FR3039279A1 - DEVICE FOR MANUFACTURING A FUNCTIONALIZED SUBSTRATE FOR DETECTING AT LEAST ONE PARAMETER OF A GIVEN ENVIRONMENT - Google Patents

Abstract

The invention relates to a device (1) for manufacturing a functionalized substrate for the detection of at least one parameter of an environment comprising a chamber divided into at least two compartments, said first and second compartments, which are distinct but contiguous. and separated from one another by a membrane (7), the first compartment (3) comprising a diamond substrate to be functionalized and the second compartment (5) comprising at least one molecule for functionalizing said substrate and for detecting said parameter of the environment.

Description

DEVICE FOR MANUFACTURING A FUNCTIONALIZED SUBSTRATE FOR DETECTING AT LEAST ONE PARAMETER OF A GIVEN ENVIRONMENT

DESCRIPTION

TECHNICAL AREA

The present invention relates to a device for the production of a functionalized substrate for the detection of at least one parameter of a given environment, which parameter of the environment may be, for example, chemical or biological species to be detected. in a given environment.

This device therefore finds application in the field of the detection of at least one environmental parameter or, more specifically, of the qualitative or quantitative analysis of such a parameter and in a wide variety of sectors, such as: -the sector medical, in particular, for the analysis of biological fluids, such as blood, urine in order to establish a diagnosis; -the agri-food sector, in particular, for the analysis and quality control of food products marketed on a large scale; -the water sector, in particular, for the analysis and control of water intended for domestic use; -the sector of research and development in chemistry, biochemistry or pharmacology. At the present time, the detection and analysis of such parameters are carried out conventionally in the laboratory by qualified personnel, who implements standardized procedures with, however, disadvantages: - long analysis periods justified by the need to move to the relevant laboratory; and - a fortiori, a lack of responsiveness.

As a variant, the user who needs to detect a parameter of an environment can also directly design the functionalized substrate intended to enable this detection, provided that he has, however, the necessary experience or know-how, because the steps of functionalization of a substrate can be complex, especially when it is a question of modifying the physicochemical characteristics of a base substrate (for example, its polarity, its hydrophilic or hydrophobic character) with a view to improving the affinity of the substrate for the target species, which one wishes to detect. In this case, experience and know-how are also justified when it comes to carrying out the functionalization step very shortly before the experiment, especially when the functionalized substrate is particularly sensitive, this may be the case especially when a substrate is functionalized with complex biological molecules (such as antibodies, oligonucleotides, DNA, RNA) dedicated to specific recognition for applications in the field, by example, clinical diagnosis.

Also, in view of the drawbacks mentioned above, the authors of the present invention set themselves the objective of developing a device for manufacturing a functionalized substrate for the detection of a parameter of an environment, which can suitable for direct on-site use (ie without the need to move to a specific analytical laboratory, for example, in the factory or outside) and when the need for detection is required, this device which can be used, for example, by a non-specialist in the field of detection.

SUMMARY OF THE INVENTION

Thus, the present invention relates to a device for manufacturing a functionalized substrate for detecting at least one parameter of an environment comprising a chamber divided into at least two compartments, said first and second compartments, which are distinct but contiguous and separated from one another by a membrane, the first compartment comprising a diamond substrate to be functionalized and the second compartment comprising at least one molecule for functionalizing said substrate and for detecting said parameter of the environment.

Also, having such a device available, a user wishing to proceed to the detection of one or more given parameters of a given environment can, by simple opening of the membrane, proceed to bringing into contact the two aforementioned compartments, this bringing into contact concretizing by a reaction of the substrate to functionalize with the functionalization molecule, the product resulting from this reaction being thus the functionalized substrate ready to be used for the detection of the parameter or parameters.

It is a device for implementing a functionalization of a diamond substrate in a simple, inexpensive, fast and suitable for use by the general public and also allowing functionalization by both simple molecules and more complex molecules (such as biological molecules, such as antibodies, immunoglobulins and integrins).

In other words, this device can make it possible to make a substrate functional in a few minutes simply by bringing it into contact with the contents of the second compartment. This functionalization can be carried out on site without any particular restriction concerning the operating parameters (for example, the temperature).

Before going into more detail in the description of this invention, the following definitions are specified.

By molecule of functionalization and detection of the parameter of the environment, is meant, conventionally, in what precedes and what follows, a molecule comprising both, at least one group capable of functionalizing said diamond substrate and at least one group capable of allowing the detection of the parameter of the environment. This molecule can also be described as a probe molecule, in that it is able to capture the presence of the parameter or parameters of the environment, which it is desired to detect.

By group capable of enabling the detection of the parameter of the environment, is meant a group capable of being modified in the presence of said parameter of the environment, this modification possibly being when the parameter of the environment is a chemical compound or a species biological, in a chemical reaction between this group and one of the groups of the chemical compound or the biological species to be detected, this chemical reaction may be, for example, a formation reaction of a covalent bond.

Functionalization is understood to mean a chemical grafting, preferably covalently, of the aforementioned functionalization molecule to the surface of the diamond substrate. In other words, it is an immobilization of the aforementioned molecule (s) on the surface of the diamond substrate by means of a covalent chemical bond, thanks to the presence on this or these molecules of at least a group capable of grafting chemically to the surface of the substrate, which implies, of course, that said substrate has, on its surface, groups capable of reacting with the aforementioned molecule (s) to form the covalent bond.

By parameter of an environment, it is meant a generally chemical entity forming part of the environment that it is desired to detect, this environment possibly being ambient air, a chemical or biological fluid (for example, a blood sample, a urine sample) and this entity can be a chemical compound, a biological species, such as an enzyme, an antibody.

As mentioned above, the first compartment comprises a diamond substrate to be functionalized. The use of a diamond substrate has many advantages: by nature, the diamond has a stable surface state, which makes it particularly suitable for storage in a device intended to be used later, all the more more if the first compartment is under a controlled atmosphere (for example, an inert atmosphere such as an atmosphere consisting of nitrogen); since it consists of carbon, some of which may be hydrogenated, the diamond lends itself to a broad spectrum of functionalization; and the intrinsic stability of the diamond makes it possible, after functionalization by the functionalization molecule or molecules, to preserve the functionalized substrate in the long term (provided, of course, that the functionalization molecule is stable in the long term).

The diamond substrate to be functionalized can take various forms, such as spherical particles (for example, whose particle diameter is nanometric, micrometric or millimetric), faceted particles or platelets. It may also be deposited on another substrate, for example in the form of thin layers, in particular to form a chip.

The diamond substrate may also be monocrystalline or polycrystalline (in which case the crystal grains may be microcrystalline or nanocrystalline grains).

The diamond substrate may also be a porous substrate.

Finally, the diamond substrate, especially when it is in the form of a film, may comprise, on its surface, patterns, and more specifically patterns forming reliefs on the surface of the substrate, these patterns being able, for example, , be obtained by lithography techniques, etching techniques, contact printing techniques (the latter being known in the English terminology "contact printing").

As specified above, the diamond substrate, present in the first compartment, is intended to be functionalized by contact with the molecule or molecules present in the second compartment, the functionalization occurring by reaction between said molecule (s) and the substrate surface, which implies that the substrate and the one or more molecules must each comprise groups capable of reacting together.

More specifically, when the functionalization consists of a covalent chemical grafting, it implies that the substrate of the first compartment comprises groups capable, during a chemical reaction, of reacting with a group of the aforementioned molecule (s) to form a link covalently, such a reaction can be any chemical reactions contributing to the formation of such a binding, such as coupling reactions (for example, coupling reactions between an amine group and a carboxylic group and its derivatives), reactions of substitution (for example, nucleophilic substitution reactions), addition reactions (for example, nucleophilic addition reactions).

More specifically, the diamond substrate may comprise, as reactive group, a group selected from the following groups: groups belonging to the category of leaving groups, which may be involved in nucleophilic substitution reactions, such groups for example, halogen atoms (such as iodine, bromine), a nonaflate group of formula -SO3- (CF2) 3-CF3, a triflate group of formula CF3-SO3-, a tosylate group of formula CH3-Phenyl-SO3- (with the groups CH3 and SO3 being in the para position), a mesylate group of formula CH3-SO3-, a fluorosulfonate group of formula F-SO3-, an oxonium group (such as a group -OH +, -ORH + with R being, for example, an alkyl group), an optionally substituted diazonium group or an ammonium group (such as tetraalkylammonium groups); hydrocarbon groups (such as the groups CH, CH 2, CH 3), in which case the hydrogenated diamond can be described, such groups being capable of reacting with a functionalization molecule comprising, for example, a group leaving the category from those listed above; groups capable of participating in condensation reactions (such as esterification reactions, amidation reactions), such groups possibly being, for example: ester groups optionally activated by a leaving group, this is that is, the ester group is converted into a group of the formula -CO-R ', R' being a leaving group, this group possibly being, for example, a group derived from the coupling between the ester group and dicyclohexyl diimide (DCC ), these ester groups being capable of reacting in amidation reactions with amine groups, which would be present in the functionalization molecule; carboxylic acid groups, these groups being capable of reacting in esterification reactions with hydroxyl groups, which would be present in this case in the functionalization molecule; acyl halide groups, such as acyl chlorides, these groups being capable of reacting in esterification reactions with hydroxyl groups, which would be present in this case in the functionalization molecule; acid anhydride groups, these groups being capable of reacting in esterification reactions with hydroxyl groups, which would be present in this case in the functionalization molecule; hydroxyl groups, these groups being capable of reacting in esterification reactions with carboxylic acid groups, acyl halides and acid anhydrides, which would be present in this case in the functionalization molecule; groups capable of participating in nucleophilic addition reactions (such as hydrazone formation reactions), such groups being able to be chosen from: ketone and aldehyde groups, these groups being capable of reacting with nucleophilic groups , which would be present in the functionalization molecule, such nucleophilic groups may be, for example, hydrazine groups, amine groups; nucleophilic groups, such as, for example, hydrazine groups, amine groups, capable of reacting with ketone or aldehyde groups, which would be present in the functionalization molecule.

In order to generate appropriate groups capable of reacting with the selected functionalization molecule (s), the diamond substrate, before its introduction, in the first compartment, can be subjected to a specific treatment to introduce groups such as those mentioned above, such a treatment that can be a surface treatment, such as those listed below: a plasma treatment, such as a plasma torch treatment, a reactive ion etching treatment (also known as RIE treatment terminology for "Reactive-ion etching"), a microwave induced plasma treatment (also known as MIP treatment for "Microwave induced plasma"), associated with a suitable gas (such as H2, O2, NH3, Cl2, F2, Ar, N2) with, optionally, a control of the atmosphere or under vacuum; an anodizing treatment; and / or an oxidizing chemical treatment or a chemical reducing treatment, according to the groups that one wishes to create on the diamond substrate.

Finally, the diamond substrate may further comprise on its surface, one or more specific zones able to be functionalized by said one or more functionalization molecules, while other zones remain non-functionalizable by said molecule or molecules, which allows, in the end, to obtain a functionalized substrate at these specific areas. A substrate of patterned substrate (which corresponds to the Anglo-Saxon terminology "patterned substrate") can be qualified. By way of example, a substrate of this type may be a substrate comprising hydrogenated local zones (ie zones comprising -CH, -CH2- or -CH3 groups) distributed at an oxidized zone (or vice versa). the hydrogenated local zones constituting the functionalizable zones, for example, by biological molecules, such as peptides, in order to constitute, after functionalization, biochips.

As for the second compartment, it comprises, as mentioned above, at least one molecule of functionalization of the substrate and detection of the parameter of the environment, this functionalization molecule may belong to the category of organic compounds. This molecule comprises, conventionally, at least one group capable of reacting with the substrate to form a covalent bond by any type of chemical reactions capable of generating this type of bond. Also, by way of example, the functionalization molecule may comprise, as reactive group, a group chosen from the following groups: groups belonging to the category of leaving groups, which may intervene in nucleophilic substitution reactions, such groups may be, for example, halogen atoms (such as iodine, bromine), a nonaflate group of formula -SO3- (CF2) 3-CF3, a triflate group of formula CF3-SO3-, a tosylate group of formula CH3-Phenyl-SO3- (with the groups CH3 and SO3 being in the para position), a mesylate group of formula CH3-SO3-, a fluorosulfonate group of formula F-SO3-, an oxonium group (such as a group -OH 2 +, -ORH + with R being, for example, an alkyl group), an optionally substituted diazonium group or an ammonium group (such as tetraalkylammonium groups), these compounds being capable of reacting with a hydrogenated diamond substrate, that is, a substrate nt, on its surface, CH, CH2, CH3 groups; hydrocarbon groups (such as CH, CH 2, CH 3 groups), such groups being capable of reacting with a substrate comprising, on its surface, a group starting from the category of those listed above; groups capable of participating in condensation reactions (such as esterification reactions, amidation reactions), such groups possibly being, for example: ester groups optionally activated by a leaving group, this is that is, the ester group is converted into a group of the formula -CO-R ', R' being a leaving group, this group possibly being, for example, a group derived from the coupling between the ester group and dicyclohexyl diimide (DCC ), these ester groups being capable of reacting in amidation reactions with amine groups, which would be present in this case on the diamond substrate; carboxylic acid groups, these groups being capable of reacting in esterification reactions with hydroxyl groups, which would be present in this case on the diamond substrate; acyl halide groups, such as acyl chlorides, these groups being capable of reacting in esterification reactions with hydroxyl groups, which would be present in this case on the diamond substrate; anhydride groups of acid, these groups being capable of reacting in esterification reactions with hydroxyl groups, which would be present in this case on the diamond substrate; hydroxyl groups, these groups being capable of reacting in esterification reactions with carboxylic acid groups, acyl halides and acid anhydrides, which would be present in this case on the diamond substrate; groups capable of participating in nucleophilic addition reactions (such as hydrazone formation reactions), such groups possibly being chosen from: ketone and aldehyde groups, these groups being capable of reacting with nucleophilic groups , which would be present on the diamond substrate, such nucleophilic groups may be, for example, hydrazine groups, amine groups; nucleophilic groups, such as, for example, hydrazine groups, amine groups, capable of reacting with ketone or aldehyde groups, which would be present on the diamond substrate; groups capable of participating in silanization reactions of the substrate, such groups possibly being, for example, alkoxysilane groups.

Depending on the groups it comprises and its origin, the functionalization molecule may belong to the category of biomolecules or the like, such as amino acids, oligopeptides, proteins (for example, antibodies, integrins, immunoglobulins). ), oligonucleotides, DNAs, RNAs, nucleic acid nucleic acids (known by the abbreviation PNA) whereby the substrate thus functionalized by functionalization molecules can thus constitute a device of the biochip type for immunobiological analyzes, a device of the biochip protein or DNA type.

In the device of the invention, at least one of the compartments advantageously comprises a liquid medium, in addition to the substrate to be functionalized in the first compartment and the functionalization molecule or molecules in the second compartment.

This liquid medium may comprise water and / or an organic solvent, especially in the first compartment and in the second compartment, which may contribute to the stability of the substrate or of the functionalization molecule or molecules.

In addition, the second compartment may comprise other adjuvants necessary for the functionalization reaction, for example, catalysts, bases, acids or adjuvants, such as buffer solutions, a physiological medium, a culture medium.

The liquid medium and any adjuvants may be included in a third compartment, which is contiguous with the first compartment and / or the second compartment and separated therefrom by a membrane, which is intended to be opened to allow contacting. liquid medium and any adjuvants with the appropriate compartment when using the device.

Alternatively, at least one of the first and / or second compartments may comprise an inert atmosphere, such as an inert gas atmosphere, such as nitrogen, argon, which atmosphere may contribute to the preservation of the atmosphere. integrity of the contents of the first and / or second compartments for several years.

As specified above, the first compartment and the second compartment are contiguous with each other but separated by a membrane, so as to physically separate the contents of these two compartments. This membrane may be, for example, a membrane made of a polymeric material.

This membrane can be either removable, that is to say it can be removed completely, when it is desired to bring the contents of the two compartments into contact or can be destroyed, in part, for example, by drilling, so as to allow the contact of the contents of the two compartments. From a practical point of view, when the membrane is removed in whole or in part, the functionalization molecule (s) is (are) brought into contact with the diamond substrate, the resulting mixture can be maintained under an inert atmosphere or in air under function of the functionalization that one wishes to obtain. The resulting mixture may also be subjected to continuous or discontinuous agitation. After a suitable contact time (for example, from 5 to 30 minutes), the substrate is separated from the mixture and optionally rinsed with water or an organic solvent identical to that used in the first compartment and / or the second compartment. The substrate may be subjected to a drying step, if the functionalization molecule attached to the substrate allows it.

The thus functionalized substrate can be directly used for the application for which it was designed. Other features and advantages of the invention will emerge from the additional description which follows and which relates to particular embodiments.

Of course, this additional description is only given as an illustration of the invention and does not in any way constitute a limitation

BRIEF DESCRIPTIOND OF THE FIGURES

FIG. 1 illustrates a device according to the invention and described in example 1.

DETAILED DESCRIPTION OF PARTICULAR EMBODIMENTS EXAMPLE 1

This example illustrates the preparation of a device according to the invention and its use, this device comprising: a first compartment comprising a diamond substrate; and a second compartment comprising a functionalization molecule, which is avidin, which is capable of capturing the presence of biotin, the device (referenced 1) being represented in FIG. 1 in the appendix, in which: the reference 3 means the first compartment; reference 5 denotes the second compartment; and the reference 7 designates the membrane separating the first compartment from the second compartment.

More specifically, the first compartment comprises a solution comprising a nanocrystalline hydrogenated diamond film.

The second compartment comprises, more specifically, a solution comprising a phosphate buffer and 0.1 mg of avidin.

The membrane is opened to allow the contents of the first compartment and the second compartment to mix for a period of 15 minutes.

Next, the nanocrystalline diamond film is separated and rinsed by simply soaking in phosphate buffered saline (known as PBS). Then the film thus obtained is kept for 6 months in air. At the end of these 6 months, the film was subjected to a test to verify its functionalization with avidin, the protocol of this test being set out below.

For this test, a solution of spherical particles of hydrogenated diamond (the particles having an average diameter of 30 nm) at a rate of 2 mg / ml is mixed with a solution comprising 1 mM biotin and a carbonate buffer at pH = 10, the volume ratio between the solution of particles and the solution comprising biotin being 1/10.

The mixture is stirred and left in contact for 15 minutes. The particles are then rinsed three times with ultrapure water to remove excess biotin.

Then, the thus rinsed particles are resuspended in ultrapure water and the resulting solution is brought into contact with the functionalized nanocrystalline diamond film for 20 minutes. The film is then dipped in phosphate buffered saline and then in water. It is then dried and analyzed by scanning microscopy, which allows to observe that the diamond particles functionalized with biotin are only detectable in the areas of the diamond film functionalized with avidin.

In summary, from this example 1, follow the following conclusions: the effectiveness of the device of the invention for functionalizing a diamond film in a very short time (20 minutes maximum); -the potential of diamond conservation over time; the reactivity of hydrogenated diamond substrates whatever their forms (nanoparticles or films); and the sterility of the diamond with respect to the grafted molecules, in that the molecules retain their original properties after grafting. EXAMPLE 2

This example illustrates the preparation of a device according to the invention and its use, this device comprising: a first compartment comprising a diamond substrate; and a second compartment comprising a functionalization molecule, which is an anti Escherichia coli IgG antibody.

More specifically, the first compartment comprises a solution comprising spherical hydrogenated diamond particles having an average diameter of a few micrometers at a rate of 5 mg / ml in water.

The second compartment comprises, more specifically, a solution comprising the anti Escherichia coli IgG antibody at a rate of 0.1 mg / ml in a phosphate buffer of pH = 8.

The membrane is opened to allow the contents of the first compartment and the second compartment to mix, the ratio of the volume of the contents of the first compartment to that of the second compartment being 1 to 10, the antibody thus being in excess.

Then the particles are rinsed.

The particles were then tested to verify the effectiveness of their functionalization by the aforementioned antibody.

To do this, after rinsing, they are resuspended in ultrapure water and the resulting solution is brought into contact with 200 μl of a solution of Escherichia coli comprising 10,000 bacteria / ml, the volume ratio between the particle solution and the Escherichia Coli solution being 50/50.

After stirring for 15 minutes, the resulting mixture is left standing for 2 minutes. The supernatant is removed and the precipitate is resuspended in 100 μl of ultrapure water and then deposited on a substrate and dried for scanning electron microscopy.

This analysis clearly shows the presence of diamond particles on the surface of bacteria, which testifies the good recognition and the assembly of particles with bacteria in a simple and fast way.

In parallel, at comparative value, a test was carried out with diamond particles functionalized with a hexyl chain - (CH 2) 5 -CH 3, which were put in contact with a solution of Escherichia coli. After analysis by scanning electron microscopy, it appears the absence of diamond particles on the surface of the bacteria.

Claims (11)

1. Device (1) for manufacturing a functionalized substrate for detecting at least one parameter of an environment comprising a chamber divided into at least two compartments, said first and second compartments, which are distinct but contiguous and separate one from the other. on the other by a membrane (7), the first compartment (3) comprising a diamond substrate to be functionalized and the second compartment (5) comprising at least one molecule for functionalizing said substrate and for detecting said parameter of the environment. 2. Device according to claim 1, wherein the functionalization molecule is a molecule comprising both, at least one group capable of functionalizing said diamond substrate and at least one group capable of enabling the detection of the environmental parameter. 3. Device according to claim 1 or 2, wherein the functionalization consists of a covalent chemical grafting of the functionalization molecule on the surface of the diamond substrate. The device of claim 2 or 3, wherein the diamond substrate comprises groups capable, during a chemical reaction, of reacting with a group of the functionalization molecule (s) to form a covalent bond. A device according to any one of the preceding claims, wherein the diamond substrate comprises, as a reactive group, a group selected from the following groups: ## STR4 ## groups belonging to the category of leaving groups selected from atoms of halogen, a nonaflate group of formula -SO3- (CF2) 3-CF3, a triflate group, a tosylate group, a mesylate group of formula CH3-SO3-, a fluorosulfonate group of formula F-SO3-, an oxonium group, a group diazonium, an optionally substituted ammonium group; hydrocarbon groups, such as CH, CH 2, CH 3 groups; groups capable of participating in condensation reactions chosen from: ester groups optionally activated by a leaving group; carboxylic acid groups; acyl halide groups; acid anhydride groups; hydroxyl groups; groups capable of participating in nucleophilic addition reactions chosen from: ketone and aldehyde groups; nucleophilic groups, such as hydrazine groups, amine groups. 6. Device according to any one of the preceding claims, wherein the functionalization molecule comprises, as group capable of functionalizing the substrate, a group selected from the following groups: groups belonging to the category of leaving groups chosen from atoms halogen, a nonaflate group of formula -SO3- (CF2) 3-CF3, a triflate group, a tosylate group, a mesylate group, a fluorosulfonate group of formula F-SO3-, an oxonium group, a diazonium group, a group optionally substituted ammonium; hydrocarbon groups, such as CH, CH 2, CH 3 groups; groups capable of participating in condensation reactions chosen from: ester groups optionally activated by a leaving group; carboxylic acid groups; acyl halide groups; acid anhydride groups; hydroxyl groups; groups capable of participating in nucleophilic addition reactions chosen from ketone and aldehyde groups; nucleophilic groups; groups capable of participating in substrate silanization reactions chosen from alkoxysilane groups. The device according to any one of the preceding claims, wherein the functionalization molecule belongs to the class of amino acids, oligopeptides, proteins, oligonucleotides, DNAs, RNAs or nucleic acid peptides. 8. Device according to any one of the preceding claims, wherein at least one of the compartments comprises a liquid medium, in addition to the substrate to be functionalized in the first compartment (3) and the functionalization molecule or molecules in the second compartment. compartment (5). 9. Device according to claim 8, wherein the liquid medium comprises water and / or an organic solvent. 10. Device according to any one of the preceding claims, further comprising a third compartment contiguous with the first compartment (3) and / or the second compartment (5) and separated therefrom by a membrane, this third compartment comprising a liquid medium as defined in claims 8 or 9. 11. Device according to any one of the preceding claims, wherein at least one of the first and / or second compartments (3, 5) comprises an inert atmosphere.