EP1440335A1

EP1440335A1 - Substrate coated with a transparent organic film and method for making same

Info

Publication number: EP1440335A1
Application number: EP02790522A
Authority: EP
Inventors: François BRENIAUX; Christophe Bureau; Patrick Chaton; Stéphane Getin
Original assignee: Commissariat a lEnergie Atomique CEA
Current assignee: Commissariat a lEnergie Atomique et aux Energies Alternatives CEA
Priority date: 2001-10-18
Filing date: 2002-10-16
Publication date: 2004-07-28
Also published as: US20050106566A1; FR2831275A1; WO2003034101A1; CN1605035A; CA2463845A1; FR2831275B1; JP2005505782A

Abstract

The invention concerns a substrate coated with a transparent organic film and a method for making same, and use thereof. The film-coated substrate is characterized in that the film is an electrically insulating organic polymer and transparent in at least a range of wavelengths, and in that the film is associated with a marker emitting at least in said wavelength range. The invention is in particular applicable in a means for detecting a chemical species, such as a biochip, in a quality control process, a certification process or an authentication process for an object.

Description

SUBSTRATE COATED WITH TRANSPARENT ORGANIC FILM AND MANUFACTURING METHOD

DESCRIPTION Technical area

The present invention relates to a substrate coated with a transparent organic film, to a process for manufacturing this substrate coated with the transparent organic film, and to its use. By “transparent” film is meant a film having remarkable optical properties of transparency (absence of optical absorption, absence of “quenching”, etc.) over at least one domain of the electromagnetic spectrum, and in particular a low optical extinction or "Coefficient k" in the wavelength range considered for transparency. This optical extinction coefficient is measured for example by spectroscopic ellipsometry or by spectrophotometry. The substrate can be an insulating, conductive or semiconductor substrate of electricity. It constitutes the support for the transparent organic film, and it is chosen according to the use or the destination of the substrate coated with the transparent organic film according to the present invention.

The present invention finds, for example, an application in the field of quality control, certification or authentication of substrates coated with transparent thin films. Indeed, it makes it possible to prove the manufacture and / or the origin of the substrate coated with the organic film of the present invention, into or onto which a fluorescent, phosphorescent or chemiluminescent marker known in very small quantities could have been purposely inserted. In this application of the present invention, the substrate is any object.

It also finds an application, for example in the field of chemisorption or physisorption detection on or in the transparent organic film of chemical, biochemical or biological species previously functionalized by a fluorescent phosphorescent or chemiluminescent marker, such as, for example , in fluorescence detection methods for microarrays with chemical or biochemical analysis, such as DNA microarrays. In this type of application, the substrate constitutes the support for the detection means.

For example, in the case of a biochip, the substrate may for example be a support of silica, gold or a composite such as Au / Si, Au / Si0 or more generally metal / substrate, and the transparent organic film a molecular means of attaching the biological probes to certain parts of the surface.

When the biochip is brought into contact with a sample solution to be analyzed, pairings take place between the DNAs of the sample and those fixed on the substrate. This fixation can, for example, be detected by having previously labeled the DNAs of the sample with a fluorescent, phosphorescent or chemiluminescent marker. In accordance with the present invention, the film is chosen to be transparent to the emission wavelength of the fluorescent, phosphorescent or chemiluminescent marker used, so as to absorb the photons emitted by this marker as little as possible, making it detectable at very low concentrations on the surface of the substrate, and minimizing the interference with the measurement. The signal / noise ratio and the lower detection limits of pairings on the biochip are thus improved. In the following description, the references in square brackets refer to the annexed list of references.

PRIOR ART With regard to the biochip application, the documents FR-A-2 787 581 (1998), FR-A-2 787 582 (1998) and US 5 810 989 (1998), describe electro-copolymerization on a silica substrate of precursor monomers of conductive polymers, such as pyrrole, with monomers functionalized by recognition molecules, in particular oligonucleotides. This technique is among the most used today for the localized fixation of recognition molecules on the studs of a biochip. In this technique, the adhesion of the conductive polypyrrole film to the substrate is exploited so as to achieve the fixing of the recognition molecules therein.

As described in the aforementioned documents, as well as in documents FR-A-2 784 188 (1998) and FR-A-2 784 189 (1998), the chip thus functionalized is brought into contact with a sample solution to be analyzed containing target molecules capable of coming to couple with the molecules of recognition of the support. In order to selectively detect the pads on which there is a coupling between the recognition molecules and the target molecules, the latter can advantageously be "labeled" with a fluorescent molecule, such as for example fluorescein or phycoerythrin which exhibits absorption at 543 n and an emission at 580 nm, the presence of which can then be detected using an appropriate optical device.

Unfortunately, polypyrrole has a significant absorption in the range of emission wavelengths of the fluorescent label used. This drawback is widely described in the literature relating to the technique in question.

Indeed, Arwin et al., In reference [1], measure on a polypyrrole film 22 nm thick on gold, an extinction index k = 0.3, for a refractive index n = 1, 45; Kim et al., In reference [2], measure an extinction k = 0.3 and an index n = 1.6 at λ = 632.8 nm, for a polypyrrole film 47 nm thick; Kim et al., In reference [3], measure on a 54 nm polypyrrole film in the oxidized state an index n = 1.45 for an extinction k = 0.28, and on a 47 nm film at the state reduces an index n = 1.6 for an extinction k = 0.21; and Guedon et al., in reference [4], measure on a polypyrrole film on gold and for thicknesses of film between 7.5 and 20 nm, an index n = 1.7 for an extinction k = 0.3 at 633 nm.

Consequently, the polypyrrole used to fix the recognition molecules absorbs a large part of the fluorescence signal from the coupled target molecules. It therefore interferes with the detection mode.

In addition, the target molecules and their markers being included in the polypyrrole, in particular because the recognition molecules have been fixed by co-polymerization, this parasitic absorption raises the value of the lower limit of detection which can be reached by this process. .

In the field of diagnostics, for example, this drawback is very annoying, since it is precisely at low concentrations of target molecules, therefore at low surface concentrations of fluorescent markers, that the biochip presents all its interest.

SUMMARY OF THE INVENTION The aim of the present invention is in particular to provide a substrate coated with a transparent thin film and a method of manufacturing this substrate coated with the film which on the one hand responds to the technical problems posed in the prior art concerning biochips, and on the other hand provides a powerful new tool in the areas of quality control, certification and 1 authentication of any objects.

The substrate coated with a film of the present invention is characterized in that the film is an electrically insulating and transparent organic polymer in at least one range of wavelengths, and in that that said film is associated with a marker emitting at least in said range of wavelengths.

Indeed, the inventors have demonstrated that, unexpectedly, the character of an electrical conductor and the high optical extinction are linked.

Thus, according to the present invention, by "transparent" means transparent to the wavelength of detection of the marker used in accordance with the present invention. According to a first embodiment of the present invention, the wavelength range within which the insulating polymer must be transparent according to the present invention is determined according to the marker used for example in the aforementioned applications.

In particular, once the marker has been chosen, and therefore the emission wavelength of the marker, it is easy to determine the extinction value of such or such polymer at the desired wavelength, for example by ellipsometry spectroscopically or spectrophotometrically, and to examine whether it can be used according to the invention.

According to this first embodiment of the present invention, it is therefore the insulating polymer which is chosen as a function of the marker.

According to a second embodiment of the present invention, after having chosen an insulating polymer, the wavelength range in which it is transparent is determined, then according to this wavelength range, a marker emitting in said range of transparency of the polymer is selected.

According to this second embodiment of the present invention, it is therefore the marker which is chosen as a function of the polymer. According to the present invention, all the insulating polymers are therefore capable of being used as transparent films over at least one range of wavelengths.

Among these, there may be mentioned, by way of nonlimiting example, vinyl polymers, co-polymers and their mixtures, crosslinked or non-crosslinked, and in particular polymers, co-polymers and their mixtures, crosslinked or non-crosslinked, acrylonitrile, methacrylonitrile, methyl methacrylate, ethyl methacrylate, propyl methacrylate, butyl methacrylate, hydroxyethyl methacrylate, hydroxypropyl methacrylate, cyano acrylate, acrylic acid, l methacrylic acid, styrene, parachloro styrene, N-vinyl pyrrolidone, vinyl halides, acryloyl chloride, methacryloyl chloride.

Mention may also be made, by way of nonlimiting example, of the crosslinked or noncrosslinked polymers chosen from polyacrylamides, polymers of isoprene, ethylene, propylene, ethylene oxide and molecules with tight cycles, lactic acid or its oligomers, lactones, ε-caprolactone, glycolic acid, polyamides, polyurethanes, parylene and polymers based on substituted parylene, oligopeptides and proteins , as well as pre-polymers, macromers or telechelics based on these polymers, as well as the co-polymers and / or mixtures which can be formed from the monomers of these polymers or of these polymers themselves. The choice of the insulating polymer to be used for the application considered, for example from the aforementioned insulating polymers, can then be determined by considerations other than those strictly related to the optical properties of the material. Thus, according to the invention, the polymer can be selected, from the range of polymers which can be used according to the invention, for example from a polymer capable of adhering to a substrate, a polymer capable of being functionalized, a polymer having properties thermo-elastic, etc.

Among the aforementioned insulating polymers, the inventors have focused their attention more particularly on vinyl polymers, since they can easily be produced by different types of reactions, for example ionic or radical, in particular in thin films, and they can also be obtained electrochemically, and be electrografted on electrically conductive or semi-conductive surfaces. The vinyl polymers are obtained by polymerization of monomers of generic formula (I) below:

in which Ri, R ₂ , R ₃ and R ₄ are hydrogen atoms or organic groups of any kind, for example from hydrocarbons chosen for example from alkanes, alkenes, alkynes; for example amides, aldehydes, ketones, carboxylic acids, esters, acid halides, anhydrides, nitriles, amines, thiols, phosphates, ethers, homo- or hetero-cyclic aromatics or any cyclic group comprising these functions, as well as any group carrying several of these functions; or by polymerization of a mixture of different monomers corresponding to formula (I) above.

In the nonlimiting examples of embodiment of the present invention set out below, the optical properties of polymethacrylonitrile (PMAN) with R _x = R ₂ = H, R ₃ = CH ₃ and R ₄ = CN, and of polymethylmethacrylate (PMMA) with Ri = R ₂ = H, R ₃ = CH ₃ and R4 = C (= 0) 0CH ₃ were examined. These two monomers can lead to insulating polymers that are electrografted on conductive surfaces, by electroreduction in an aprotic organic medium. According to the invention, in a particular application, the vinyl polymers can be grafted, optionally selectively and localized, on the conductive or semiconductive surfaces of a substrate, by electro-grafting of vinyl monomers, which makes them interesting as substitutes for polypyrroles in a biochip application.

These polymers can be deposited on all types of surfaces according to the application of the present invention, by the various methods known to those skilled in the art for depositing thin films of polymers, such as coating techniques by centrifugation (“spin- coating ”); dipping; vaporization under ultra-vacuum; CVD deposits; chemical surface polymerization, as described, for example, in patents US4421569, US5043226 or even US5785791; photochemical grafting of polymers on surfaces, such as described for example in patent applications WO-A-9908717 and WO-A-9916907; grafting of polymers under irradiation of particles or photons; chemical grafting on an oxide or other polymer surface, either directly , or via chemical coupling agents (such as thiols, silanes, etc.); deposits resulting from polymerization caused by initiators, in particular radicals, obtained in situ by electrochemistry; ... etc.

According to a particular embodiment of the invention, it is possible for example to obtain a deposit of transparent polymer in a thin layer by polarizing a conductive or semiconductive surface in a solution or in a gel containing in particular diazonium salts and polymerizable monomers by way radical.

According to an advantageous embodiment, such polymer films are grafted onto the surface of the substrate, in particular in thin films, that is to say with thicknesses less than a micrometer, for example between 1 and 100 nm.

Other thicknesses are possible within the scope of the present invention as defined in the appended claims. They may be either films of preformed polymers grafted onto the surface, for example chemically, electrochemically or even photochemically, in one or more stages, or films constructed directly on the surface from precursor monomers, initiated for example by chemical, electrochemical or photochemical route.

This grafting can be carried out, according to the physicochemical characteristics of the process using the present invention, on insulating, conductive or semi-conductive surfaces of electricity.

According to a preferred embodiment, ultra-thin films of transparent polymers can be obtained on electrically conductive or semi-conductive surfaces by electro-grafting of vinyl monomers, for example as described in patent application EP- A- 038 244.

According to the present invention, the marker can be any marker provided that it can be associated with an electrically insulating organic polymer, in accordance with the present invention, said polymer having to be transparent in at least the range of emission wavelengths of the marker.

For example, the marker can be a fluorescent, phosphorescent or chemiluminescent marker.

It may for example be a marker chosen from fluororescein, substituted fluororesceins such as fluorescein diacetate, 5- and 6-carboxyf luoresceins, 5- and 6 -carboxyf luorescein diacetate, ester of succinimidyle of 5- and 6- carboxyf luoresceins ... etc. ; rhodamine and substituted rhodamines; coelenterazine and substituted coelenterazines; aequorin; luciferin and substituted luciferins; bromo chloro indoxyl phosphates (BCIP); luminol; Orange Nonylacridin (NAO); 5, 5 ', 6, 6' -tetrachloro-1, l ', 3, 3' -tetraethyl-benzimidazolyl carbocyanine chloride; iodide of ^' 4- (4- ditetradecylaminostyryl) -N-methylpyridinium, perchlorate of 1, 1' -dioctadecyl-3, 3, 3 ', 3' -tetramethyl indocarbocyanine, hydroxyethanesulf onate of 3.3'- dihexa decyloxa carbocyanine, bis- (1, 3-dibarbituric acid) -trimethine oxanol, tetrazolium salts, calcium, potassium complexes, anthraquinone, anthracene, pyrene, doxorubicin, phycoerythrin, porphyrins, phthalocyanines and more generally organometallic complexes, fluorescent proteins and in particular GFP, Green Fluorescent Proteins (KF Sullivan, SA Kay, “Methods in cell biology: Volume 58: Green fluorescent proteins”, Académie Press, 1999), salts of fluorescent minerals (and in particular uranium salts) as well as any molecule having a fluorophore group. A chemiluminescent marker is a marker which emits fluorescence when it is placed in the presence of another molecule. These markers are known to a person skilled in the art, it may for example be the biotin-avidin couple commonly used in molecular biology.

The quantity of markers can be very low taking into account the choice of the associated electrical insulating organic polymer. For example, the marker can be at a concentration of the order of nano- to μmolar, for example from 1 nmolar to 10 μM.

According to the invention, the term “associated marker” means a marker mixed with the film of insulating organic polymer, or fixed on the monomer (s), for example previously functionalized (s) used for the manufacture of the polymer film, or fixed to the surface of the film directly or indirectly, or trapped in the polymer film during the manufacture of the latter on the surface, or simply deposited on the film by means of a solution of the marker.

For example, in quality control, certification or authentication applications, the marker can be mixed with the insulating organic polymer, or inserted after deposition of the polymer by soaking in a solution of a swelling solvent for the polymer containing the marker, or inserted during synthesis. by carrying out the polymerization in the presence of the marker in the synthesis medium, or inserted during the synthesis by carrying out the polymerization with monomers or co-monomers chemically functionalized with the marker, or deposited on the polymer by means of a marker solution.

For example in applications relating to biochips, the marker can be associated by grafting directly onto the film, or indirectly onto recognition molecules grafted onto the film of insulating organic polymer.

Thus, the present invention makes it possible, for example, to solve the problems of the aforementioned prior art relating to biochips, by replacing the polypyrrole commonly used by an insulating polymer in accordance with the present invention, and for example by a vinyl polymer. Indeed, as demonstrated below in the embodiments of the present invention, these insulating polymers have extinctions 10 to 100 times weaker than conductive polymers such as polypyrrole, which considerably reduces their interference with a marker in the detection methods using a chip, for example a biochip.

Consequently, the present invention makes it possible to manufacture detection chips much more sensitive than those of the prior art, by lowering the lower detection limit for example compared to the chips using polypyrrole or another conductive polymer. These detection chips can be manufactured by any known means, except that the conductive films deposited on the substrates will have to be replaced by insulating films chosen in accordance with the present invention. The present invention also relates to the use of such organic thin films transparent.

In general, such a coating is capable of harboring a marker, for example fluorescent, phosphorescent or chemiluminescent chosen in the field of the electromagnetic spectrum corresponding to its zone of transparency, and of authorizing the detection of this very marker, and above all, when this marker is present at very low concentrations in or on the organic film, and this thanks to the optical transparency properties of the insulating organic polymer film chosen.

Thus, the present invention relates to the use of an electrically insulating and transparent organic polymer film in at least one range of wavelengths associated with a marker emitting at least in said range of wavelengths in a method detection of a chemical species.

The insulating organic polymer film of the present invention can indeed be functionalized with molecules for recognition of the chemical species such as nucleic acids, proteins, antigens, antibodies, synthetic organic molecules etc. For example, in a biochip detection method, the chemical species may be DNA.

The insulating organic polymer film of the present invention can also serve to encapsulate molecules, for example bioactive molecules such as doxorubicin, whose molecular structure is such that these molecules are fluorescent. In this application, the molecule has two properties of its own only: that linked to its bioactivity and that linked to its fluorescent character.

More generally, the present invention relates to the use of the electrically insulating and transparent organic polymer film of the present invention in at least one range of wavelengths associated with a marker emitting at least in said range of lengths d waves in a means of detecting a chemical species. As explained above, the detection means can be a biochip, such as a DNA chip, a protein chip, a chemical sensor, etc.

The present invention also relates to the use of the electrically insulating and transparent organic polymer film of the present invention in at least one range of wavelengths associated with a marker emitting in said range of wavelengths in a chosen process among a quality control process, a certification process or an object authentication process.

In the quality control of an industrial process for depositing thin insulating polymer films, for example, it is essential to be able to characterize the thickness of the coating. On thin films, in particular when these films have a thickness of less than 100 nm, it is necessary to use expensive and slow techniques such as profilometry or ellipsometry to obtain reliable thickness measurements. In addition, rapid measuring devices often have a lower detection limit greater than one micron. In addition, when the samples to be qualified are of complex shape (microbeads, grids, powders ... etc), the measurements, for example by profilometry or ellipsometry, are delicate. By combining, according to the present invention, a polymer film and a marker, for example fluorescent, phosphorescent or chemiluminescent, it is possible to very easily obtain an indirect measurement of the thickness of the film by measuring the intensity of fluorescence emitted.

This can be achieved, for example, by subjecting the object on which the polymer film associated with the marker according to the present invention has been deposited to irradiation by a light source whose spectrum contains at least the absorption wavelength of the fluorophore, and by measuring the intensity of the fluorescence emitted, over the wavelength of emission of the fluorophore.

For this, it suffices to carry out beforehand a calibration curve, or standard curve, on which the fluorescence intensity of a film associated with a fluorescent marker is brought as a function of the thickness of the film measured by profilometry or ellipsometry. for different flat samples covered with the polymer film according to the industrial deposition process in question, the marker being associated with the film at a chosen concentration and identical for all the samples.

From this standard curve, the measurement of the fluorescence intensity emitted by the same polymer film produced by the industrial process in question and comprising the chosen concentration and identical to the aforementioned samples as a marker, suffices to determine whether the film has the required thickness, that is to say whether it meets the specifications in terms of thickness, from the knowledge of the area of said object.

If this area is not known, the same protocol provides at least one means of online control of the reproducibility of the industrial method of depositing a polymer film.

The thickness of the films according to the present invention can therefore be checked in a very short time from a standard curve.

In this quality control application, the present invention exploits the active properties of the coating due to the intervention of a fluorophore.

The present invention can also be used in the certification or authentication of an object. For this, it suffices to deposit on said object a film of an organic electrically insulating and transparent polymer in at least one range of wavelengths, associated with a marker emitting at least in said range of wavelengths according to the present invention. Thus, by a simple fluorescence measurement, it is possible to determine if the object is an authentic object, that is to say comprising the film associated with the marker, or if it is a copy of said object.

Establishing a counterfeit is often tricky, since many processes differ as much on the part treated, as by the nature of the interface that has been built between the polymer film and the surface of the untreated part. This interface being buried under the film which has been deposited, it becomes difficult to analyze it through the film deposited, especially when its thickness is greater than 10 nm. In this case, a holder of an industrial property title relating to a process for depositing thin films can advantageously mark the films of its manufacture with a marker, for example fluorescent, phosphorescent or chemiluminescent, in accordance with the present invention, in concentration sufficiently weak that a measuring device such as that described above is necessary for its detection. The aforementioned applications are only to be considered as an illustration, and neither these applications nor the procedure by which the insulating polymers constituting the film are deposited on the surface of the substrates can not constitute a limitation to the application of the present invention.

Those skilled in the art will, in fact, be able to measure, on other applications, the importance of the present invention by associating one or more organic coatings with one or more polymers. electrical insulator (s) having low optical extinctions in a wavelength range and an emitting marker in said range.

Other advantages and characteristics of the present invention will appear to those skilled in the art on reading the examples below given by way of illustration and not limiting with reference to the attached figures.

Brief description of the figures

- Figure 1 is a diagram illustrating the principle of ellipsometry with a rotating polarizer used to measure the thickness of the insulating polymer films according to the present invention.

- Figures 2 and 3 are spectra of ellipsometric measurements of gold, as a substrate, for different angles of incidence in degrees: tan (Ψ) = f (λ (nm)) (fig.2) and cos (Δ) = f (λ (nm)) (fig. 3), (λ = wavelength in nm).

FIG. 4 is a graph grouping together measurements of index (I) and (N) and of optical extinction coefficient (E) and (K) carried out on a single gold substrate (Gold) and on a covered substrate of an organic film, measured at an angle of 75 ° for all wavelengths between 300 and 800 nm.

- Figures 5 and 6 are ellipsometric spectra of a platinum substrate without organic polymer film obtained from measurements carried out on a spectral range extending from 300 to 800 nm with a step of 5nm: tan (Ψ) = f (λ (nm)) with an incidence of 75 ° (fig. 5) and cos (Δ) = f (λ (nm)) with an incidence of 75 ° (fig. 6).

FIG. 7 is a graph grouping together measurements of index (I) and (N) and of optical extinction coefficient (E) and (K) carried out on a platinum substrate without organic film, measured at an angle of 75 ° for all wavelengths between 300 and 800 nm, as well as values given by the "Handbook of Optical Constants of Solids" edited by ED Palik.

- Figures 8a), 9a), 10a) and lia) represent ellipsometric spectra of a platinum substrate covered with a PMAN film obtained from measurements made between 55 and 75 ° with a step of 5 °, the spectral range extending from 300 to 800 nm with a step of 5 nm: tan (Ψ) = f (λ (nm)), respectively for the samples AuMAN7, AuMAN24, Au2401 and Au2301. - Figures 8b), 9b), 10b) and 11b) represent ellipsometric spectra of a platinum substrate covered with a PMAN film obtained from measurements made between 50 and 75 ° with a step of 5 °, the spectral range extending from 300 to 800 nm with a step of 5 nm: with an incidence of 75 ° (fig. 5) and cos (Δ) = f (λ (nm), respectively for the samples AuMAN7, AuMAN24, Au2401 and Au2301.

FIG. 12 is a graphical representation of the reflection measurements (in%) of the samples with a gold substrate coated with different electrically insulating and transparent organic polymer films according to the invention as a function of the wavelength (in nm) .

- Figures 13a) and 13b) are spectra of ellipsometric measurements made between 50 and 75 ° with a step of 5 °, the spectral range extending from 300 to 800 nm with a step of 5 nm, on a platinum substrate coated with a conductive organic polymer film based on diazonium salts (sample 0101Pt6), for different angles of incidence in degrees: tan (Ψ) multiangles = f (λ (nm)) (fig.l3a)) and cos (Δ) multiangles = f (λ (nm)) (fig.l3b)). - Figures 14a) and b) are spectra of ellipsometric measurements made between 50 and 75 ° with a step of 5 °, the spectral range extending from 300 to 800 nm with a step of 5 nm, on a platinum substrate coated with a conductive organic polymer film based on diazonium salts (sample 0101Ptl4), for different angles of incidence in degrees: tan (Ψ) multiangles = f (λ (nm)) (fig.l4a)) and cos (Δ) multiangles = f (λ (nm)) (fig.l4b)). FIG. 15 represents the results of spectrophotometric measurements intended to determine the reflection of a sample of platinum coated with an organic conductive film based on diazonium salts (samples OlOlPtβ and 0101Ptl4), on the range 400 to 800 nm with a reference wavelength of 560 nm.

FIG. 16 represents the results of spectrophotometric measurements intended to determine the losses of a sample of platinum coated with an organic conductive film based on diazonium salts (samples 0101Pt6 and 0101Ptl4), on the range 400 to 800 nm with a reference wavelength of 560 nm.

- Figure 17 is a dipolar modeling diagram of a surface fluorophore: it represents a dipole of dipole moment m placed above a surface x. Three environments are distinguished, the environment of the dipole, zone 1 of index i, here the superstrate; a stack of thin layers, zone 2 of index n ₂ ; and the substrate, zone 3 of index n ₃ . - Figures 18a) and 18b) represent modeling (signal (ua) = f (thickness of the polymer (in nm)) of the fluorescence signal of a fluorophore dipole placed on the surface of an organic film in two orientations, parallel (//) (figure 18a)) and perpendicular (_L) (figure 18b)) to the surface. In these figures, case 1: n = 1, 5 and k = 0.02; case 2: n = 1.5 and k = 0.003; case 3: n = 1.5 and k = 0.0 (extreme value); case 4 models a typical conductive organic film, like those obtained from diazonium salts: n = 1.5 and k = 0.4. In these figures, the abscissa axis represents "e" the thickness of the polymer film, and the ordinate axis "s" the signal.

- Figures 19a) and 19b) are schematic representations of gold substrates coated on the one hand with a polypyrrole film (Figure 19a)) in accordance with the prior art, and on the other hand with a polymer film organic insulator (ManAull) (Figure 19b)) according to the present invention.

- Figure 20a) is a snapshot (objective x50 - exposure time: 200 ms) of the fluorescence of a drop

(0.5 μl of a 1 μM solution) of fluorophore Cydctp (trademark) from the company Amersham deposited on a gold substrate, on the gold zone.

- Figure 20b) is a snapshot (objective x50 - exposure time: 200 ms) of the fluorescence of a drop

(0.5 μl of a 1 μM solution) of fluorophore Cydctp (trademark) from the company Amersham deposited on a polypyrrole film deposited on a gold substrate, on the zone corresponding to the polypyrrole. - Figure 20c) is a snapshot (objective x50 - exposure time: 200 ms) of the fluorescence of a drop (0.5 μl of a 1 μM solution) of Cydctp fluorophore

(trademark) of the company Amersham deposited on a film of polymethacrylonitrile (MAN) deposited on a gold substrate (AuManll sample), on the zone corresponding to the deposition of MAN.

EXAMPLES

EXAMPLE 1 Techniques for Measuring and Modeling the Optical Properties of the Sample

The extinction measurements are carried out by spectroscopic ellipsometry. It is a non-destructive optical technique which makes it possible to characterize, by determination of the index and the thickness, deposits of thin materials by exploiting the modifications which these subject to the polarization of the light.

When a beam is reflected on the surface of a sample, its polarization state is changed. Indeed, in oblique incidence, the electric field of a light wave breaks down in two proper directions, one of which is perpendicular to the plane of incidence (S wave) and the other parallel to this plane.

(P). These two waves interact differently with the surface of the sample and show the reflection coefficients, in amplitude, r _s and r _p depending on whether they are S or P waves.

The change in polarization state, which results from the difference in amplitude and phase behavior of the S and P waves, can then be characterized by p according to the following equation eql: p = L = tan (Ψ) e ^m eql ^r s tan (ψ) represents an amplitude ratio and Δ a phase difference between the polarizations S and P.

The ellipsometer used is the GESP5 (trademark) of the company SOPRA. It makes it possible to measure the parameters tan (ψ) and cos (Δ) as a function of the wavelength, hence the term "spectroscopic ellipsometry", and / or of the angle of incidence θ of the light beam from analysis. The operating principle of the rotating polarizer ellipsometer used is shown in Figure 1.

In this figure, S represents a light source, s and p the polarization vectors of the incident light, P and A of the rotating polarizers, and D a detector.

By means of a modeling of the sample, the characteristics of the latter can be calculated using regression algorithms on the measurements given by the ellipsometer. To characterize the samples, thickness e, index n and extinction k, we proceeded by regression of the ellipsometric measurements using a smoothing software by nonlinear regression in the complex plane with a non-dispersive law, then with a model. of Lorentz oscillators.

The consistency of the results was checked with spectrophotometric measurements.

Example 2: Samples examined The measurements were carried out on eight different samples, the characteristics of which are summarized in Table 1.

Three distinct series of samples were analyzed: - a series of PMAN films electro-grafts on gold, of thickness varying between 9 and 150 nm. These films are obtained by electro-reduction of a 2.5 M solution of methacrylonitrile in anhydrous acetonitrile on a gold electrode, in the presence of 5 × 10 ⁻² M of tetraethylammonium perchlorate (TEAP) as the support electrolyte. The electro-reduction takes place in voltametric conditions between -0.3 and -2.6 V / (Ag ⁺ / Ag) at 100 mV / s, in non-separated compartments, with a large surface platinum counter electrode. The different thicknesses are obtained by varying the number of voltammetric scans;

- two films of PMMA electro-grafts on gold, one 100 nm thick, the other thick (thickness> 0.5 μm). These films are obtained by electro-reduction of a methyl methacrylate solution, under the same conditions as for PMAN films;

- two films obtained by electro-reduction of a 10 ^" 3M solution of para-nitrophenyl diazonium tetrafluoroborate (PNPD) in anhydrous acetonitrile on a platinum electrode, in the presence of 5 × 10 ^-2 M TEAP as a support electrolyte. Potential sweeps are applied from + 0.3 V / (Ag ⁺ / Ag) to -2.9 V / (Ag ⁺ / Ag), at -200 mV / s. Two films, of respective thicknesses 3 and 30 nm are obtained by this protocol. This last series of samples was chosen because the organic films obtained by electro-grafting of PNPD are conductors of electricity, unlike the films of the first two series. In addition, the ellipsometric measurements will also be compared with those obtained on a polypyrrole film as a conductive polymer.

The thicknesses of the different films reported in Table I below are measured by profilometry. In this table are also reported the arithmetic roughness, measured by profilometry, according to two peak paths: 500 μm and 2 mm.

Table I: References and characteristics of the samples used in the exemplary embodiments

In this table, polymethacrylonitrile or polymethylmethacrylate films are insulating, and nitrobenzoic films are conductors.

ELLIPSOMETRIC MEASUREMENTS OF SUBSTRATES. Characteristics of the gold layer (substrate). The ellipsometric spectra of gold are measured for different angles of incidence. This way of proceeding makes it possible to judge the quality of the final result according to the results obtained for these different values, between 50 ° and 75 ° with a step of 5 °. The measurement spectra range from 300 nm to 800 with a measurement step of 5 nm. They are shown in Figures 2 and 3 attached.

The measurement of the Gold was carried out on the MANAull sample, on the part not soaked in the reaction mixture and on which no organic film was deposited.

We are looking for the index and extinction values of this layer of gold, its thickness greater than one micron makes it possible to assimilate it, "ellipsometrically" to a substrate. We then proceed to an inversion, equation resolution, of the data to derive the n and the k from the gold. This operation is carried out for each measurement angle. The indices measured at an angle of incidence of 75 ° for all the wavelengths between 300 and 800 nm are shown in the appended FIG. 4.

For comparison, the measurements made on a gold substrate alone were also reported, independently of the samples carrying a grafted organic film, which makes it possible to control the stability of the measurements. Characteristics of the platinum layer (substrate).

The ellipsometric spectra of Platinum are measured under the same conditions as for Gold, measurement angle between 50 ° and 75 ° with a step of 5 ° and the spectral range extends from 300 to 800 nm with a step of

5 nm.

The Platinum measurement was carried out on the OlOlPtδ sample, on the part not soaked in the reaction mixture and on which no organic film was deposited.

Figures 5 ((tanΨ) = f (λ (nm)), incidence 75 °) and 6 ((CosΔ) = f (λ (nm)), incidence 75 °) are graphical representations of the results obtained from these measures.

We are looking for the index and extinction values of this Platinum layer, its thickness greater than a micron allows it to be assimilated, from an ellipsometric point of view, to a substrate. We then invert the measurement data at 75 ° to derive the n (index) and k (extinction coefficient). FIG. 7 groups together the index (I) and extinction (E) results thus obtained. In this graph, values given by the “Handbook of Optical Constants of Solids” edited by E.D. Palik have also been represented.

For the characterizations of the samples, we will take as reference values of the Platinum the results given by the ellipsometric measurement. ELLIPSOMETRIC MEASUREMENTS OF SAMPLES.

The measurements are carried out on the spectral range 300-800 nm and at θ = 60 °.

Ellipsometric results of samples on gold substrate.

In order to be more precise in terms of results, the AuMAN7, MANAu24, Au2301 and Au2401 samples were measured for variable angles.

The measurements carried out made it possible to construct the spectra represented in FIGS. 8a) and b) for AuMAN7; in Figures 9a) and b) for AuMAN24; in Figures 10a) and 10b) for Au2401; and lia) and b) for Au2301. Figures a) correspond to tan (Ψ) multi-angle measurements, and Figures b) to cos (Δ) multi-angle measurements.

Spectrophotometric results of samples on gold substrate.

The samples were measured using a manual "Lambda9m" spectrophotometer (trademark) to determine the Reflection, the

Transmission and Losses of each of them over the wavelength range: 400-800 nm with the reference wavelength: 560 nm. We note that all the samples on Gold have the same spectra and that at 560 nm: 72.5% <R <84.2%;

T≈O and 157% <P <27.5%. These results are collated in Table 2 below: Table 2: Reflection (R), transmission (T) and losses (P) at 560nm for the samples on gold

FIG. 12 appended is a graphic representation of the reflection measurements (in%) carried out on these samples with a gold substrate coated with different electrically insulating and transparent organic polymer films according to the invention as a function of the wavelength (in nm).

We note that all the samples on gold have the same spectra and that at 560 nm: 72.5% <R <84.2%; T≈O and 15.7% <P <27.5%. In particular, it can be seen that these insulating films have little loss over a wide range of wavelengths. This observation will become even clearer when compared to the results obtained with the conductive PNPD films. Ellipsometric results of samples on platinum substrate.

FIGS. 13a) and 13b) represent the spectra of the ellipsometric measurements carried out between 50 and 75 ° with a step of 5 °, the spectral range extending from 300 to 800 nm with a step of 5 nm, on the samples 0101Pt6, for different angles of incidence in degrees: tan (Ψ) multiangles = f (λ (nm)) (fig.l3a)) and Cos (Δ) multiangles = f (λ (nm)) (fig.l3b)). FIGS. 14a) and b) represent the spectra of the ellipsometric measurements carried out between 50 and 75 ° with a step of 5 °, the spectral range extending from 300 to 800 nm with a step of 5 nm, on the samples 0101Ptl4, for different angles of incidence in degrees: tan (Ψ) multiangles = f (λ (nm)) (fig.l4a)) and Cos (Δ) multiangles = f (λ (nm)) (fig.l4b)).

Spectrophotometric results of samples on platinum substrate. The samples were measured with a manual "Lambda9m" spectrophotometer (trademark) in order to determine the Reflection, Transmission and Losses of each of them over the wavelength range: 400-800 nm with the length d 'reference wave: 560 nm.

We note that the samples on platinum have very different spectra. Reflections, transmission and losses are summarized in Table 3 below. It is observed that the losses in PNPD films, conductive polymers have a much greater weight than in the case of insulating films made on gold substrates.

Table 3: Reflection (R), transmission (T) and losses (P) at 560nm for samples on stage

FIG. 15 represents the reflection and FIG. 16 represents the losses of the platinum samples coated with a conductive polymer 0101Pt6 and 0101Ptl4, over the range 400 to 800 nm with a reference wavelength of 560 nm.

CHARACTERIZATION OF SAMPLES.

To characterize the samples (e, n and k), we proceeded by regression of the ellipsometric measurements using the SporX software (trademark) with a non-dispersive law, then with Lorentz oscillators. Consistent results were obtained on the one hand between the two models, and on the other hand with spectrophotometric measurements.

The results of these characterizations are summarized in Table 4.

Very low extinction coefficients are measured for vinyl insulating films (k <0.02), whereas conductive PNPD films give extinctions at least 10 times higher. By way of comparison, the polypyrrole films with a thickness close to that of the samples measured (20 nm) have an extinction coefficient of 0.3 to 0.5, measured under the same conditions.

Table 4: Optical characteristics of the samples

We are now trying to characterize the signal which would be emitted by a fluorophore adsorbed on the surface of the film.

We first present the expected benefit due to the low extinctions, then we show, on a simple test, that the fluorescence rendering is actually better on PMAN films on gold than on a polypyrrole film of the same thickness.

The fluorophore is modeled as being an electric dipole, as shown in FIG. 17, that is to say the smallest possible source in the context of electromagnetic theory. This approach does not harm the general application of the question, since we can calculate the Green's function of the problem and recompose any more complex electromagnetic source as superposition of basic dipoles. It is further assumed that the electromagnetic fields will not be quantified. The comparison of theoretical models to model fluorescence and luminescence in laser cavities, showed almost similar results whether the point of view is classical or quantum.

On the basis of the results of Table 4, the following cases are simulated: case 1: n = 1.5 / k = 0.02; case 2: n = 1.5 / k = 0.003; case 3: n = 1.5 / k = 0, (extreme value); the last case models a typical conductive organic film, such as for example the films obtained from diazonium salts (samples 0101Pt6 and 0101Ptl4): case 4: n = 1.5 / k = 0.4.

The fluorophore used is phycoerithrin, absorption = 543 nm and emission = 580 nm, its altitude relative to the surface is 10 nm. The fluorophore is bathed in a liquid environment.

Two different orientations are taken into account for the dipoles: one parallel and the other perpendicular to the surface. The thickness of the polymer is varied and the signal emitted in the ambient environment of the fluorophore is calculated (case of a very open fluorescence analysis microscope).

Figure 17 is a dipolar modeling diagram of a surface fluorophore: it represents a dipole of dipole moment m placed above a surface x. Three environments are distinguished, the environment of the dipole, zone 1, here the superstrate; a stack of thin layer (s), zone 2; and the substrate, zone 3. nι, 8ι; n ₂ , ε ₂ and n ₃ , ε ₃ are respectively the index and the dielectric permittivity of zones 1, 2 and 3; d represents the distance from the dipole to the surface of the polymer and t represents the thickness of the polymer.

Figures 18a) and 18b) represent modeling (signal (ua) = f (polymer thickness (in nm)) of the fluorescence signal of a fluorophore dipole placed on the surface of an organic film in two orientations, parallel (figure 18a)) and perpendicular (figure 18b)) to the surface. In these figures, case 1: n = 1.5 and k ≈ 0.02; case 2: n = 1.5 and k = 0.003; case 3: n = 1.5 and k = 0.0 (extreme value); case 4 models a typical conductive organic film, such as for example the films obtained from diazonium salts (samples 0101Pt6 and 0101Ptl4): n = 1.5 and k = 0.4.

In view of the results shown in Figures 18a) and 18b) attached, there is a gain of about 25 on the signal with the present invention using a film "MAN" with a parallel orientation of fluorophores, the most likely case because of the photo-selection phenomenon, and about 100 for a perpendicular orientation.

The predictions of this model are now tested in a simple way by examining the fluorescence signal of a drop of fluorescent marker on a PMAN film (AuMANll sample) deposited on a gold substrate and comparing it to the same signal on a polypyrrole film of the same thickness deposited on a gold substrate. The fluorophore used is Cy ₃ dctp (trademark) (Amersham) at 1 μM. The volume of the drops is 0.5 μl.

Figures 19a) and 19b) are schematic representations of gold substrates coated on the one hand with a polypyrrole film (figure 19a)), and on the other hand with an insulating organic polymer film (ManAull) (figure 19b)) in accordance with the present invention.

Figures 20a), b) and c) are the pictures (objective x50 - exposure time: 200ms) of the fluorescence on the gold zone (figure 20a)), on the polypyrrole film (figure 20b)) and on the polymethacrylonitrile (MAN) film obtained in this example.

The bright spots on the three photographs are either agglomerates of fluorophores or fluorescent dust. These points do not correspond to fluorophores close to the surface, that is to say a few nm, which is the case for biochips for example, but to balls of a few tens to a few hundred nm. It is therefore not on these luminous points that the comparison must be made, but on the "continuous background" which surrounds these points.

It is observed that with identical exposure times, photographic receptors of the same sensitivity, the same magnifications carried out with the same optics, and even when the marker concentration is very low (lμm), the fluorophores which are in "direct" contact "With the surface are not" turned off "in the case of the AuMANll samples (photo c)), while they are on the gold zones (photo a)) and on the area of the conductive organic coating (photo b)). Here, therefore, the superior transparency properties of the insulating PMAN films on the conductive films are directly observed here, as illustrated in these figures 20.

EXAMPLE 3 Obtaining a Deposition of Polymethacrylonitrile Film by Electro-initiation in the Presence of Diazonium Salts

Is carried out, on the same gold surfaces as those of Example No. 1, a deposition of polymethacrylonitrile films by electro-initiation from diazonium salts. These films are obtained by electro-reduction of a 2.5 M solution of methacrylonitrile in anhydrous acetonitrile in the presence of 5 × 10 ⁻² M of tetraethylammonium perchlorate (TEAP) as support electrolyte and of 10 ⁻³ M of 4-nitrophenyl diazonium tetrafluoroborate. Electro-reduction takes place in voltammetric conditions between +0.3 and -1.5 V / (Ag ⁺ / Ag) at 100 mV / s, in non-separated compartments, with a large surface platinum counter electrode. The number of scans is adjusted so as to obtain films with a thickness of the order of 100 nm.

The optical measurements reveal a coefficient k <0.02, as observed with the polymethacrylonitrile films obtained by direct electro-grafting (see Table 4). Example 4: Reproducibility control procedure

Polymethacrylonitrile films of variable thickness truffled with phycoerithrin are made on identical gold slides. For this, the gold plates are polarized in voltammetric conditions between +0.3 and -1.0 V / (Ag ⁺ / Ag) at 100 mV / s, in non-separated compartments, with a platinum counter electrode of large area, in a solution containing 2.5 M methacrylonitrile in anhydrous acetonitrile, 5 × 10 ⁻² M of tetraethylammonium perchlorate

(TEAP) as support electrolyte, and 10 ^" 3M of 4-nitrophenyl diazonium tetrafluoroborate and 1 μM of phycoerithrin. The thickness of the films obtained, measured by ellipsometry, is 50 ± 5 nm, ie an accuracy of 10% .

Each slide is then irradiated at 543 nm, and the resulting fluorescence intensity is measured at 580 nm. Identical fluorescence intensities are measured on all the slides, with a dispersion of the measurements less than 15%.

Example 5: Quality control procedure

PMAN films are deposited

(polymethacrylonitrile) of increasing thickness according to the procedure of Example 4 on gold slides of 1 cm ² .

The different thicknesses, between 5 and 150 nm are obtained by varying the number of voltammetric scans.

The fluorescence measurement of the coatings obtained makes it possible to draw a calibration curve giving the thickness as a function of the intensity / cm ² . A 84 nm PMAN deposition is then carried out (measurement by ellipsometry) on a gold surface of 5 cm ² . The fluorescence measurement indicates, from the calibration curve, a thickness of 75 nm, which makes it possible to validate a correct control without the aid of a direct measurement of the thickness.

REFERENCE LIST

[1] Arwin et al., Synthetic Metals 6 1983: "Dielectric Function of Thin Polypyrrole and Prussian Blue Films by Spectroscopie Ellipsometry.

[2] Kim et al., Journal of the Electrochemical Society, 138 (11), 1991: "Real Time Spectroscopie Ellipsometry: In Situ Characterization of Pyrrole Electropolymerization" [3] Kim et al., Bulletin of the Korean Chemical Society, 17 (8), 1996: "Polypyrrole Film Studied by Three-Parameter Ellipsometry".

[4] Guedon et al., Analytical Chemistry, 22, 6003-6009, 2000: "Characterization and Optimization of a Real-Time, Parallel, Label-Free, Polypyrrole-Based DNA Sensor by Surface Plasmon Resonance Imaging".

Claims

1. Substrate coated with a film characterized in that the film is an organic electrically insulating and transparent polymer in at least one range of wavelengths, and in that said film is associated with a marker emitting at least in said range wavelengths.

2. A substrate coated with a film according to claim 1, in which the film comprises an insulating polymer chosen from vinyl polymers.

3. A substrate coated with a film according to claim 1, in which the film comprises an insulating polymer chosen from crosslinked or non-crosslinked polymers of acrylonitrile, methacrylonitrile, methyl methacrylate, ethyl methacrylate, propyl methacrylate. , butyl methacrylate, hydroxyethyl methacrylate, hydroxypropyl methacrylate, cyano acrylate, acrylic acid, methacrylic acid, styrene, parachloro styrene, N-vinyl pyrrolidone, and halides vinyl.

4. A substrate coated with a film according to claim 1, in which the film comprises a crosslinked or non-crosslinked insulating polymer chosen from polyacrylamides, polymers of isoprene, ethylene, propylene, oxide of ethylene and tight cycle molecules, lactic acid or its oligomers, lactones, ε-caprolactone, glycolic acid, aspartic acid, polyamides, polyurethanes, parylene and polymers based on substituted parylene, oligopeptides and proteins, as well as pre-polymers, macromers or telechelics based on these polymers, as well as the co-polymers and / or mixtures which can be formed from these polymers.

5. A substrate coated with a film according to claim 1, in which the film is a vinyl polymer obtained by polymerization of a monomer of formula (I) below:

(I) in which R _x , R ₂ , R ₃ and R ₄ are independently hydrogen atoms or organic groups such as the hydrocarbons chosen from alkanes, alkenes, alkynes; amides, aldehydes, ketones, carboxylic acids, esters, acid halides, anhydrides, nitriles, amines, thiols, phosphates, ethers, homo- or hetero-cyclic aromatics or any cyclic group comprising these functions, as well as any group carrying several of these functions, or of a mixture of different monomers corresponding to said formula (I).

6. A film-coated substrate according to claim 1, wherein the film is polymethacrylonitrile or polymethylmethacrylate.

7. The substrate coated with a film according to claim 1, in which the marker is chosen from a fluorescent marker, a phosphorescent marker or a chemiluminescent marker.

8. Use of an electrically insulating and transparent organic polymer film in at least one wavelength range associated with a marker emitting at least in said wavelength range in a method for detecting a chemical species.

9. Use according to claim 8, in which the insulating organic polymer film is functionalized with molecules for recognition of the chemical species.

10. Use according to claim 8, wherein the chemical species is DNA.

11. Use of an electrically insulating and transparent organic polymer film in at least one range of wavelengths associated with a marker emitting at least in said range of wavelengths in a means for detecting a chemical species.

12. Use according to claim 11, in which the detection means is a biochip.

13. Use of an electrically insulating and transparent organic polymer film in at least one wavelength range associated with a marker emitting in said wavelength range in a process chosen from a quality control process, a process certification or an object authentication process.

14. Use according to any one of claims 8 to 13, in which the film is a vinyl polymer obtained by polymerization of a monomer of formula (I) below:

(I) in which Ri, R ₂ , R ₃ and R ₄ are independently hydrogen atoms or organic groups such as the hydrocarbons chosen from alkanes, alkenes, alkynes; amides, aldehydes, ketones, carboxylic acids, esters, nitriles, amines, thiols, phosphates, ethers, homo- or hetero-cyclic aromatics or any cyclic group comprising these functions, as well as any group carrying several of these functions, or of a mixture of different monomers corresponding to said formula (I).

15. Use according to claim 14, in which the film is polymethacrylonitrile or polymethylmethacrylate.

16. Use according to claim 14, in which the marker is chosen from a fluorescent marker, a phosphorescent marker or a chemiluminescent marker.