGN probes according to claim 16 is used,

- in that this support is shaped into an affinity sensor comprising at least one structure comprising at least one semiconductor material Se, coated on at least one of its faces with at least one insulating layer Is, the latter presenting on its surface the OGN probes,

- in that these OGN probes are placed in contact with a LC conductive liquid medium comprising the target OGN sequences,

- and in that we apply the following operating mode: a) select unmarked OGN probes, b) ensure that the Fermi level of the Se corresponds substantially to, or passes through the intrinsic surface level of the Se, c) subject the Se to periodic illumination comprising photons whose energy is > the band gap energy of the Se, d) directly or indirectly measure the variations ΔVbp of the 5 flat band potential Vbp of the Se, induced by a charge effect phenomenon directly and essentially linked to the specific pairings of the target OGN sequences of the LC conducting medium with their complementary ligands of the OGN probe(s), excluding: 10 (i) variations resulting from possible effects of charges and/or charge transfers caused by chemical reactions catalyzed by enzymes and in which consumption of part of the substances to be detected occurs, (ii) and variations in the photoresponse linked to the appearance in the LC medium of at least one tracer product capable of being revealed through variations in pH or Redox potential, and/or through markers, preferably of the type of those absorbing or emitting radiation (fluorescent, radioactive, colored , eg). 20 - and interpret the signals collected in terms of identification and/or dosage of LC target OGN sequences.