METHOD FOR IMMOBILISING A COMPOUND OF INTEREST ON A SUBSTRATE IN A GIVEN PATTERN AND KIT FOR IMPLEMENTING SAME

Abstract

A method for immobilizing a compound of interest on the surface of a substrate in a given pattern using a printing pad. The printing pad is made from a polymer material with a face having a hollow profile that geometrically matches the pattern. The compound of interest is deposited on the surface of walls of a recess. A solution of a compound, capable of forming a link with the substrate and a link with the compound of interest, is confined inside the recess between the substrate and the face of the pad, in a solvent capable of penetrating into the polymer material. The confinement is carried out at a temperature and for a period sufficient to allow the solvent to penetrate the polymer material.

Claims

1-12. (canceled)

13. A method for immobilizing a compound of interest on a surface of a substrate according to a given pattern, comprising successive steps of: on a printing pad made from a polymer material comprising a printing face having a profile of recesses that is geometrically complementary to said given pattern, deposition of said compound of interest on a surface of walls of at least one recess; confinement of a solution of a linker compound in a solvent inside said recess between said substrate and said printing face of the pad, said linker compound capable of simultaneously forming a bond with said substrate and a bond with said compound of interest, and the solvent capable of penetrating into said polymer material; and wherein said confinement is carried out at a temperature and for a period of time to allow the solvent inside said recess to penetrate into said polymer material.

14. The method as claimed in claim 13, wherein the printing pad is made from an elastomeric material.

15. The method as claimed in claim 13, wherein the solvent is chosen from a group consisting of toluene and tetrahydrofuran.

16. The method as claimed in claim 13, wherein the linker compound is a phosphorus-comprising dendrimer having a central nucleus and comprising, at its periphery, a functional group capable of forming a covalent bond with said substrate and a functional group capable of forming a covalent bond with said compound of interest.

17. The method as claimed in claim 13, wherein the confinement is carried out for a period of time of between 10 seconds and 15 minutes.

18. The method as claimed in claim 13, wherein said given pattern constitutes a diffraction grating.

19. The method as claimed in claim 13, wherein said compound of interest is chosen from a group consisting of a nucleic acid molecule, a peptide, a protein, a polysaccharide and a lipid.

20. The method as claimed in claim 13, wherein said substrate is made from glass.

21. The method as claimed in claim 13, wherein said substrate is made from silicon.

22. The method as claimed in claim 13, wherein said substrate is made from plastic.

23. A method of fabricating biochips comprising a step of using a method as claimed in claim 13.

24. The method of fabricating biochips as claimed in claim 22, wherein the biochips are DNA biochips.

25. A kit for performing steps of a method for immobilizing a compound of interest on a surface of a substrate according to a given pattern as claimed in claim 13, comprising: a printing pad made from a polymer material comprising a printing face having a profile of recesses that is geometrically complementary to said given pattern; and a compound capable of simultaneously forming a bond with said substrate and a bond with said compound of interest.

26. The kit as claimed in claim 25, comprising a solid or semi-solid substrate.

27. The kit as claimed in claim 25, comprising instructions for performing the steps of the method for immobilizing said compound of interest on the surface of said substrate according to said given pattern.

28. A kit as claimed in claim 25, wherein a solution of said compound capable of simultaneously forming the bond with said substrate and the bond with said compound of interest is in a solvent, the solvent capable of penetrating into said polymer material.

29. The kit as claimed in claim 25, comprising reagents to pretreat said substrate to facilitate am attachment of the linker compound on the surface of said substrate.

30. The kit as claimed in claim 29, comprising silanization reagents.

Description

[0096] The characteristics and advantages of the invention will emerge more clearly in the light of the implementation examples below, provided simply by way of illustration and which are in no way limiting with respect to the invention, with the support of FIGS. 1 to 10, in which:

[0097] FIG. 1 shows diagrammatically the various steps of a method according to one particular embodiment of the invention;

[0098] FIG. 2 shows diagrammatically various variants of printing pads that can be used in a method according to the invention;

[0099] FIG. 3 shows images, obtained by a fluorescence scanner, of the substrate after carrying out a method according to the invention by means of a pad comprising a single recess with a circular cross-section, the compound of interest being a fluorescent oligonucleotide and the linker compound a phosphorus-comprising dendrimer, respectively before and after a step of treating the substrate in order to reduce the imine functions present between the oligonucleotide and the dendrimer and between the dendrimer and the substrate, (a) the pad having been inked with a solution of the oligonucleotide, (b) the pad having been inked with a solution without oligonucleotides, (c) the pad not having been inked;

[0100] FIG. 4 shows a graph representing the fluorescence intensity measured for each of the substrates the images of which are shown in FIG. 3;

[0101] FIG. 5 shows images, obtained by a fluorescence scanner, of the substrate after carrying out a method according to the invention by means, respectively, of two pads having a profile of recesses comprising line-shaped recesses (lines of width 15 m with a pitch of 30 m for the pad T1, and lines of width 10 m with a pitch of 20 m for the pad T2), the compound of interest being a fluorescent oligonucleotide and the linker compound a phosphorus-comprising dendrimer;

[0102] FIG. 6 shows an image, obtained by fluorescence microscope, of the substrate after carrying out a method according to the invention by means of a pad having a profile of recesses comprising line-shaped recesses of width 500 nm with a pitch of 1 m, the compound of interest being a fluorescent oligonucleotide and the linker compound a phosphorus-comprising dendrimer;

[0103] FIG. 7 shows images of the substrate of FIG. 6, obtained by atomic force microscopy, (a) viewed from above and (b) in perspective view;

[0104] FIG. 8 shows images obtained by atomic force microscopy, (a) viewed from above and (b) in perspective view, of the substrate after carrying out a method according to the invention by means of a pad having a profile of recesses comprising line-shaped recesses of width 20 m with a pitch of 40 m, the compound of interest being a fluorescent oligonucleotide and the linker compound a phosphorus-comprising dendrimer;

[0105] FIG. 9 shows images obtained by atomic force microscopy, viewed from above, of the substrate after carrying out some of the steps of a method according to the invention, not using a compound of interest, by means of a pad having a profile of recesses comprising recesses with a circular cross-section of diameter 20 m, (a) the linker compound being 1,2-polybutadiene-NH.sub.2 and the solvent being toluene, (b) the linker compound being a phosphorus-comprising dendrimer and the solvent being ethanol; and

[0106] FIG. 10 shows a graph representing the fluorescence intensity measured for DNA biochips obtained by means of a method in accordance with the invention, or by means of a prior art method by microcontact printing, after hybridization of the oligonucleotide probe immobilized on the biochip with a fluorescent complementary target oligonucleotide, for various concentrations of oligonucleotide probe (1, 2 and 5 M).

[0107] The various steps of a method according to one embodiment of the invention, for the immobilization of a compound of interest on a solid or semi-solid substrate in a given pattern, are shown in FIG. 1.

[0108] This method uses a pad 10, made from elastomeric polymer material, for example from crosslinked PDMS. This pad 10 can be produced by any method that is conventional in itself. For example, it can be produced by means of a mold, for example made from polyurethane, from silicon or from epoxy resin, of appropriate shape, by filling this mold with a precursor of the material constituting the pad 10 in liquid form, and curing, in particular by heat-crosslinking.

[0109] The pad 10 is formed of a membrane 11, placed in a carrier structure 14, for example made from PLEXIGLASS. A plurality of recesses 13 are made in one face, called printing face, 12 of this membrane 11, according to a profile that is geometrically complementary to the desired pattern for the immobilization of the compound of interest on the substrate.

[0110] In a first phase, the method according to the invention comprises the deposition of the compound of interest on the surface of the walls of the recesses 13. This deposition can be carried out by the succession of the following steps.

[0111] In a first step, shown in 20 in FIG. 1, a drop 30 of a solution of the compound of interest in a solvent is deposited on the printing face 12 of the pad 10.

[0112] The drop 30 is then removed from the pad 10 having thus been inked, and said pad is dried, in particular under a stream of nitrogen, so as to obtain, as indicated in 21 in FIG. 1, a pad 10 the surface of the printing face 12 of which, including in the recesses 13, is coated with a layer 31 of the compound of interest, which remains attached to this surface. In FIG. 1, for better visibility of the layer 31, the latter is represented with a thickness that is much greater than its actual thickness. Likewise, the relative sizes of all the elements represented in FIG. 1 are not representative of the reality, some elements being artificially enlarged so as to facilitate understanding.

[0113] The next step, shown in 22 in FIG. 1, consists in removing the compound of interest from the zones of the printing face 12 distinct from the recesses 13. For this purpose, the printing face 12 of the pad 10 is applied onto a solid surface such as a glass slide 23, according to microcontact printing technology. The molecules of the compound of interest in contact with the slide 23 are transferred onto the latter. The compound of interest then remains solely present on the surface of the walls of the recesses 13.

[0114] The next phase of the method according to the invention consists of a confinement of a solution of a linker compound between the substrate 40 and the pad 10, in the recesses 13.

[0115] For this purpose, as indicated in 24 in FIG. 1, a drop 32 of a solution of the linker compound in the solvent, for example tetrahydrofuran or toluene, is deposited on substrate 40, for example by means of a pipette 25. The printing face 12 of the pad 10 is then applied, as indicated in 26, against the surface of the substrate 40.

[0116] This operation has the effect of trapping a volume 33 of the solution of the linker compound between the substrate 40 and the pad 10, in the recesses 13, as shown in 27 in FIG. 1. The linker compound is placed there in the presence of the layer 31 of the compound of interest arranged at the surface of the walls of the recesses 13. The confinement is maintained until the solvent has penetrated through the pad 10. During this confinement phase, the molecules of the linker compound are forced to assemble to the substrate 40. Simultaneously, the molecules of compound of interest are transferred from the surface of the pad 10 onto the molecules of linker compound.

[0117] At the end of this confinement phase, after removal of the pad 10, a stack of molecules of the linker compound 34 and of molecules of the compound of interest 31 is obtained on the substrate 40, as indicated very diagrammatically in 28 in FIG. 1. The compound of interest is thus immobilized on the substrate 40 in a pattern that is the inverse of the profile of recesses 13 of the pad 10.

[0118] The implementation of all of these steps has advantageously been simple and fast.

[0119] FIG. 2 shows diagrammatically various examples of profiles of recesses 13 of pads 10 that can be used in accordance with the present invention.

[0120] In a first example, shown in (a) in FIG. 2, the pad 101 has a single recess 13. The compound of interest is then immobilized on the substrate 40 in the form of a spot.

[0121] In a second example and a third example, both shown in (b) in FIG. 2, two distinct pads 102, 103 show, respectively, the following profiles of recesses: a network of recesses of circular cross-section and of diameter 5 m, with a pitch of 10 m (pad 102); a network of recesses of circular cross-section and of diameter 20 m, with a pitch of 20 m (pad 103).

[0122] In a fourth example and a fifth example, both shown in (c) in FIG. 2, four distinct pads 104, 104 and 105, 105 show, respectively, the following profiles of recesses: a network of recesses in the form of lines of width 15 m, with a pitch of 30 m (pads 104, 104); a network of recesses in the form of lines of width 10 m, with a pitch of 20 m (pads 105, 105).

[0123] In a sixth example, shown in (d) in FIG. 2, sixteen distinct and identical pads 106 show a profile of recesses consisting of a network of recesses in the form of lines of width 500 nm with a pitch of 1 m.

[0124] Various examples of implementation of the method according to the invention are described below, in the context of the fabrication of DNA biochips, in which are immobilized, on the substrate 40, as compounds of interest, oligonucleotide probes intended for the detection, in a given sample, of complementary target oligonucleotides.

[0125] A. Materials and Methods

[0126] Biological Material

[0127] All of the oligonucleotides used in the examples below are used in a phosphate buffer solution (Na.sub.2HPO.sub.4) at 0.3 M, pH 9. Their respective sequences are the following:

TABLE-US-00001 Oligonucleotidelabeledwithafluorophore (SEQIDNO:1): F1:5-[NH.sub.2]-TAT-ACT-CCG-GGA-AAC-TGA-CAT-CTA-G- [Cy5]-3 Oligonucleotideprobe(HSP12genefragment) (SEQIDNO:2): S: 5-[AmC6F]-AATATGTTTCCGGTCGTCTC-3

[0128] wherein AmC6F represents a spacer consisting of a chain comprising six carbon atoms and ending with an NH.sub.2 amine function.

TABLE-US-00002 Fluorescentlylabeledcomplementarytarget oligonucleotide(CC)(HSP12genefragment) (SEQIDNO:3): 5-[Cy5]-GAG-ACG-ACC-GGA-AAC-ATA-TT-3 Fluorescentlylabelednon-complementarytarget oligonucleotide(NC)(SEQIDNO:4): 5-[Cy5]-TTT-AGC-TTT-TGC-TGG-CAT-ATT-TGG-GCG-GAC- A-3

[0129] Products and Solvents [0130] For each of the products and solvents of commercial origin used, the suppliers are indicated in table 1 below.

TABLE-US-00003 TABLE 1 Commercial origin of the products/solvents used Product/solvent Supplier Sodium phosphate (NaH.sub.2PO.sub.4) Sigma-Aldrich Sodium borohydride (NaBH.sub.4) Sigma-Aldrich SSC buffer solution (saline sodium Corning citrate: 0.3M sodium citrate, 3M NaCl) Sodium dodecyl sulfate (SDS) Corning 3-Aminopropyltriethoxysilane (APTES) Sigma-Aldrich Tetrahydrofuran (THF) Sigma-Aldrich Ethanol (EtOH) Sigma-Aldrich Isopropanol Sigma-Aldrich PDMS (SYLGARD 184) Corning [0131] The 4.sup.th-generation (G4) phosphorus-comprising dendrimers, corresponding to general formula (III) below, are obtained in the following way.

##STR00003##

[0132] In a first step, the N-methyldichlorothiophosphorhydrazide (IV), a fundamental synthon for obtaining the dendrimer, is synthesized according to the reaction scheme:

##STR00004##

[0133] This is carried out by dropwise addition, under argon, of a solution of methylhydrazine (1.9 equiv.) in chloroform CHCl.sub.3, to a solution of trichlorothiophosphine (1 equiv.) in chloroform, while maintaining the temperature of the mixture at 60 C. throughout the addition.

[0134] The mixture is then left to slowly return to ambient temperature overnight, while maintaining the stirring. The following day, the reaction is controlled by .sup.31P{1H} NMR and left to stir, if necessary, for a further one to two days. The monomethylhydrazine hydrochloride obtained is then filtered under argon using a filtering hollow tube.

[0135] The N-methyldichlorothiophosphorhydrazide is stored in solution in chloroform at low temperature (20 C.) and is subsequently used as it is.

[0136] In the next step, the dendrimer with free aldehyde ends (V), which is a precursor of the phosphorus-comprising dendrimers (III), is prepared:

##STR00005##

[0137] To do this, hexachlorocyclotriphosphazene (1 equiv.), 4-hydroxybenzaldehyde (6.6 equiv.) and distilled THF, taken under argon, are mixed in a round-bottomed flask under argon. This mixture is stirred until the solids have completely dissolved.

[0138] Potassium carbonate (12 equiv.) is then added spatula by spatula, and the mixture is left to stir overnight at ambient temperature.

[0139] The following day, the reaction is controlled by .sup.31P{1H} NMR. The potassium carbonate is filtered through filter paper and the filtrate is concentrated in a rotary evaporator to give a white solid. At ambient temperature, the solid is taken up in methanol, filtered through a sinter funnel and rinsed twice with methanol and twice with ether. The dendrimer corresponding to general formula (V) above, termed 0 generation dendrimer, called DP0, is then obtained.

[0140] The fourth-generation phosphorus-comprising dendrimer, used to functionalize the glass slides, said dendrimer being called DP4 and corresponding to general formula (III) above, is then obtained by repeating one and the same sequence of two reactions, until the 4.sup.th generation is obtained:

[0141] 1) DPn (n representing the dendrimer generation, and n=0 to 3) (1 equiv.), CHCl.sub.3 and the solution of N-methyldichlorothiophosphorhydrazide prepared as described above (7, 13, 27 and 53 equiv., respectively) are mixed under argon.

[0142] After stirring for 2 h for the low generations, and 3 h for the high generations, at ambient temperature, the reaction is controlled by .sup.31P{1H} NMR.

[0143] The mixture is concentrated by half under reduced pressure, by means of a rotary evaporator, transferred into a dropping funnel and added dropwise to a large volume of pentane, in order to precipitate the product.

[0144] The precipitate is filtered off using a hollow tube. The solid is taken up in a minimum amount of chloroform, precipitated once again in a 4/1 pentane/diethyl ether mixture and filtered off using a hollow tube. 1.sup.st-, 2.sup.nd-, 3.sup.rd- and 4.sup.th-generation dendrimers with chlorine ends, called respectively DP1, DP2, DP3, DP4, are thus obtained.

[0145] 2) The DPn (n=1 to 4) (1 equiv.) and 4-hydroxybenzaldehyde (13, 28, 55 and 110 equiv., respectively), followed by distilled THF taken under argon, are introduced under an argon atmosphere at ambient temperature. Cesium carbonate (20, 40, 60 and 120 equiv., respectively) is then added spatula by spatula. The mixture is left to stir at ambient temperature for 16 h (overnight).

[0146] The following day, the reaction is controlled by .sup.31P{1H} NMR. The salts are removed by filtration through filter paper for the low generations and then using a centrifuge, and the filtrate is evaporated under reduced pressure to give a white solid.

[0147] The solid is dissolved in a minimum amount of chloroform and added dropwise to a large volume of a pentane/ether mixture in order to precipitate the product. The precipitate is filtered off through a sinter funnel. The solid is taken up in chloroform, precipitated again and filtered off. The 1.sup.st-, 2.sup.nd-, 3.sup.rd- and 4.sup.th-generation dendrimers with aldehyde ends, called DP1, DP2, DP3 and DP4, are thus obtained. [0148] Substrate

[0149] The epoxysilane slides are obtained from NEXTERION Slide E, Schott.

[0150] The glass slides are obtained from Delta Microscopies.

[0151] They are modified by silanization, as follows.

[0152] The slide is first of all washed in a 2.5 M alcoholic sodium hydroxide solution (50 g of NaOH in a mixture of 200 ml of milliQ H.sub.2O and 300 ml of 96% EtOH), for 30 min at temperature with stirring at 25 rpm. After a return to neutrality by means of three successive washes with milliQ water with stirring at 23 rpm, the slide is immersed in 96% ethanol for 5 min, and is then immersed in the silanisation bath comprising 3-aminopropyltrimethoxysilane (APTES) at 5% v/v in 96% ethanol EtOH. The slide is left in this bath for 30 min with stirring at 23 rpm at ambient temperature. It is then rinsed several times by immersing it for 5 min in a bath of 96% EtOH, then in a bath of absolute EtOH, still with stirring at 23 rpm. It is then dried by centrifugation (8 min at 500 rpm). Finally, the slide is kept in an oven at 120 C. for 1 h in order to ensure crosslinking of the silane-based coating on the slide.

[0153] Printing Pads Production

[0154] The pads are made from polydimethylsiloxane (PDMS, SYLGARD 184). The PDMS is a mixture of two components: an oligomer (silicone) and a crosslinking agent. These are mixed in proportions of 10/1 weight/weight. This mixture is then deposited on silicon molds with various types of patterns, degassed, and then placed at 80 C. for 6 h in order for the PDMS to crosslink.

[0155] Inking of the Printing Pads with the Compound of Interest

[0156] The inking of the pad is carried out by deposition of a drop of solution of compound of interest on the pad for 1 min. The drop is then removed and the pad is dried under a nitrogen stream.

[0157] Removal of the Compound of Interest from the Zones of the Printing Face of the Pad Distinct from the Recesses

[0158] The inked pad is brought into contact with a glass slide for 1 min in order to ensure transfer of the compound of interest from the pad to the slide.

[0159] Surface-Functionalization of the Substrate by the Microcontact Printing Technique (Prior Art)

[0160] Pads are made from polydimethylsiloxane (PDMS, SYLGARD 184). The pad is inked by deposition of a drop of solution of compound of interest on the pad for 1 min. The drop is then removed and the pad is dried under a nitrogen stream.

[0161] The inked pad is then brought into contact with a glass slide for 1 min for transfer of the compound of interest from the surface of the pad to the slide according to the patterns of the pad.

[0162] Functionalization by Confinement of the Dendrimers

[0163] A drop of 60 l of solution of G4 phosphorus-comprising dendrimers at 58 M in tetrahydrofuran (THF) is trapped under the structure of the PDMS pad (optionally inked with compound of interest), then all of the solution is confined on the silanized glass slide until the solvent has penetrated into the polymer material forming the pad (5 min at ambient temperature). During the confinement step, the dendrimers are forced to assemble on the slide according to the pattern that is geometrically complementary to the profile of recesses of the pad.

[0164] Deposition of the Oligonucleotide Probes on Slides for DNA Biochip Design (Prior Art)

[0165] The oligonucleotide probe is diluted to various concentrations (1, 5, 10, 20 M) in a phosphate buffer solution (0.3 M Na.sub.2HPO.sub.4, pH 9). 63 examples of each concentration are deposited in the form of spots with an automated depositing device (Q-Array mini, Genetix) using hollow needles. Each spot measures approximately 150 m in diameter. The deposition is carried out at a relative humidity of 50% and a temperature of 22 C.

[0166] Reduction of the Imine Functions after Deposition

[0167] After drying overnight in a humid atmosphere, the imine functions present between the dendrimers and the oligonucleotide probes and between the surface of the silanized substrate and the dendrimers are reduced for 3 h using an aqueous solution of sodium borohydride (NaBH.sub.4, 3.5 mg/ml). They are then rinsed three times in a bath of milliQ water for 5 min and, finally, dried under a nitrogen stream or by centrifugation. This step makes it possible to covalently bond the oligonucleotide probes to the dendrimers and the dendrimers to the substrate. The reduction step also makes it possible to convert the aldehyde functions of the dendrimers into inert alcohol functions, thus contributing to the reduction of the background noise.

[0168] Biochip Hybridization Protocols

[0169] After reduction, the oligonucleotide probes are brought into contact with the (fluorescently labeled) complementary target oligonucleotide CC at a concentration of 100 nM in the 5SSC buffer, 0.1% SDS, for 30 min at 37 C.

[0170] With regard to the hybridization with non-complementary targets, the oligonucleotide probes are brought into contact with the non-complementary target oligonucleotide NC, which is also fluorescently labeled and identical in size to the complementary target oligonucleotide CC. It is used at the same concentration (100 nM).

[0171] After the hybridization step, the slides are washed twice (3 min) in a bath of 2SSC, 0.2% SDS, then once (3 min) in a bath of 0.1SSC with stirring (1200 rpm). Finally, the slides are dried under a nitrogen stream.

[0172] Reading of Fluorescence

[0173] Each slide is analyzed using a fluorescence scanner (INNOSCAN 700, Innopsys) using two excitation wavelengths (532 nm and 635 nm). The photomultipliers (PMTs) of each wavelength are regulated according to the hybridization intensities so that there is no saturation of the fluorescent signal.

[0174] Unless otherwise indicated, the scanner parameters are the following: PMT 635: 100%, PMT 532: 100%, light: 50, contrast: 15, balance: 0.

[0175] Data Processing

[0176] For each spot the average fluorescence intensity, from which the intensity of the background noise is subtracted, is calculated with the dedicated software of the fluorescence scanner (Mapix, Innopsys). The fluorescence intensity after hybridization for each experiment is the average of all of the spots per probe concentration.

[0177] Fluorescence Microscopy Image

[0178] The fluorescence microscopy images were obtained with the Zeiss LSM 510 NLO microscope. Laser wavelength : 633 nm.40 immersion objective.

[0179] Atomic Force Microscopy Analysis

[0180] The analysis of the substrates by atomic force microscopy was carried out by means of an AFM Brucker Catalyst Mode SCANASYST Air microscope, with the following parameters: fo: 50-90 Hz, k: 0.4 N/m.

[0181] Detection by Light Diffraction

[0182] The diffraction signal is collected before and after the step of incubating the biochip with the target molecule, by means of a diffraction scanner which makes it possible to determine the intensity of a 1.sup.st-order diffraction beam of a grating of lines of 500 nm with a pitch of 1 m. The diffraction scanner parameters are the following: power (p): 1 mW, gain (g): 0.

[0183] The TIFF images from the diffraction scanner are analyzed with the Mapix software (Innopsys), which makes it possible to determine the average or median intensity of all the pixels of a precise zone of the image.

[0184] Any modification of the periodic arrangement of the gratings, in particular an increase in the height and in the width of the lines of the grating, linked to the interactions of the target molecules on the networks of probe molecules, causes variations in the diffracted signal intensity. These variations are quantified by calculating the gain according to the following formula:

[00001]$\mathrm{Gain}.Math..Math.\left(\mathrm{as}.Math..Math.\%\right)=\frac{I_1-I_0}{I_0}100$

[0185] wherein I.sub.1 represents the intensity of the 1.sup.st-order diffraction beam of a grating, measured after interaction with the target molecules, minus the background noise around the grating; and I.sub.0 represents the intensity of the 1.sup.st-order diffraction beam of a grating, measured before interaction with the target molecules, minus the background noise around the grating.

EXAMPLE 1

[0186] In this example of implementation of the method according to the invention, a pad comprising a single recess with a circular cross-section 1.5 cm in diameter was used.

[0187] The compound of interest is the F1 oligonucleotide having an amine function at its 5 end and labeled with a Cy5 fluorophore at its 3 end, at a concentration of 10 M.

[0188] After inking of the pad with this compound of interest, the confinement of the solution of G4 phosphorus-comprising dendrimers, between the silanized glass slide and the pad, is carried out.

[0189] The slide is then subjected to a step of reducing the imine functions present between the dendrimers and the oligonucleotide.

[0190] Controls in which the pad is inked by means of a solution without oligonucleotide, or in which the pad is not inked prior to the confinement phase, are also carried out.

[0191] The images obtained by the fluorescence scanner are shown in FIG. 3. A fluorescent spot with a substantially circular cross-section is observed therein for the slides obtained in accordance with the present invention (a). In comparison, no fluorescence is observed for the controls.

[0192] The method according to the invention thus made it possible, in a single step, which moreover is very short, that is to say 5 minutes, to immobilize the oligonucleotide on the glass slide in the desired pattern, by means of the phosphorus-comprising dendrimers.

[0193] The fluorescence intensity was also measured. The results obtained are shown in FIG. 4. They confirm the efficiency of the immobilization of the compound of interest on the substrate by means of the method according to the invention. Furthermore, the presence, after reduction of imine functions, of fluorescence at an intensity greater than the controls confirms the bonding of the compound of interest on the dendrimers.

EXAMPLE 2

[0194] In this example, the method according to the invention was applied to the immobilization of the compound of interest on the substrate according to micrometric patterns.

[0195] Two pads T1 and T2 were used, said pads having a profile of recesses comprising recesses in the form of lines, said lines having a width of 15 m with a pitch of 30 m for the pad T1, and said lines having a width of 10 m with a pitch of 20 m for the pad T2. The pitch is defined throughout the present description as the distance between the non-contiguous edges of two adjacent lines, that is to say as the sum of the width of a line and of the width of the zone which separates it from the adjacent line.

[0196] The compound of interest is the F1 oligonucleotide having an amine function at its 5 end and labeled with a Cy5 fluorophore at its 3 end, at a concentration of 10 M.

[0197] After inking of the pad with this compound of interest, the confinement of the solution of G4 phosphorus-comprising dendrimers, between the silanized glass slide and the pad, is carried out.

[0198] The images obtained by the fluorescence scanner are shown in FIG. 5. A grating of fluorescent lines reproducing negatively the profile of recesses of the pads is observed therein, both for the pad T1 and for the pad T2.

EXAMPLE 3

[0199] In this example, the method according to the invention was applied to the immobilization of the compound of interest on the substrate according to a nanometric pattern.

[0200] A pad having a profile of recesses comprising recesses in the form of lines having a width of 500 nm with a pitch of 1 m was used. This type of profile allows the fabrication of biochips suitable for detection by diffraction.

[0201] The compound of interest is the F1 oligonucleotide having an amine function at its 5 end and labeled with a Cy5 fluorophore at its 3 end, at a concentration of 10 M.

[0202] After inking of the pad with this compound of interest, the confinement of the solution of G4 phosphorus-comprising dendrimers, between the silanized glass slide and pad, is carried out.

[0203] The image obtained by the fluorescence microscope is shown in FIG. 6. A grating of fluorescent lines reproducing negatively the profile of recesses of the pad is found therein.

[0204] The slide was also examined by atomic force microscopy. The images obtained are shown in FIG. 7, viewed from above (a) and in perspective view (b). It is observed therein that the pattern obtained consists of lines having a width corresponding to a stack of dendrimers. Thus, after the solvent has been evaporated off and has been discharged through the PDMS, the dendrimers fill the space located inside the recesses of the pad.

EXAMPLE 4

[0205] In this example, a pad having a profile of recesses comprising recesses in the shape of lines having a width of 20 m with a pitch of 40 m was used.

[0206] The compound of interest is the F1 oligonucleotide having an amine function at its 5 end and labeled with a Cy5 fluorophore at its 3 end, at a concentration of 10 M.

[0207] After inking of the pad with this compound of interest, the confinement of the solution of G4 phosphorus-comprising dendrimers, between the silanized glass slide and the pad, is carried out.

[0208] The slide obtained was examined by atomic force microscopy. The images obtained are shown in FIG. 8, viewed from above (a) and in perspective view (b). It is observed therein that the pattern obtained consists of lines, the dendrimers having stacked along the walls of the recesses of the pad, so that they reproduce the outline of the walls of these recesses. This probably results from the fact that, when the ratio between the width of the recesses and the amount of dendrimers used is high, the amount of dendrimers confined in these recesses is insufficient to fill all of the space therein. The dendrimers then stack in a directed manner along the walls of the recesses.

EXAMPLE 5

[0209] In this example, 1,2-polybutadiene-NH.sub.2 (average molar mass 15000 g/mol) at 1 mg/ml in toluene, or the G4 phosphorus-comprising dendrimers at 1 mg/ml in ethanol, were used as linker compound.

[0210] Ethanol does not have the capacity to penetrate into the PDMS. For this solvent, the confinement was carried out at 80 C. for 15 min.

[0211] The pad has a profile of recesses comprising recesses of circular cross-section with a diameter of 20 m.

[0212] For this example, no compound of interest was used.

[0213] For each of the solutions of the linker compound, the confinement of the solution between the pad and an epoxysilane slide was carried out.

[0214] After confinement for 5 min, or 15 min for ethanol, the slides obtained were examined by atomic force microscopy. The images obtained are shown in FIG. 9, viewed from above, for (a) the 1,2-polybutadiene-NH.sub.2 in toluene, (b) the G4 phosphorus-comprising dendrimers in ethanol.

[0215] It is observed therein that, when the solvent is toluene, which is capable of penetrating into the PDMS, and the linker compound is 1,2-polybutadiene-NH.sub.2, the method according to the invention allows the immobilization of the linker compound on the substrate, in the form of a network of cylinders of diameter substantially equal to 20 m. The stacking of the linker compound did indeed occur along the walls of the recesses of the pad.

[0216] On the other hand, when the solvent is ethanol, no pattern can be observed on the substrate. No ordered immobilization of the linker compound on the substrate occurred.

EXAMPLE 6DNA BIOCHIP

[0217] A DNA biochip, suitable for detection both by fluorescence and by diffraction, was fabricated using a method in accordance with the invention, by means of G4 phosphorus-comprising dendrimers as linker compound.

[0218] The compound of interest is the S oligonucleotide probe.

[0219] The pattern formed on the silanized glass slide is a diffraction grating formed of lines of width 500 nm with a pitch of 1 m.

[0220] After reduction of the imine functions present between the dendrimers and the oligonucleotide probe, the slide is incubated, under hybridization conditions, with, on the one hand, the complementary target oligonucleotide CC, and, on the other hand, the non-complementary target oligonucleotide NC as negative control. These target oligonucleotides are both labeled with the Cy5 fluorophore.

[0221] At the end of the incubation step, an analysis of the slide with a fluorescence scanner makes it possible to measure, after incubation with the complementary target oligonucleotide CC, a fluorescence of intensity 729 (AU) and, after incubation with the non-complementary target oligonucleotide NC, a fluorescence of intensity 28 (AU) (average results obtained on 10 different interactions per condition).

[0222] This demonstrates in particular that the hybridization of the oligonucleotide probe immobilized on the substrate in accordance with the invention to the complementary oligonucleotides present in a sample, is possible and efficient.

[0223] With regard to the detection by diffraction, the diffraction gain obtained is 10.7% after incubation with the complementary target oligonucleotide CC, and 2.3% after incubation with the non-complementary target oligonucleotide NC (average results obtained on 10 different interactions per condition).

[0224] Thus, the diffraction gain is positive for the hybridization with a perfectly complementary target oligonucleotide, whereas it is negative after incubation with a non-complementary target oligonucleotide. This result validates the adequacy of the biochips fabricated in accordance with the invention, with respect to light diffraction detection techniques.

EXAMPLE 7

[0225] In this example, the performance levels of the method according to the invention in the field of application of fluorescence DNA biochips, compared with the prior art technique for fabricating biochips by microcontact printing, was evaluated.

[0226] For carrying out the method according to the invention, the linker compound is the G4 phosphorus-comprising dendrimer, and the solvent is THF. The pattern formed on the silanized glass slide is a spot 1.5 cm in diameter.

[0227] The deposition of the compound of interest by microcontact printing is carried out in a manner that is conventional in itself, on a silanized glass slide on which G4 phosphorus-comprising dendrimers have been attached beforehand.

[0228] For the two techniques (method according to the invention and microcontact printing), the compound of interest is the S oligonucleotide probe, used at the various following concentrations: 1, 2 and 5 M.

[0229] After reduction of the imine functions present between the dendrimers and the oligonucleotide probe, the slides are placed in the presence, under hybridization conditions, of the complementary target oligonucleotide CC.

[0230] The fluorescent signals were analyzed for each slide after this incubation in the presence of the complementary target oligonucleotide CC.

[0231] The results obtained are shown in FIG. 10. For each concentration of oligonucleotide probe tested, a strong increase in the fluorescent signals is observed therein for the slides obtained in accordance with the method according to the invention, compared with the slides obtained by the microcontact printing technique of the prior art.