Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J31/00—Catalysts comprising hydrides, coordination complexes or organic compounds
  • B01J31/02—Catalysts comprising hydrides, coordination complexes or organic compounds containing organic compounds or metal hydrides
  • B01J31/12—Catalysts comprising hydrides, coordination complexes or organic compounds containing organic compounds or metal hydrides containing organo-metallic compounds or metal hydrides
  • B01J31/14—Catalysts comprising hydrides, coordination complexes or organic compounds containing organic compounds or metal hydrides containing organo-metallic compounds or metal hydrides of aluminium or boron
  • B01J31/143—Catalysts comprising hydrides, coordination complexes or organic compounds containing organic compounds or metal hydrides containing organo-metallic compounds or metal hydrides of aluminium or boron of aluminium
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J31/00—Catalysts comprising hydrides, coordination complexes or organic compounds
  • B01J31/16—Catalysts comprising hydrides, coordination complexes or organic compounds containing coordination complexes
  • B01J31/1608—Catalysts comprising hydrides, coordination complexes or organic compounds containing coordination complexes the ligands containing silicon
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
  • C07B47/00—Formation or introduction of functional groups not provided for in groups C07B39/00 - C07B45/00
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2231/00—Catalytic reactions performed with catalysts classified in B01J31/00
  • B01J2231/30—Addition reactions at carbon centres, i.e. to either C-C or C-X multiple bonds
  • B01J2231/32—Addition reactions to C=C or C-C triple bonds
  • B01J2231/323—Hydrometalation, e.g. bor-, alumin-, silyl-, zirconation or analoguous reactions like carbometalation, hydrocarbation
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
  • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
  • C07B2200/11—Compounds covalently bound to a solid support
• • C—CHEMISTRY; METALLURGY
  • C40—COMBINATORIAL TECHNOLOGY
  • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
  • C40B40/00—Libraries per se, e.g. arrays, mixtures
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
  • Y02P20/00—Technologies relating to chemical industry
  • Y02P20/50—Improvements relating to the production of bulk chemicals
  • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S436/00—Chemistry: analytical and immunological testing
  • Y10S436/807—Apparatus included in process claim, e.g. physical support structures

Definitions

• the present invention relates to a process for functionalizing solid supports, to the functionalized solid supports obtained by implementing such a process, to their uses, in particular for the immobilization of biological molecules, such as nucleic acids, polypeptides, lipids, carbohydrates and hormones.
• the supports carrying immobilized biological molecules are advantageously used for the detection and the recognition of biological species, but other applications of these supports, such as chemical synthesis or modification on support or even the enhancement of biological reactions on support, are also possible.
• These supports must in particular allow the reproducible immobilization of the biological molecules of interest, insofar as a reproducible immobilization is conditional on a detection itself reproducible. These supports must also allow the immobilization of the biological molecules of interest in a sensitive manner.
• the sensitivity of a functionalized solid support depends on the immobilization rate and the method of detection of a signal, but also and above all on the level of background noise (non-specific signal). Reducing background noise improves the signal-to-noise ratio.
• the background noise essentially comes from the non-specific absorption of molecules other than the biological molecules of interest that one wishes to immobilize. and therefore should be limited.
• the immobilization step must be carried out in a well localized and homogeneous manner on the surface of the studs, in order to avoid any risk of burrs or halos.
• the immobilization of biological molecules on solid supports is generally carried out in two distinct stages:
• the reaction with the surface involves amino functions.
• the surface fixation of such functions is mainly ensured by thiourea or amide type bonds.
• thioureas has in particular been described in the article by Guo Z. et al., Nucleic Acid Research, 1994, 22, 5456-5465 and consists in activating amino surfaces by sulfur molecules such as for example phenylenediisothiocyanate, then react the molecules on the surface thus activated.
• amide bonds are obtained by reactions of amines on carboxylic acids in general by chemical modification of an amino support (for example by succinic anhydride or a polymerization reaction) as that is described for example in the American patents 5,919,523, 5,667,976 and 6,043,353.
• Direct functionalization on solid support with carboxylic acids can be carried out by the formation of self-assembled layers of acid alkanethiols on gold deposits (BONCHEVA M. et al., Langmuir, 1999, 15, 4317-4320; HUANG E. et al, Langmuir, 2000, 16, 3272-3280) or else by hydrosilylation reactions on silicon supports (Strother T. et al., J. Am. Chem. Soc, 2000, 122, 1205 -1209); however, in the latter case, the binding of the biomolecules involves complexation reactions.
• oligonucleotides with a dialdehyde group in order to allow their attachment to hydrazide functions.
• the functionalized supports are, in this case, polyacrylamide or nitrocellulose gels which form thick and therefore three-dimensional networks which have the advantage of increasing, for the same surface, the density of the immobilized strands. These gels nevertheless have certain limits concerning the size of the deposited pads and the size of the DNA strands which can be hybrids (Yershov G. et al, Proc. Natl. Acad. Sci. USA, 1996, 93, 4913-4918).
• the present invention firstly relates to a process for functionalizing a solid support comprising on the surface hydroxyl or hydride functions, comprising the following steps: a) grafting by covalent fixation (silylation or hydrosilylation) of at least one bifunctional molecule comprising a protected carboxylic acid function of formula (I) below:
• A represents a group allowing the covalent fixing of the bifunctional molecule of formula (I) on the hydroxyl or hydride functions of the support,
• R represents a protecting group for the carboxylic acid function
• - X represents a C 2 -C 8 hydrocarbon chain, linear or branched, saturated or unsaturated, optionally interrupted by one or more heteroatoms chosen from N, O and S
• b) deprotection of the carboxylic acid functions which have not been deprotected during step a) of grafting characterized in that step a) is carried out at a temperature between 50 and 200 ° C with a compound of formula (I) in which A is a mono functional group and in that said method further comprises a step c) of passivation of the residual hydroxyl or hydride functions of the support which has not reacted with the said molecule (s) of formula (I).
• the hydrocarbon chain of the bifunctional molecule of formula (I) comprises from 2 to 18 carbon atoms; such a chain length (less than 20 carbon atoms) does not lead to a solid support comprising an organized self-assembled monolayer (SAM). Indeed, according to A. ULMAN, Chem. Rev., 1996, 96, 1533-1554, the formation of SAM on solid supports using organosilicon compounds comprising a tri-functional end (of the alkyltrichlorosilane type) is possible when the hydrocarbon chain contains at least 20 carbon atoms .
• SAM self-assembled monolayer
• the use of molecules of formula (I), in which the group A allowing the fixing of these on the support is monofunctional makes it possible to obtain mono or sub-monomolecular layers densely reproducible.
• the molecules of formula (I) which are fixed to the surface of the support each have their anchoring point directly on the surface, unlike the di- or tri-functional molecules which often lead to thick, rough and poorly grafted layers. defined.
• the solid supports which can be functionalized according to this process are organic (plastic materials) or mineral surfaces, preferably porous or flat, of the metal oxide, silica and its derivatives types (glass, quartz,
• the monofunctional group A can in particular be chosen from dialkyl (dialkylamino) silane, dialkylhalosilane groups, diphenyl (dialkylamino) silane, diphenylhalosilane, [(monoalkyl),
• the protective group R of the molecules of formula (I) above can be chosen from the groups described in Protective groups in organic synthesis (TW GREENE and a, 2 nd edition, Wiley Interscience), such as for example a C ⁇ alkyl radical. -C or a cyclic radical.
• the molecules of formula (I) are preferably chosen from the organosilicon compounds of formula (la) below:
• a 3 represents a C ⁇ -C alkoxy, dialkyl (C ⁇ -C 4 ) amino radical or a halogen atom such as chlorine,
• - R represents a protective group chosen from C ⁇ -C 4 alkyl radicals and cyclic radicals such as the benzyl radical,
• - n is an integer between 2 and 18.
• C ⁇ -C alkyl radicals defined for Ai and A 2 mention may in particular be made of methyl, ethyl, propyl and butyl radicals, the methyl radical being particularly preferred.
• dialkyl (C ⁇ -C 4 ) amino radicals defined for A3 mention may especially be made of the dimethylamino radical.
• the compounds of formula (la) are more particularly chosen from those in which:
• - R represents a t-butyl radical
• a 3 represents a dimethylamino radical
• the corresponding molecule ((CH 3 ) 2 N-Si (CH 3 ) - (CH 2 ) ⁇ o-C- (0) -0-t-butyl), which n is not commercial, can be prepared according to conventional methods of esterification of undecylenic acid (Trost BM et al, "Comprehensive organic synthesis", 1991, 6, Pergamon Press; Staab HA et a, "Azolides in organic synthesis and biochemistry ", 1998, Wiley Weinheim) and hydrosilylation (Fleming L,” Comprehensive organic synthesis ", volume III, Chapter 13).
• the molecules of formula (I) are preferably chosen from alkenes, alkynes, aldehydes, peroxides or even organometallic compounds. Among these compounds, the methyl or t-butyl esters of undecylenic acid are particularly preferred.
• step a) is a silylation step, it can just as well be carried out in the organic, aqueous phase, catalyzed or not, as in the vapor phase.
• step a) advantageously comprises the following steps: i) removal of the contaminants from the solid support and hydroxylation of its surface, ii) introduction into a solvent chosen from non-polar hydrocarbon solvents, polar solvents and their mixtures, under an inert atmosphere, of an organosilicon compound of general formula (la) as defined above, iii) the silanization of the support obtained in step i) by immersion in the solution prepared in step ii) and iv) the annealing of the silanized support obtained in step iii), after evaporation of the solvent under an inert atmosphere and at a temperature between 50 and 200 ° C, preferably at 140 ° C, for a period of between 2 and 72 hours, and v) cleaning and drying the modified support obtained in step iv).
• contaminants of the solid support is meant any compound such as grease, dust or the like, present on the surface of the support and which is not part of the chemical structure of the support itself.
• step i) can be carried out using one or more solvents and / or oxidants and / or hydroxylants (for example a sulfochromic mixture), a detergent (for example Hellmanex ® ), photochemical ozone treatment or any other appropriate treatment.
• the concentration of the organosilicon compound of formula (la) in the solvent is preferably between 10 ° and 1 mole / liter.
• step a) is a hydrosilylation step
• said step a) preferably comprises the following steps: i) removal of the contaminants from the solid support and the hydration of its surface, ii) washing the support in an anhydrous alcohol such as anhydrous methanol, iii) bringing the support into contact with a deoxygenated alkene previously under argon, iv) hydrosilylation of the support by adding d '' a catalyst (Speier or Karstedt catalyst, ethyldichloroaluminium) for 2 to 24 hours at a temperature between 90 and 200 ° C.
• d '' a catalyst Speier or Karstedt catalyst, ethyldichloroaluminium
• the grafting step a) makes it possible to fix a quantity of molecules of formula (I) of between 1 and 8 ⁇ mol / m 2 of substrate.
• the grafting step must leave intact the protection of the carboxylic acid of the bifunctional molecule of formula (I) in order to ensure better ordering of the grafted layer, the grafting density, as well as the reproducibility of the reaction.
• a deprotection of the carboxylic acid functions occurs during certain hydrosilylation reactions or else when a chlorosilane is used as compound of formula (la).
• the grafting step a) is complete, it is therefore necessary to deprotect the carboxylic acid functions which would not have been deprotected during the grafting step.
• the deprotection step b) can be carried out under mild conditions according to a method consisting in forming a silyl ester, easily hydrolysable in water, preferably by reacting the support with a silane of the following formula (II):
• esters can be obtained:
• an iodotrialkylsilane such as iodotrimethylsilane in an anhydrous organic solvent (such as chloroform or dichloromethane) at room temperature or by heating to a temperature of approximately 60 ° C.
• step c) of passivation is therefore carried out simultaneously by step b).
• step b) is a thermal deprotection.
• This deprotection method is applicable to certain carboxylic acid esters such as isopropyl and t-butyl esters.
• This thermal deprotection can be carried out by treating the functionalized support at a temperature between 250 and 280 ° C. for 30 to 60 minutes, in an inert atmosphere (argon or nitrogen for example).
• This thermal deprotection step carried out after the silylation or hydrosilylation step, makes it possible to generate in situ, by simple raising of the temperature, the carboxylic acid function at the surface.
• this thermal deprotection step can therefore be carried out simultaneously with step a), by simply raising the temperature at the end of step a).
• the thermal deprotection can be carried out locally, by raising the temperature locally, for example using a laser beam or any other suitable means, so as to define activatable zones surrounded by zones in which the carboxylic acid functions remain protected.
• the deprotection of the carboxylic acid functions is preferably carried out by formation of a silyl ester, since this method makes it possible both to generate carboxylic acid functions under mild conditions and to block the residual hydroxyl functions of the support ( passivation step of the support), which subsequently makes it possible to limit the non-specific adsorption of the molecules on the support. It is in fact the non-specific adsorption of biological molecules which is responsible for the background noise during detection.
• the step of passivation of the hydroxyl or hydride functions of the support is then carried out by, for example, reacting the latter with a silane of formula (II) as described above or by any other method conventionally known to those skilled in the art, such as for example by reaction of the support with hexamethyldisilasane or alternatively by a method of strong adsorption of small molecules such as methanol.
• the functionalized supports are washed with water and dried for example by a jet of compressed air.
• the subject of the invention is also the functionalized solid support obtained by implementing the functionalization method according to the invention and as described above.
• These functionalized supports allow immobilization reproducible biological molecules of interest while limiting the non-specific adsorption of molecules and therefore increasing the signal / noise ratio during detection. They also make it possible to limit the burrs and the halos of the localized deposits by controlling the spreading of the drops of reagents.
• These functionalized supports can in particular be used for the immobilization, by covalent fixation, of biological molecules of interest carrying amino or hydroxyl functional groups, such as nucleic acids, polypeptides (proteins, enzymes), lipids, carbohydrates or hormones.
• amino or hydroxyl functional groups such as nucleic acids, polypeptides (proteins, enzymes), lipids, carbohydrates or hormones.
• Nucleic acids both oligonucleotides as DNAs or RNAs, or nucleic acids with a backbone or modified bases, such as peptide nucleic acids (PNA: Peptide Nucleic Acids) which involve peptide bonds in place of phosphodiester bonds.
• PNA Peptide Nucleic Acids
• the present invention therefore also relates to a method of immobilizing biological molecules on a functionalized solid support, characterized in that it comprises the following steps: a) the preparation of a functionalized solid support comprising terminal carboxylic acid functions in the form ester according to the process as defined above, b) deprotection and activation of the terminal carboxylic acid functions, c) bringing the modified solid support obtained in step b) or c) into contact with one or more solutions applied locally, in one or more solvents, of the biological molecule (s) to be immobilized, said biological molecules carrying at one of their ends an amine function or a hydroxyl function or a spacer arm functionalized by a primary amine function, d) evaporation of the solvent in order to cause the covalent fixing of the biological molecule (s) at the functional level ns carboxylic acid, e) inactivation of activated carboxylic acid functions which have not reacted with biological molecules using an amine in the gas phase or in solution, and f) washing the solid support
• Step b) of activation of the carboxylic acid functions can for example be carried out using a solution of N-hydroxysuccinimide, carbonyldiimidazole or carbodumide, or else any other suitable activation reagent known from 1 'Man of the trade.
• this activation step can be carried out in the presence of carbonyldiimidazole (0.1 M) in anhydrous tetrahydrofuran (THF), in about two hours at room temperature.
• This type of activation makes it possible in particular to obtain activated surfaces which are very reactive with respect to nucleophilic reagents (amines, alcohols, water).
• nucleophilic reagents amines, alcohols, water.
• the biological molecules are reacted in anhydrous solvents in the presence of a tertiary amine such as triethylamine to ensure the presence of the amino form of the functional group of biological molecules.
• the concentration of the solution (s) of biological molecules is preferably between 10 "6 to 10 " 3 mol / 1.
• any aqueous or anhydrous solvent allowing good dissolution of the latter and subsequent control of the evaporation of the solution could be used.
• the nature of the solvent must of course be chosen as a function of the nature of the biological molecule to be isolated.
• Each solution of biological molecules to be immobilized can be deposited at a determined location on the support by suitable means of microdeposition.
• anhydrous DMSO for example to immobilize oligonucleotides
• the oligonucleotides are preferably present in the solution at a concentration of between 10 "5 and 10 " 3 mol / 1.
• the DNA strands and the oligonucleotides are soluble in aqueous solution in all proportions.
• the immobilization step is preferably carried out in aqueous condition.
• the activation of the carboxylic acid functions by N-hydroxysuccinimide makes it possible to work in an aqueous condition.
• step d) of immobilization of the biological molecules, by evaporation of the solvent is crucial to the good progress of the immobilization process because it allows the covalent grafting of the biological molecules of interest on the functionalized support.
• the immobilization method according to the invention preferably uses very dilute solutions of biological molecules (of the order of 10 "6 to 10 " 3 mol / 1) the detection of which would then be impossible if the covalent bonding was not performed.
• this evaporation step is carried out slowly, that is to say for a time of between 1 and 10 hours approximately.
• the activated carboxylic acid functions which have not reacted with the biological molecules are then inactivated, using an amine in the gas phase or in solution (step e) ).
• This inactivation step makes it possible to avoid the problems of smearing of the drops, of contamination or of pollution during the following steps, it can also be called "capping".
• this inactivation step is carried out by reacting, in the gas phase, methylamine or dimethylamine, preferably dimethylamine, for 15 to 45 minutes .
• This inactivation step can easily make it possible to process several supports simultaneously.
• the inactivation step is carried out only on predefined areas of the same support, the areas not to be inactivated being previously protected for example by drops of water, organic solvents or mineral oil.
• the deactivation of unprotected zones then makes it possible to create a network of activated or reactivable zones and chemically inert zones.
• This embodiment of the method makes it possible to obtain spatially resolved supports which can, for example, be obtained by depositing drops of water on a surface comprising activated carboxylic acid functions. (for example with carbonyldiimidazole) followed by the step of inactivation of the surfaces accessible by an amine in the gas phase, then by rinsing of the support.
• the zones inactivated by the drops of water can then be reactivated before the step of covalent fixing of the biological molecules.
• the final washing step f) is intended to desorb the molecules which would not be covalently attached to the functionalized support according to the invention in order to obtain a reproducible density of biological molecules attached to the surface.
• This washing step is preferably carried out by subjecting said support to successive increasingly stringent washing steps, ranging for example from washing with water at a temperature of about 80 ° C. for about an hour to washing. in an aqueous solution of sodium dodecyl sulfate (SDS) concentrated, for example at 10%, at a temperature of approximately 80 ° C. for approximately 1 hour, followed optionally by sonication for a few minutes.
• SDS sodium dodecyl sulfate
• the reuse of supports is conditioned by obtaining reproducible signals. This washing step plays a role with regard to the reuse of these supports since it makes it possible to guarantee that the supports will not release molecules during the following possible stages of detection.
• this washing step has the effect of eliminating a certain number of biological molecules which are not covalently attached, and consequently of reducing the immobilization rate, therefore the intensity of the detection signal, it also makes it possible to decrease the non-specific adsorption rate and therefore the background noise during detection.
• the overall assessment of this washing step is therefore the improvement of the signal / noise ratio as well as the reproducibility of the measurement.
• the present invention also relates to the solid supports obtained by implementing the immobilization process according to the invention, that is to say the solid supports on which the biological molecules of interest are immobilized by covalent fixation.
• These solid supports can be used as analysis tools (diagnosis, sequencing, etc.) as well as preparation tools (isolation, separation of complex molecules) or for coating surfaces with properties specific (coatings for chromatography, "active" coatings such as antistatic, antibacterial coatings, etc.).
• immobilized biological molecules are enzymes, - as biosensors ("immunoassays", DNA analysis technique based on “microarrays” for example sequencing by SBH hybridization, single nucleotide polymorphism (SNP) and biomedical diagnosis).
• biosensors DNA analysis technique based on “microarrays” for example sequencing by SBH hybridization, single nucleotide polymorphism (SNP) and biomedical diagnosis.
• solid supports modified according to the present invention is particularly advantageous for the preparation of DNA chips, namely supports on which are fixed, covalently, a set of DNA of known sequences in a very precise order, these chips being reusable many times.
• DNA chips make it possible, by hybridization of the DNAs immobilized on the support with target nucleic acids or oligonucleotides, to determine the sequence of these target molecules or to follow the expression of genes.
• the applications are numerous: discovery of new genes, new drugs, carrying out diagnostics, toxicity studies, etc.
• a further subject of the present invention is therefore a nucleic acid or polypeptide chip, characterized in that it is obtained by the method of immobilization of biological molecules according to the present invention, in which said biological molecules are acids nucleic acids or polypeptides.
• the nucleic acid or polypeptide chips according to the invention have the advantage of being stable and therefore of being able to be reused many times, in particular the DNA chips can be reused in numerous cycles of hybridization and denaturation.
• the invention also comprises other arrangements which will emerge from the description which follows, which refers to an example of functionalization of a solid support, to an example of immobilization of oligonucleotides as well as to a example showing the reproducibility of the immobilization process according to the invention.
• Glass slides (76 x 26 mm) are immersed in a solution of sulfuric acid and hydrogen peroxide (7/3 v / v) at 80 ° C for one hour. The slides are then rinsed thoroughly with ultra pure water and then dried with a gas blower. They are finally placed in a glass reactor.
• the reactor containing the glass slides is heated to 140 ° C. for one hour under vacuum and then for one hour under a sweep of dry inert gas (argon, nitrogen).
• the reactor is then cooled in an ice-water bath and 150 ml of pentane are injected so as to cover the surface of the samples. Then injected 300 ⁇ l of silane.
• the pentane is then evaporated under vacuum, then the reactor is heated to 140 ° C and swept by a stream of inert gas (argon or nitrogen) for 12 hours.
• inert gas argon or nitrogen
• the functionalized glass slides obtained above in Example 1 are dried under an inert atmosphere at 140 ° C for 2 hours. After cooling, they are immersed in a solution of dichloromethane (DCM) previously dried over alumina, then iodotrimethylsilane is injected in an amount sufficient to reach a concentration of 0.1M. The hydrolysis is carried out overnight at room temperature. At the end of the reaction, the slides are washed with deionized water and dried with a jet of compressed air.
• DCM dichloromethane
• the slides are dried at 140 ° C under argon for two hours.
• the oligonucleotides (25 mothers, carrying a spacer arm functionalized by an amine function (C 6 -NH)), are solubilized in PBS phosphate buffer (Phosphate-Buffered Saline) of pH 8.5. Surfaces of 1 cm 2 are covered with 50 ⁇ l of oligonucleotide solution. After slow evaporation of the PBS buffer at a temperature of 50 ° C., the slides are inactivated by blocking (capping) of the acid functions which would not have reacted with the amino functions of the spacer arms of the oligonucleotides. To do this, the slides are treated with methylamine in the gas phase for 30 minutes.
• PBS phosphate buffer Phosphate-Buffered Saline

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