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• • B—PERFORMING OPERATIONS; TRANSPORTING
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  • B01J2219/00612—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports the surface being inorganic
• • B—PERFORMING OPERATIONS; TRANSPORTING
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  • B01J2219/00614—Delimitation of the attachment areas
  • B01J2219/00621—Delimitation of the attachment areas by physical means, e.g. trenches, raised areas
• • B—PERFORMING OPERATIONS; TRANSPORTING
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  • B01J2219/00626—Covalent
• • B—PERFORMING OPERATIONS; TRANSPORTING
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  • B01J2219/00632—Introduction of reactive groups to the surface
  • B01J2219/00637—Introduction of reactive groups to the surface by coating it with another layer
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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  • B01J2219/00718—Type of compounds synthesised
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• • B—PERFORMING OPERATIONS; TRANSPORTING
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  • B01J2219/00718—Type of compounds synthesised
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  • B01J2219/00722—Nucleotides
• • B—PERFORMING OPERATIONS; TRANSPORTING
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• • C—CHEMISTRY; METALLURGY
  • C40—COMBINATORIAL TECHNOLOGY
  • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
  • C40B40/00—Libraries per se, e.g. arrays, mixtures
  • C40B40/04—Libraries containing only organic compounds
  • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
• • C—CHEMISTRY; METALLURGY
  • C40—COMBINATORIAL TECHNOLOGY
  • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
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  • C40B40/10—Libraries containing peptides or polypeptides, or derivatives thereof
• • C—CHEMISTRY; METALLURGY
  • C40—COMBINATORIAL TECHNOLOGY
  • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
  • C40B60/00—Apparatus specially adapted for use in combinatorial chemistry or with libraries
  • C40B60/14—Apparatus specially adapted for use in combinatorial chemistry or with libraries for creating libraries

Definitions

• 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.
• polymer probes oligonucleotides and / or peptides
• the revelation of these pairings can be carried out using fluorescent, colored, radioactive markers or by electronic transductance.
• 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.
• OGN sequences oligo - nucleotide and / or peptide
• the invention also relates to a method for detecting and / or identifying target OGN sequences using biosensors manufactured by the above-mentioned method.
• the OGN sequences correspond to poly- preferably oligo-nucleotide and / or peptide sequences.
• it can be: - oligonucleotides each carrying an ATCG nitrogen base,
• PNA Peptide Nucleic Acid
• 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.
• a support 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.
• OGN probes oligonucleotide sequences
• the 5 'ends of the nucleotides are protected by DMT (dimethoxytrityl) and the 3' ends by ⁇ -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.
• FODOR et al corresponding to the article Science vol 251, 767 - 773, 1991; discloses an in situ synthesis localized on the support, photochemically.
• 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.
• NVOC nitrotératryloxycarbonyl
• DMT protective group
• 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.
• 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.
• 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.
• 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.
• 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.
• reaction regions on the support are those 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.
• the reaction regions can be formed by three-dimensional wells delimited by walls as shown in FIG. 12.
• OGN sequences of oligonucleotides are synthesized in all the reaction regions.
• 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 em ⁇ nucleotide.
• Patent No. 5,658,802 of the United States of America 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.
• PCT application WO 94/27,719 concerning a method and a device for producing a chemical reaction network on the surface of a support.
• 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.
• 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 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.
• 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 ;
• 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 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 ⁇ m 2 and 10 x 10 ⁇ m 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.
• APN Nucleic Acid Peptide
• 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.
• 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.
• 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.
• positive resin is meant in the sense of the invention a resin made very soluble by irradiation.
• 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.
• 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.
• an organic solvent may, for example, be acetone, ⁇ -butyrolactone, Propylene-Glycol-Methyl-Ether-Acetate (PGMEA), acetonitrile or even other solvents known to those skilled in the art.
• PMEA Propylene-Glycol-Methyl-Ether-Acetate
• acetonitrile even other solvents known to those skilled in the art.
• FIGS. 1 and 2 provide other details useful for the non-limiting description of the support and its production.
• 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.
• the silanizing agent is glycidoxypropyltrimethoxy-silane.
• silanizing agents mention may be made of glycidoxypropyldimethoxysilane, aminopropyltriethoxysilane, or alternatively carboxymethyltriethoxysilane.
• 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.
• 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.
• 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.
• the carrier gas used for saturation can be: argon or helium.
• this gas flow is such that it determines an overpressure in the reaction chamber relative to the surrounding atmosphere.
• the gas flow determines a pressure of 0.01 to
• 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.
• OGN for example oligonucleotides
• fluids gases, solvents, etc.
• 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
• non-localized liquid 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%.
• 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.
• gas preferably neutral
• 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.
• 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
• 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.
• FIG. 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. 7 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.
• 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;
• 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.
• 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.
• microwells of height varying between 0.1 to 1000 ⁇ m, preferably from 50 to 500 ⁇ m with surfaces of between 10 ⁇ 10 ⁇ m and 1000 ⁇ m ⁇ 1000 ⁇ m.
• 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 device of 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.
• 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.
• 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).
• chassis and / or gantries The various constituent elements of this device are, in practice, supported by chassis and / or gantries and / or adapted bases.
• 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.
• 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.
• the reaction chamber 5 is composed of a lower part 5 ⁇ 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 5 ⁇ 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 ®).
• the localized supply means 6 comprise a projection system.
• the projection system is advantageously chosen from the following systems:
• the projection system is of type 1-a. and comprises at least one nozzle 6 ⁇ for spraying or depositing localized liquids.
• This piezoelectric nozzle 6 ⁇ is connected, via a conduit 6 2 , to one or more localized liquid tanks 11.
• piezoelectric systems 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.
• 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
• 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 (6 ⁇ ), mechanical parts (20) produced from of materials inert towards non-localized liquid reagents. It can be stainless steel, polytetrafluoroethylene (PTFE), polyamide (NYLON ® ).
• PTFE polytetrafluoroethylene
• NYLON ® polyamide
• 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 neutral gas (28) for example argon
• overpressure e.g.: 0.5 bar
• 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 ⁇ m.
• 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.
• 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.
• the quantity (30) delivered is adapted to a support 1 with microwells 4 with a surface area of 400 ⁇ 400 ⁇ m and a height of 400 ⁇ m.
• the elements (20) (21) (29) can be, for example, the constituent elements of a microsyringe with a diameter less than 100 ⁇ m.
• 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.
• the elastomer valve can be replaced with a fluorinated elastomer valve (KALREZ ® or CHEMRAZ ® ).
• 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.
• the non-localized supply means 8 comprise a supply conduit 8 ⁇ for liquid not located in the lower part 5 ⁇ 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 8 ⁇ . 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 solenoi
• 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 9 ⁇ connecting an outlet orifice 9 2 formed in the lower part 5 ⁇ of the enclosure 5 and the effluent tank 13.
• the pipe 9 ⁇ 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 10 ⁇ , 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 10 ⁇ 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 10 ⁇ comprise solenoid valves 10 7 and 10 8 controlled by the central unit 16 which thus controls the flow of saturation gas.
• 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.
• 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.
• the displacement means have sufficient chemical resistance to the reagents used for the synthesis of OGN, in particular the solvent (s) used for saturation.
• 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 6 ⁇ .
• 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 5 ⁇ relative to the projection nozzle 6 ⁇ .
• Such displacement means make it possible to supply the reaction wells 4 of the support 1 with localized liquid.
• FIGS. 5 and 6 are enlarged photographs of a support (1) comprising several tens of microwells (4) each having a surface of 150 ⁇ 150 ⁇ m and a height of approximately 100 ⁇ m. 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.
• 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.
• the amplitude of the mean displacements of microdeposition / bellows is limited to the deflections of the bellows, ie a few centimeters.
• Figs. 7, 8 and 9 show a second embodiment of the device according to the invention.
• 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.
• 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.
• 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 ® ).
• TEFLON ® PTFE
• steel stainless perfluorinated elastomers
• PALREZ ® perfluorinated elastomers
• CHEMRAZ ® perfluorinated elastomers
• polyamides NYLON ®
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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 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.
• 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 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.
• 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.
• 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.
• 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 °.
• 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.
• 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.
• 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.
• 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
• 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.
• 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 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
• 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,
• 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.
• 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 ⁇ m and a height of approximately 415 ⁇ m.
• 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
• the crosslinking of the resin is sufficiently advanced for the superstructure to be inert with respect to the synthesis of oligonucleotides.
• 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:
• 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'):
• 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 X ⁇ X 2 X 3 ⁇ ACG is the OGN L226 of biological interest.
• 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.

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• 2000
  • 2000-09-27 WO PCT/FR2000/002671 patent/WO2002026373A1/fr active Application Filing
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