PRINTING AN ADHESIVE PATTERN ON A ROT-PROOF BACKING

Description

TECHNICAL FIELD

[0001] The present invention relates to the field of grafting a protein onto a substrate, according to an optically defined pattern. BACKGROUND

[0002] The publication of international application number WO2013 / 135844 (hereinafter "STUDER" or "the publication") discloses a device for microstructured protein grafting onto a substrate or for photochemical printing. In the publication, an aqueous solution of a benzophenone (BP) and a protein is illuminated in specific areas according to a pattern above a substrate, and a durable transfer of the protein to the illuminated areas is achieved, thus creating the print. However, the process described in the publication performs a protein transfer onto the substrate in the presence of BP and simultaneously with the illumination. This device involves combining, at the same location, an illumination device that projects an image of a pattern onto the substrate and a microfluidic device that delivers an aqueous solution containing both a protein and a BP.This results in a problem of clutter in the printing system as well as a risk of damaging the protein by the combined action of benzophenone and the light from the lighting device.

[0003] Other examples of photochemical processes for grafting proteins onto a substrate are described in patent document WO 2006 / 084482 and in the scientific publication "An electrogenerated poly(pyrrole-benzophenone) film for the photografting of proteins" by Cosnier et al.

[0004] Ideally, it would be useful to print a pattern that is simply adhesive for the protein, without any protein adhering to it, onto a substrate using the lighting system. A real pattern would then be revealed on the substrate upon contact with an aqueous solution of a protein, for example, a fluorescent one. The protein would preferentially bind to the illuminated parts of the adhesive pattern to form the real pattern. However, such a solution, for a given protein, depends on the availability of a process capable of producing a latent or subsequently revealed adhesive pattern that is printed onto a substrate coated with an antifouling layer for proteins. An antifouling layer for proteins is defined as a layer made of a material that prevents protein adhesion to that layer within the timescale of the intended printing process.

[0005] Such a substrate, coated with its antifouling layer, or antifouling substrate, can consist of a rigid support, such as optical-grade glass transparent to the light from the lighting system, or a soft support, such as PDMS. The glass or PDMS is coated with a polymer brush material, or a polymer that adheres to the support via chains of molecules, such as PEG and polyNipam. In antifouling substrates of this type, the polymer chains are attached to the support at one end and free at the other, like the bristles of a brush.

[0006] Other techniques such as photolithography applied to an antifouling substrate for proteins through a mask using laser ablation of patterns of antifouling materials on an antifouling substrate, allow in the prior art to obtain antifouling supports with patterns allowing the subsequent selective grafting of a protein onto the substrate, according to the illuminated areas of the substrate from which the antifouling material or polymer brush has been removed by light energy.

[0007] It is estimated that material ablation is caused by substrate illumination and that the resulting level differences allow for a recessed image of the subsequent actual image. When these differences are observed using phase-contrast optical spectroscopy, which is sensitive only to the optical path, the adhesive pattern can be attributed either to antifouling material ablation or to a change in the material's nature, altering its refractive index and providing subsequent preferential adhesion of proteins to the illuminated polymer chain regions. Other techniques that allow observation of the latent image (notably atomic force microscopy, ellipsometry, X-ray analysis, etc.) can, in some cases, demonstrate that the latent image is due to complete ablation of the PEG layer.Such ablation techniques therefore do not allow for the creation of concentration gradients, as the ablation of the PEG or antifouling layer is a priori total.

[0008] Ultimately, it would be desirable to have a process for producing a polymer-based dirt-repellent or brush-resistant substrate with adhesion that varies proportionally or continuously with the brush's exposure to illumination, according to a pattern, without molecules necessarily adhering to the brush simultaneously with illumination. Rather, it would be preferable for these molecules to adhere to the brush at a later time. GENERAL PRESENTATION

[0009] The following definitions apply to this application: "Adhesive pattern" means a surface pattern in which certain molecules, including proteins (and especially antibodies), nanobeads, DNA (deoxyribonucleic acid) or RNA strands, or bacteria, are stably distributed over time on a substrate coated with an anti-adhesive, antifouling, or brush-resistant polymer layer, outside of said adhesive pattern. Since the pattern is defined outside of an anti-adhesive or antifouling zone or set of zones, an adhesive pattern can also be defined on a substrate as a set of zones or patterns that are more adhesive to the molecules of interest than the complementary surface area of ​​all the zones on the substrate.A difference in adhesion effect, necessary for the existence of a pattern, can be predicted for a polymer brush, without contact with an aqueous solution of a molecule, by at least two techniques available in the prior art: . Atomic force microscopy reveals a reduction in the length of the polymer chains in the brush, such a reduction causing a decrease in the anti-adhesive effect or an increase in the adhesion effect in those areas. Phase-contrast microscopy reveals a variation in the optical path length through the brush in the more adhesive areas, such a variation being associated with a change in the adhesion effect.

[0010] "Polymer brush": refers to a nanometric layer (i.e., one whose thickness is on the nanometric scale, typically between 1 nm and 100 nm) that is antifouling, particularly for proteins, nanobeads, DNA strands, and bacteria. Such a nanometric layer is present on the surface of a substrate to form an antifouling surface. It is estimated, as of the date of this application, that such a brush consists of a set of polymer chains grafted onto the surface of a substrate. This set extends over a thickness zone of between 1 nm and 20 nm on the substrate surface for PEG and between 1 nm and 30 nm for polyNIPAM. It is estimated that between 1nm and 20nm, such a brush possesses anti-adhesion or anti-fouling properties, particularly for proteins, nanobeads, DNA strands or bacteria.A layer of polyethylene glycol or "PEG" or a layer of poly(N-isopropylacrylamide) or polyNIPAM are examples of polymer brushes.

[0011] "Thickness" for a polymer brush refers to the distance from the substrate to the free ends of the polymer chains that make up the brush. For example, with PEG, the thickness of the coating is controlled by the length of the PEG chains, that is, the number of ethylene glycol monomers composing these chains. These chains can be angled relative to the substrate, compressed, or modified in any way similar to the action on the bristles of a brush to create a texture or variation in thickness on the free surface of the brush.

[0012] In this context, the invention relates to a method for printing an adhesive pattern on a brush of a polymer extending onto the surface of a support in a nanometric anti-fouling layer, as defined in claim 1.

[0013] In variations of the process: The layer thickness is between 1 nm and 20 nm. The wavelength is chosen between 300 nm and 400 nm. The polymer is polyethylene glycol (PEG). The polymer is polyNIPAM. The substrate is glass. The substrate is polydimethylsiloxane (PDMS). The surface energy of the illumination imparted to the PEG layer is between 10 mJ / mm² and 1000 mJ / mm². The surface energy of the illumination imparted to the polyNIPAM layer is between 100 mJ / mm² and 10000 mJ / mm². The Young's modulus of the PDMS substrate is less than 15 kPa.

[0014] The invention also relates to a method as above, for imprinting a pattern of a protein onto the polymer brush, comprising the following additional steps: Rinse to remove contact between the layer and the first solution. Then, place the layer in contact with a second aqueous solution containing the protein. The invention also relates to a method as above for imprinting a pattern of nanobeads onto the polymer brush, comprising the following additional steps: rinse to remove contact between the layer and the first solution; place the layer in contact with a second solution containing the nanobeads.

[0015] The invention also relates to a method as above, for printing a pattern of DNA strands onto the polymer brush, comprising the following additional steps: rinse to remove contact between the layer and the first solution; place the layer in contact with a second solution containing the DNA strands.

[0016] The invention also relates to an application of the process for printing an adhesive pattern, to the creation of an adhesive pattern exhibiting an adhesion gradient to the surface of the support, by spatial variation of the surface energy. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The invention will be better understood in connection with the list of figures below, in which: There figure 1 The cross-sectional diagram shows an antifouling substrate composed of a glass support and a layer of antifouling material, specifically designed for proteins, nanobeads, or DNA strands—that is, a polymer brush grafted or attached to the support. The substrate is coated with a drop of an aqueous solution containing benzophenone, covering all or part of the polymer brush. A region AB of the benzophenone-coated layer is illuminated through the support (through the droplet would also be feasible) by radiation with wavelengths in the absorption spectrum of benzophenone, i.e., radiation between 300 nm and 400 nm. figure 2 represents the rinsed substrate of the droplet figure 1 and exhibiting a latent image, visually represented by a depression in the surface of the antifouling material at zone AB. Such a material is suitable for imprinting, in particular, a pattern of a protein, nanobeads, or strands of DNA or RNA according to a pattern corresponding to the surface of zone AB. Indeed, illumination of the polymer brush in the presence of benzophenone makes the polymer brush potentially adhesive in zone AB and allows the subsequent adhesion of, in particular, a protein, nanobeads, or DNA strands, by subsequently bringing the polymer brush layer into contact with, respectively, a solution of the protein, a solution of nanobeads, or a solution of DNA strands. Illumination across a set of zones such as AB thus allows the creation of an adhesive pattern or an adhesive pattern of molecules on a polymer brush, for molecules to which the brush is normally non-adhesive. DETAILED DESCRIPTION OF EXAMPLE(S)

[0018] In a first embodiment, it is disclosed with reference to the figure 1 for the reference numbers in parentheses, an antifouling substrate composed of a glass support (1) and a layer (2) of a polymer brush material which is, in this first mode, PEG or polyNIPAM.

[0019] In this first mode, a beam (3) illuminates the layer (2) along a region AB (AB), here through a support (1) chosen to be transparent to the beam used. A drop (4) of an aqueous benzophenone solution is deposited on the layer (2), covering the region AB (AB). Equivalently, it would be possible to illuminate the layer through the drop (4), along the same region AB.

[0020] The radiation used includes at least one wavelength within the absorption spectrum of benzophenone, a spectrum which, usefully in practice, extends between 300 nm and 400 nm. Preferably, radiation with a wavelength shorter than 390 nm will be used within this range; in this case, the exposure time of the layer to the radiation will be minimized.

[0021] The less absorption of benzophenone at the chosen wavelength, the greater the power of the light source must be, or the longer the exposure time of the illuminated area must be, the radiation dose received, equal to the product of the light power by the exposure time to light, being the parameter governing the achievement of the effect of the invention.

[0022] Since no protein to be grafted is in solution, the radiation will, if necessary, be of a higher power than that which would destroy a protein to be grafted later, and will be limited only by the surface energy density that the layer can absorb without degradation. However, the presence of benzophenone allows PEG to be treated with optical powers 10 to 100 times lower than those required for ablation or masking techniques.

[0023] An energy density between 10 mJ / mm² and 1000 mJ / mm² can be used to create an adhesive pattern on PEG. The invention can therefore use a source producing 2 mW of illumination on a 400-micron square at a wavelength of 372 nm ultraviolet from a semiconductor laser. For polyNipam on a PDMS substrate, a usable energy density is between 100 mJ / mm² and 10,000 mJ / mm². The same semiconductor laser source can also be used by simply multiplying the exposure times for PEG by 10.

[0024] In a first step of the process of this embodiment, the antifouling substrate is brought into contact with a drop of aqueous benzophenone solution, and then in a second step an AB area of ​​the antifouling layer of the substrate is illuminated with the ultraviolet light source.

[0025] Any optical system that allows the energy from the source to be focused onto area AB or onto a set of areas simultaneously is usable, and such systems are known from the prior art. A microscope with a micromirror array is thus a viable option for implementing the illumination system in this embodiment. Similarly, the droplet can be replaced by a film of aqueous benzophenone solution brought into contact with the layer and then rinsed after illumination using known microfluidic methods.

[0026] There figure 2 This represents the polymer brush formed in a nanometric layer, rinsed of the benzophenone solution droplet and visually endowed with a latent pattern, also nanometric in depth, in zone AB. This latent pattern, in order to be revealed, requires subsequent contact with molecules or molecular assemblies capable of adhering to the substrate at this point in the polymer brush (proteins, nanobeads, DNA strands, bacteria, etc.). The adhesion of these molecules according to the latent pattern then occurs, in the presence of these molecules in aqueous solution, at the level of the areas of the layer that were illuminated (here AB) with benzophenone. The adhesion of molecules occurs without the input of light energy. The molecules are simply adsorbed onto the polymer brush at the level of the latent or adhesive pattern. A real pattern of molecules is thus formed on the brush.In particular, if the molecules are fluorescent, it is possible to then create an image of them using techniques known from the prior art to prove the result of the adhesion.

[0027] However, even without contact with an aqueous solution, such as a protein solution, it is possible to predict, after illumination of the brush, that the effect of the invention will be achieved, regardless of the subsequent creation of an actual pattern. This can be done by measuring, after illumination, whether there are nanometer-deep pits in the brush at the illuminated locations using an atomic force microscope (AFM), or by observing whether there are optical path length variations in the brush at these same locations using phase-contrast microscopy. It is thus possible to select, without further experimentation, polymer brushes suitable for the process of the invention, in particular those for which a reduction in the length of the polymer chains of the brush is observed after illumination in the presence of benzophenone.

[0028] In a second embodiment of the invention, the device of the figure 2is brought into contact with an aqueous solution of a protein or an aqueous solution of nanobeads. The choice of protein or nanobead type is made from among those likely to adhere to the substrate in order to obtain the most durable, realistic image possible.

[0029] Thus, with this second method, it is possible to obtain a real image of the AB region, for example, using a fluorescent protein, but more generally, a pattern of a protein on the antifouling substrate for the protein that was used. Furthermore, the illumination properties of the antifouling substrates allow for fluorescence with a value that varies continuously with the illumination or the dose of optical radiation received by the AB region, and more generally, a concentration of proteins, nanobeads, or DNA strands that varies continuously with the illumination in this region, even if this region corresponds to the resolution limit of the optical illumination system, without resorting to binary dot densities to simulate varying protein concentrations.

[0030] It is thus possible to apply the invention to the creation of adhesion gradients in a direction of concentration for example of a protein, nano-beads or DNA strands, along the surface of the substrate or the antifouling layer, by aligning end to end several AB type zones, and by varying the surface energy delivered to these zones, for example by illuminating them with variable surface areas (in J / m2), during the step of illuminating the polymer brush in the presence of benzophenone or of printing the latent image or adhesive pattern.

[0031] For example, a continuously variable adhesive effect for proteins was obtained by variable dose illumination in the presence of benzophenone on a PEG brush, for a thickness reduction of between 0nm (no adhesion or off-pattern area) and 2nm (maximum adhesion) for PEG polymer brushes with an estimated thickness of 5nm outside the adhesion areas.

[0032] In the embodiments presented, a range of benzophenone concentration in millimoles per liter of aqueous solution (mmol / l) from 5mmol / l to 50mmol / l was used.

[0033] The invention is likely to have industrial application in the field of substrate production for printing adhesive patterns of a protein onto a polymer brush.

Claims

1. A process for printing an adhesive pattern on a polymer brush extending at the surface of a support (1) and forming a nanometric anti-fouling layer (2), the process comprising the following steps: - placing the layer (2) in contact with a first aqueous solution (4) containing a benzophenone, - then illuminating the layer with a radiation (3) at a wavelength within the absorption spectrum of the benzophenone, according to the pattern and according to a surface energy, and rinsing to eliminate the contact between the layer (2) and the first aqueous solution (4), thereby creating an adhesive pattern which is a zone on the polymer brush that is more adhesive for molecules of interest than the supplementary surface of the zone on the polymer brush, the adhesive pattern being a hollow pattern in the polymer brush and not being covered by the molecules of interest.

2. The process of claim 1, wherein the thickness of the layer (2) is between 1 nm and 20 nm.

3. The process of claim 1 or 2, wherein the wavelength is chosen between 300 nm and 400 nm.

4. The process of any one of claims 1 to 3, wherein said polymer is a polyethylene glycol (PEG).

5. The process of any one of claims 1 to 3, wherein said polymer is a poly(N-isopropylacrylamide).

6. The process of any one of claims 1 to 5, wherein said support (1) is made of glass.

7. The process of any one of claims 1 to 5, wherein said support (1) is made of PolyDiMethylSiloxane (PDMS).

8. The process of any one of claims 1 to 7, wherein an adhesive pattern having an adhesion gradient at the surface of the support (1) is produced by spatial variation of the surface energy of the illumination.

9. The process of any one of claims 1 to 8, wherein the length of the polymer chains of the polymer brush is reduced at the location of the adhesive pattern.

10. The process of any one of claims 1 to 9, for printing a pattern of molecules of interest on the polymer brush, comprising the following additional step: - placing the layer (2) in contact with a second aqueous solution containing the molecules of interest.

11. The process of claim 10, for printing a pattern of molecules of interest on the polymer brush, wherein the molecules of interest are proteins, nanoshells, DNA strands, RNA strands or bacteria.

12. The process as claimed in claim 10 or 11, wherein, when the layer (2) is brought into contact with the molecules of interest, the molecules of interest are adsorbed on the layer (2) at the adhesive pattern, the adsorption of the molecules of interest taking place without provision of light energy.