ITPI20100017A1 - METHOD FOR THE PRODUCTION OF MINIATURIZED BIOSENSORS, AND BIOSENSORS OBTAINED WITH THIS METHOD - Google Patents

Description

â € œMETHOD FOR THE PRODUCTION OF MINIATURIZED BIOSENSORS, AND BIOSENSORS OBTAINED WITH THIS METHODâ €,

DESCRIPTION

Scope of the invention

The present invention refers to a method for producing miniaturized biosensors, and to the biosensors obtained by means of this method.

These biosensors find their main application in the biological and biomedical fields, in particular in the production of:

ï £ § biochips capable of detecting the presence of certain analytes in biological samples;

- supports for the sequential synthesis of oligopeptides or polypeptides;

ï £ § devices for protein targeting studies.

Notes on the known technique - Technical problem

Schematically, biosensors include:

ï £ § a support suitable to be brought near a sample;

ï £ § a layer of a biological component adhered to a surface of the support; this biological component is able to interact with any analytes in the sample by binding to it / them in a stable manner, in particular the biological component can be formed by biomolecules such as antibodies or proteins, but also microorganisms or cells;

ï £ § a transducer that converts the interaction between the biological component and the analyte into a detectable physical signal; in particular electrochemical, electro-optical, acoustic and mechanical transducers are known.

Currently, supports made of inorganic materials, such as gold, silicon, glass, or polymeric supports, such as polystyrene, silicones, PTFE are used for the production of biosensors.

Some known production methods involve the use of supports having hydrophobic surface characteristics, and the application on such supports of biomolecules such as proteins, in a purely physical adsorption process in which the affinity of portions of the biomolecules is exploited to such surfaces.

However, the interactions between biomolecules and support are not very stable and non-selective: in the adsorption process, and during the use of the biosensor, a competition is created between desired and unwanted species, and the biomolecules that are intended to be adsorbed on the support. they can be removed by other species, or by surfactant substances.

In particular, proteins can undergo inactivation and / or denaturation in the adsorption process. There is in fact a very close relationship between the behavior of proteins and their structure. Proteins, used as biological interaction material in biosensors, can best express their affinity for analytes if they are left free to assume the conformations that they naturally compete with, in particular, if they have the possibility of organizing themselves in the tertiary and quaternary supramolecular structures .

Other methods of manufacturing miniaturized sensors for diagnostics involve the use of supports consisting of semipermeable membranes or sol-gel porous matrices, in which the biomolecules are trapped; the diagnostic devices thus obtained can, however, interact only with analytes whose molecular dimensions and chemical characteristics allow permeation into the pores of the membrane or porous matrix.

In still other methods, the biomolecules are immobilized on the support by means of ionic or covalent bonds. To this end, the surface of the support must undergo a laborious preliminary functionalization or activation treatment, i.e. functional groups must be created at the surface level, which are able to interact with the biomolecules, forming with them such ionic or covalent bonds.

In particular, in the case of inorganic supports, the surface is generally activated by preparing functionalized self-assembling monolayers. For example, W00125780 reports a method for manufacturing sensors for environmental use by depositing layers of polymeric particles on a support connected to two electrodes or on an acoustic sensor. The particles can be made up of non-reactive or reactive polymers thanks to the presence of carboxylic, hydroxyl, amino groups, enzymes, proteins, dyes, and more.

In the case of polymeric supports, the activation of the surface of the support can be obtained by functionalization, using treatments with plasma, corona discharge or ultraviolet light. For example, in WO2007104107 a method is reported for activating by plasma treatment inert polymeric materials, so as to make it possible to immobilize biomolecules on the surface. The polymeric material must consist of a hydrophilic surface layer and an underlying cross-linked region.

However, these processes involve not negligible costs and production times, with production costs of the biosensors not always acceptable. Furthermore, the immobilization of the biomolecules is carried out in such a way that the biomolecules, in particular proteins, assume different configurations from those they would have in the free state, due to the fact that they are chemically bound to the surface of the substrate and, at the same time, to also undergo interactions of a physical type with the surface itself, of an electrostatic type, due to the presence of polar groups in the molecules of the support, and due to the presence of hydrophilic and hydrophobic portions of such molecules. The biosensors obtained, therefore, are not able to always express satisfactory performances.

WO2007044669 describes a method for producing compositions for tissue engineering applications, release of active ingredients, diagnostic applications and more, comprising a biocompatible matrix or implant and one or more functional groups suitable for complexing one or more bioactive molecules fixed to the matrix or to the plant, in which the concentration of the functional group of the matrix can be controlled independently of the mechanical strength of the matrix. The method provides for fixing to said matrix or implant one or more of these functional groups suitable for complexing therapeutic, prophylactic or bioactive molecules, in which the functional groups comprise one or more reactive functional groups.

WO0034781 refers to a low cost method and system for detecting analytes present in a sample, such as bacteria, yeast, fungi, antibodies, antigens, allergens, enzymes, hormones, polysaccharides, proteins, and others, by means of elements capable of amplify the optical diffraction. The system includes a biosensitive device on which specific receptors for the analytes are physically connected by microcontact printing, according to a predetermined distribution. The supports can comprise polymeric materials, generally free of reactive functional groups. These materials, for example polystyrene microparticles, are bonded to the analyte before contact with the sensor. This procedure may involve the risk of compromising the biological functionality of the system.

WO9207006 reports a method for coating a solid surface with a nonionic hydrophilic polymer layer, covalently bonded to a biopolymer, to immobilize biomolecules; this method involves binding a cationic polymer to the hydrophilic chains and therefore the biomolecule to the hydrophilic chains before depositing the polymeric layer on the solid surface, which must necessarily expose negative charges on the surface. The method involves considerable limitations in the choice of the type of substrate and of the reaction medium; in particular it requires to produce a specific coating for each biomolecule to be used.

The application US20050158850 describes biosensors, and a method for their production, comprising a metal layer coated with a hydrophobic polymer on whose surface a binder is deposited on which biomolecules can be subsequently immobilized, according to a predetermined distribution. In particular, the functional groups present on the hydrophobic polymer and the Y groups are activated in sequence with carbodimide and divinylsulfones. The poly (methyl methacrylate) surface can be hydrolyzed with NaOH to form carboxyl groups on the surface. According to this document, the biomolecules are linked through a process comprising four stages after hydrolysis, with considerable complications and difficulties of transposition on an industrial scale. Furthermore, the method is only suitable for substrates comprising a substrate that exposes a metal surface.

The application US20050042363 reports the use of macroporous polymeric films deriving from methacrylic monomers (glycidyl methacrylate, hydroxyethyl methacrylate, PEG methacrylate, PEG dimethacrylate, and others) for the production on glass supports and the use of these films for immobilization of biomolecules. For the binding of the biomolecules functional groups are not used, obtained by adding to the polymerization mixture further reagents than those indicated above. The presence of porosity in the polymer film places limitations on the size of the biomolecule and the analyte, and also involves difficulties in applying the common techniques for controlling the quality of the surface.

Summary of the invention

It is therefore an object of the present invention to provide a method for producing miniaturized biosensors to detect the presence of certain components in biological samples, in particular to produce biosensors suitable for making diagnostic devices, which allows to apply a biological interaction material on a support in a manner stable.

It is a particular object of the present invention to provide such biosensors in which the biological interaction material cannot be easily removed from the support when exposed to the biological sample, in particular to an aqueous and / or flowing medium.

It is also an object of the present invention to provide such a method, which allows to apply the biological interaction material on the support in a reproducible way, in which it is therefore possible to predetermine the distribution of the molecules of the biological interaction material, and of the respective functional groups, on the support surface.

It is also an aim of the invention to provide such a method in which the biomolecules, in particular proteins, are stably bound to the support, without significantly modifying their biological activity, i.e. without the biomolecules undergoing appreciable denaturation and / or inactivation due to the effect of € ™ application on the surface of the support.

It is another object of the invention to provide such miniaturized devices, in which proteins are used as materials for interaction with the biological sample, which guarantee better performance than devices of the known art.

It is also an object of the present invention to provide a method for producing such biosensors in a simpler and more economical way than known methods.

It is also an object of the invention to provide such miniaturized diagnostic devices which have a lower cost than known devices.

It is another particular object of the present invention to provide such a method in which the surface of the support can be made in a simple and economical way, in particular, by depositing a solution of a polymeric material.

It is a further object of the present invention to provide biosensors which do not involve significant limitations to the molecular dimensions and chemical characteristics of the analytes.

These and other purposes are achieved by a method for producing biosensors capable of interacting with a biological sample or with one of its components, each biosensor comprising:

A support having a surface comprising a polymeric material; ï £ § a biological interaction material, the biological interaction material being applicable to the support to form a biological interaction layer capable of creating an interaction with the sample or with one of its components when the biosensor is placed in a proximity of the sample,

the method including the steps of:

ï £ § preparation of the support;

ï £ § application of the biological interaction material on the surface of the support;

wherein the step of applying the biological interaction material to the surface of the support comprises a creation of a chemical bond between a functional group of the biological interaction material and a functional group of the polymeric material of the support, and

ï £ § regulation of hydrophilicity / hydrophobia of the surface of the support, so that the interaction created between the biological sample or component and the biological interaction layer applied on the surface of the support has an intensity substantially equal to an interaction between the sample or the component and the biological interaction layer free, i.e. not bound to the support.

The chemical bond between the functional group of the molecular material of the biological interaction layer and the functional group of the polymeric material of the surface of the support can be of the covalent or ionic type.

In particular, the biological interaction layer comprises a molecular material of biological origin, that is, formed by biomolecules.

Advantageously, the biomolecules are proteins. According to another aspect, the molecular material can comprise molecules formed by monomer units linked by peptide bonds.

In particular, proteins are chosen from antibodies, antibody fragments, antigens, parts of antigen, enzymes, glycoproteins, a combination of such proteins.

Alternatively, the material of biological origin can be a nucleic acid, such as DNA, or a fragment thereof.

The polymeric material of the surface of the support can be a polymeric material of natural origin, in particular it can include polysaccharides.

The hydrophilicity of the surface of the support can be determined by studying the behavior of water when it is placed in contact with this surface. In other words, it is possible to classify the surface according to a scale of hydrophilicity / hydrophobia based on the value that statistically assumes the angle formed by a drop of water in static conditions with the surface, as better described in more detail below. .

In particular, the polymeric material of the support is a synthetic polymer, and the step of regulating the hydrophilicity / hydrophobia of the surface of the support provides a polymerization step, according to a predetermined molar ratio, of a plurality of monomers having respective hydrophilic to obtain a polymer having a predetermined hydrophilicity, dependent on this molar ratio.

In particular, the synthetic polymer can comprise units of the styrenic type.

Preferably, the styrene-type units are derived from chloromethylstyrene (CMS). Each chloromethylene group provides a reactive site, or a precursor thereof, capable of chemically binding a biomolecule, in particular a protein, with the polymer, creating a stable bond between the biological interaction layer and the surface of the support, by means of a reaction in which alkyl chloride undergoes a substitution process by a nucleophilic group.

In particular, the synthetic polymer is a polymer of CMS and styrene:

Chloromethylstyrene Styrene

The step of regulating the hydrophilicity / hydrophobia of the surface of the support then provides a step of polymerization of CMS with styrene, in which CMS and styrene have a predetermined molar ratio. Polymerization with styrene allows to modulate the concentration of reactive sites supplied by chloromethylene groups and therefore allows to modulate the hydrophilicity of the substrate surface as a function of the biomolecule (s) expected to form the biological interaction layer, without significantly changing the properties of the substrate surface and without making the polymer soluble in aqueous media. Furthermore, CMS and styrene are easily available and low cost monomers, which facilitate the preparation of biosensors in an economical way.

.

CMS-Styrene polymer

Preferably, the CMS and styrene polymer has a content of units derived from CMS ranging from 0 to 15% mol, in particular this content ranges from 0.1 to 10%. This concentration range of units derived from CMS contains concentration values which correspond to densities of reactive sites and hydrophilic values suitable for stably binding many biomolecules useful for forming the biological interaction layer, in particular for stably binding most of the proteins, to the at the same time without the biomolecules losing their natural conformation, and therefore their useful properties.

Alternatively, the synthetic polymer comprises units of the (meth) acrylic type, preferably units deriving from a methacrylic ester, ie from a methacrylate. The ester type bonds in the side chain of the polymer can in fact be easily hydrolyzed into carboxylic groups, which are able to bind directly with functional groups of biomolecules, typically with the amino functional groups present in the side chains of amino acid residues, or to undergo a further activation process as indicated below.

The methacrylic type unit can be a unit derived from glycidyl methacrylate (GMA). In particular, the polymer is a polymer of GMA and a monomer selected from hydroxyethyl methacrylate (HEMA) and methyl methacrylate (MMA), or preferably a combination of HEMA and MMA:

.

GMA HEMA MMA

The step of regulating the hydrophilicity / hydrophobia of the surface of the support then provides a step of polymerization of GMA with HEMA and / or MMA, in which GMA and HEMA and / or MMA have a predetermined molar ratio.

GMA-HEMA-MMA polymer

The epoxy side groups provided by the GMA units are themselves capable of reacting with the nucleophilic groups of proteins; moreover, they can easily undergo a cleavage both in an acidic environment and in a basic or neutral environment, forming hydrophilic side chains, such as to attenuate the purely physical interactions between biomolecules and polymer. In this way, the only interactions between the biomolecules and the surface of the support are defined by the reactive sites, according to an appropriate density or surface distribution, and there are limited phenomena of denaturation of the proteins; moreover, the surface distribution of the reactive sites, depending on the concentration of particular monomer units, is reproducible with relative ease, which makes the characteristics of the biological interaction layer and the series production of the biosensors according to the invention reproducible. HEMA also has the purpose of providing hydrophilic units, but also of contributing to the processability of the polymer, in particular in the case in which it must be placed on the surface of a heterogeneous substrate, for example glass. The MMA has the purpose of controlling the interactions of the polymer both with organic solvents and with aqueous media, therefore it acts as a solubility regulator. Solubility in organic solvents is a useful feature for process reasons, allowing the polymer to be deposited as a solution, while solubility in aqueous media is to be avoided as many biological samples contain aqueous media.

Alternatively, but not exclusively, the methacrylic type unit can be a unit derived from a polyethylene glycol methacrylate (PEGMA), comprising a side chain derived from a polyethylene glycol (PEG) having a predetermined molecular weight:

.

Polyethylene glycol methacrylate (PEGMA)

As in the case of the epoxy groups of GMA, the side chains derived from the PEG, strongly hydrophilic, are able to reject biomolecules, for example proteins, minimizing the phenomena of purely physical adsorption. The terminal hydroxyl groups of the PEG provide reactive sites for binding the biomolecules, upon activation treatment, as described below.

In particular, the polymer further comprises monomer units derived from a monomethoxy polyethylene glycol methacrylate (MPEGMA), ie a methyl ether of PEGMA, having a predetermined molecular weight. The choice of the concentration of monomer units deriving from MPEGMA and PEGMA in the polymer allows to assign the quantity of hydroxyl groups, which constitute the reactive sites of the polymer of the surface of the support; an appropriate choice of the percentage of MPEGMA and PEGMA units therefore allows to regulate the hydrophilicity of the polymer and, therefore, the properties that depend on it, such as the solubility in aqueous media and the reactivity towards biomolecules, in the terms seen for the others classes of synthetic polymers described above.

Monomethoxy polyethylene glycol methacrylate (MPEGMA)

In particular, the polymer comprising units deriving from PEGMA and MPEGMA also comprises a hydrophobic alkyl (meth) acrylate (AlMA), in particular a monomer selected from MMA and a butyl methacrylate (BuMA), in particular n-butyl methacrylate (n- BuMA), or a combination of MMA and a BuMA;

n-butyl methacrylate (n-BuMA)

the step of regulating hydrophilicity / hydrophobia of the surface of the support, and of the other characteristics, then provides a step of polymerization of PEGMA and of at least one such further monomer of the methacrylic type, in particular MMA and / or n-BuMA, in which the PEGMA and the further monomers of the methacrylic type have a predetermined molar ratio. In fact, the introduction of AlMA in the formulation allows to obtain insoluble products in aqueous media, so that the obtained biosensor can come into contact with a biological environment without losing its characteristics, i.e. without being dispersed or washed away from the medium. biological. N-BuMA provides a less hydrophilic side chain than MMA, so it also acts as a regulator of hydrophilicity.

Preferably, the PEGMA polymer has a content of units derived from PEGMA comprised between 2 and 10% mol. Furthermore, the polymer preferably has a content of units derived from an AlMA between 30% and 80%. With these concentrations of respective monomer units, hydrophilicity and density of reactive sites are obtained, suitable for binding stably and without compromising the characteristics of many biomolecules, in particular most of the proteins, as explained above.

Advantageously, the step of regulating the hydrophilicity / hydrophobia of the surface of the support provides for a step of introducing hydrophilic spacer groups, such spacer groups being lateral or terminal groups with respect to the main chain of the polymer of the surface of the support, in particular spacer groups comprising glycol units. o polyglycolic, ie units deriving from the PEG; therefore, in the case of units derived from PEGMA, the lateral group also acts as a spacer, depending on the length of the PEG chain, ie the molecular weight of the PEG from which the PEGMA derives. These effects can be regulated by suitably choosing this molecular weight, in particular the step of regulating the hydrophilicity of the surface of the support then comprises a step of choosing this molecular weight. In summary, the use of comonomers and / or spacers capable of reducing the concentration of reactive sites, modifying the surface characteristics of the substrate and removing the biomolecules from the surface allows to overcome the drawbacks associated with too high a number of reactive groups. and / or a very hydrophobic surface which can cause irreversible inactivation of biomolecules.

The polymer that forms part of the surface of the support can also be a widely available commercial or natural polymer, chosen from: ï £ § a polyester, in particular polyethylene terephthalate (PET);

ï £ § a polyamide, in particular nylon;

ï £ § a polyurethane;

- a poly (meth) acrylate, in particular polymethyl methacrylate (PMMA);

ï £ § a polyolefin;

• a vinyl copolymer, in particular an ethylene-acrylic acid copolymer, or an ethylene-vinyl alcohol copolymer, or an ethylene-maleic anhydride copolymer;

ï £ § a polysaccharide, in particular cellulose;

ï £ § a silicone;

ï £ § a combination of such polymers.

The synthesis polymers described above, as well as the commercial polymers indicated above are low cost materials, natively endowed with reactive functionality and further activated by resorting to simple surface chemical treatments or bulk processes, which are easily implemented on an industrial scale. The method according to the invention therefore allows to obtain biosensors with a commitment of economic resources lower than that required by the methods of the known art.

Reactive groups capable of interacting with biomolecules can therefore already be present in the side chains or be introduced by means of suitable synthesis procedures in the side chains, but also in the main chain or in the terminal groups. The introduction of the reactive groups can be carried out either by mass modification of preformed polymeric materials or by surface activation after their preparation in forms suitable for use as substrates.

In one embodiment of the method, the support preparation step involves a step of depositing the polymeric material on a base, so as to form the surface of the support using known deposition techniques such as solution casting, spin coating, dip coating or spray coating; in the first case the base can comprise a rigid material such as glass, silicon or a plastic or cellulosic material, on which said polymeric material is applied.

In another embodiment, said support preparation step provides for providing a support having a surface consisting of said polymeric material, in particular an integral support made of said polymeric material, for example in the form of a film of said polymeric material .

The polymeric material of the surface of the support can therefore be a surface layer deposited on a substrate of another material, or it can constitute the support itself. The polymeric materials used in the method according to the invention can be used directly as supports, both rigid and flexible, or as a surface coating of conventional supports, such as for example glass or silicon.

The step of regulating hydrophilicity / hydrophobia can comprise a step of chemical transformation of a portion of the chain of the polymeric material, in which the functional groups of the polymeric material of the support are created or transformed. The chemical transformation phase can provide functional groups able to combine with the functional groups of the biological interaction material, in particular with the functional groups of amino acids.

In particular, the functional groups of the polymeric material are selected from halogens;

ï £ § epoxides;

ï £ § hydroxyls;

ï £ § amides;

ï £ § amines;

ï £ § foreign;

ï £ § carboxyls;

ï £ § bunkers;

ï £ § urethanes

ï £ § a combination of these functional groups.

Thanks to the presence of surface reactive groups such as those indicated above, or those introduced by chemical reaction, the surface of the support is able to form ionic or covalent chemical bonds with biomolecules such as proteins, in particular antibodies, antibody fragments, antigens , parts of antigen, enzymes, glycoproteins, but also polysaccharides as well as oligopeptides in general and nucleic acids, for example DNA, or relative fragments. This feature guarantees the possibility of biofunctionalizing the surface of the support in a lasting and reproducible way.

in particular, the phase of creation or transformation of functional groups in the polymeric material is carried out on the surface of the support. This can occur both in the case of supports integrated into the polymeric material and in the case of supports in which the polymeric material is deposited on a substrate of another material forming the surface of the support.

Advantageously, the step of regulating hydrophilicity / hydrophobia of the surface of the support comprises a step of activating the functional groups in the polymeric material of the surface of the support, by means of at least one reaction step of the polymeric material with a reagent having at least two functional groups adapted to react with the polymer material.

In particular, by means of this reaction the reagent introduces into the polymeric material functional groups selected from:

ï £ § primary or secondary amino groups;

ï £ § aldehyde groups;

ï £ § carboxyl groups.

ï £ § epoxy groups

ï £ § isocyanate groups

Preferably, the step of activating the functional groups of the polymeric material is performed after the deposition step of the polymeric material on the support, and the reagent can be a homobifunctional reagent, i.e. formed by molecules each having two functional groups of the same type, without cross-linking and / or inactivation reactions of the reagent occur. In fact, since the polymer is already deposited to form the surface of the substrate, the probability that both functional groups react with the polymeric material are reduced compared to the case of mass activation.

In particular, the activation step of the functional groups comprises a reaction of an amine having at least two amino functional groups with a polymer provided with functional units derived from a compound selected from:

ï £ § a monomer comprising a halogen atom bonded to an alkyl carbon, such as vinyl chloride and CMS, and / or

It is a monomer comprising an epoxy group such as GMA. The diamine can advantageously comprise a molecular spacer, in particular a spacer having hydrophilic characteristics. In this way, as already observed, amino groups are obtained, available to combine with the carboxylic groups of biomolecules such as proteins, suitably spaced from the hydrophobic surface of the substrate, so as to limit the phenomena of non-covalent adsorption, and therefore avoid the denaturation of the biomolecule. Preferably, this spacer having hydrophilic characteristics is derived from a polyethylene glycol.

The diamine is preferably a Î ±, Ï ‰ -diamine containing a portion of a hydrophilic linear molecular chain which acts as a molecular spacer, in particular the portion of the chain consists of oxyethylene units in sequence, in a number, for each molecule, between two (as in the case of Î ±, Ï ‰ -diaminodiethylene glycol) (diam-DEG)

Î ±, Ï ‰ -diaminodiethylene glycol (diam-DEG)

and a number such that the diamine has a molecular weight close to 1500, for example 36 or 37 oxyethylene units, as in the case of Î ±, Ï ‰ -di (3-aminopropyl) polyethylene glycol (diam-PEG).

Î ±, Ï ‰ -di (3-aminopropyl) polyethylene glycol (diam-PEG)

Activation of a functional group of a monomer unit

derived from GMA by reaction with diam (DEG)

If a polyamine is used, it can be polyethyleneimine (PEI). In this case, the marked effect in terms of increased hydrophilicity can be explained by considering that this compound possesses a considerable amount of reactive groups complementary with the chloromethyl styrene polymer, such as to form a substantially uniform layer of PEI, so that the final characteristics of the support depend heavily on PEI and not on CMS polymer. In other words, CMS functionalization provides a method to expose a PEI surface, characterized by a degree of hydrophilicity suitable for combining biomolecules as proteins without causing denaturation.

Polyethyleneimine (PEI) Alternatively, the activation step of the functional groups involves a reaction of a dialdehyde with a polymer comprising primary amino groups such as those previously obtained by activation with polyamines.

Preferably, the dialdehyde is the glutaric aldehyde. This transformation introduces on the surface of the support imino-aldehyde functional groups suitable, for example, to combine with the amino groups of proteins.

Glutaric aldehyde

Alternatively, the functional group activation step involves a reaction of a diisocyanate derivative with a polymer comprising hydroxyl groups such as those containing units derived from PEGMA or HEMA or amino groups such as those previously obtained by activation with polyamines.

In particular, the diisocyanate derivative is 2,4-diisocyanatotoluene. This transformation introduces on the surface of the support isocyanate functional groups suitable, for example, to combine with the amino groups of proteins.

O O

C C

N N

2,4-diisocyanatotoluene

In particular, the diisocyanate derivative is 2,4-diisocyanatohexane. This transformation introduces on the surface of the support isocyanate functional groups suitable, for example, to combine with the amino groups of proteins.

OR

C N

N C

OR

2,4-diisocyanatohexane

In particular, the diisocyanate derivative is PEG-diisocyanate. This transformation introduces on the surface of the support hydrophilic PEG spacers and isocyanate functional groups suitable, for example, to combine with the amino groups of proteins.

OR

C O N

N n O C

OPEG-diisocyanate

Alternatively still, the activation step comprises a treatment step with a compound comprising a dicarboxylic acid or preferably, with a corresponding anhydride thereof. In particular, this anhydride is succinic anhydride. In this way, carboxylic groups are made available for the subsequent combination of functional groups of the biomolecules, in particular with amino groups as in the case of proteins. This compound comprising a carboxylic acid in the form of anhydride can also be a copolymer of maleic anhydride and an alkyl vinyl ether, in particular of methylvinylether and / or isobutylvinylether.

In this last case the copolymer can constitute both the surface layer deposited on a substrate and the support itself.

a) b)

Copolymers of maleic anhydride: a) with methyl vinyl ether; b) with isobutyl vinyl ether

For example, these copolymers are able to combine with the hydroxyl groups of natural or semisynthetic polysaccharides such as cellulose and its derivatives allowing an effective activation of this natural polymer used as material of the surface of the support towards the subsequent phase of application of the material. of biological interaction on the surface.

Preferably, the activation step with dicarboxylic reagents also provides for a subsequent transformation step of the carboxylic groups introduced into the polymer of the support into reactive ester groups, that is, capable of undergoing an amidation in the presence of amino functional groups of the biological interaction material, in particular of biomolecules, in particular of the amino groups of proteins. This transformation can be used to increase the affinity of the carboxyl groups introduced into the polymer of the surface of the support towards the nucleophilic, amine groups present on the macromolecule. It can advantageously be carried out by treating the polymer containing carboxylic groups with 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide (EDC)

1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC)

and, in a second step, with N-hydroxysuccinimide (NHS) which forms with the functional group supplied by EDC a reactive ester group in the sense indicated above.

N-hydroxysuccinimide (NHS)

By operating in two steps, the control of the reaction is facilitated and the biological interaction material, in particular the protein, is prevented from transforming by interacting directly with EDC.

The hydrophilic / hydrophobic regulation phase also allows to optimize the properties of the surface of the support from the point of view of wettability and from the morphological point of view, according to the deposition techniques of the biological interaction layer used, in order to form biosensors of micrometric dimensions.

Preferably, the step of applying the biological interaction material on the surface of the support is carried out with an automated technique, in particular chosen between ink jet printing and micropipetting. These deposition techniques, the success of which is made possible by the careful choice of the surface properties of the support, make it possible to easily produce miniaturized devices capable of simultaneously detecting a very large number of analytes.

According to another aspect of the invention, the objects indicated above are achieved by a biosensor obtained through the method described above.

Brief description of the drawings

The invention will be illustrated below with the following description of its embodiments and examples, given by way of non-limiting example, with reference to the attached drawings in which:

Figure 1 schematically shows the behavior of a drop of an aqueous medium on a hydrophilic surface and on a hydrophobic surface;

Figure 2 schematically shows a technique for characterizing the hydrophilicity / hydrophobia of a surface, on the basis of the behavior represented in figure 1.

Description of preferred embodiments

With reference to Figures 1 and 2, a technique is described which allows to give a quantitative evaluation of the hydrophilic / hydrophobic character of a surface of a support of a biosensor according to the invention. As shown in Figure 1, the hydrophilic or hydrophobic character of the surface 1 of the support 2 can be evaluated by the shape assumed by a drop of water 3 placed on the surface 1, more precisely by a measurement of the static contact angle Î ̧ formed between the Drop-air interface and the surface-drop interface. The greater the angle Î ̧, the less is the hydrophilicity of the surface 1 of the support 2. The measurement of the contact angle Î ̧ can be carried out with the equipment schematically shown in figure 2, comprising a syringe 4 connected to a metering pump, not shown, capable of depositing a plurality of drops 3, a light source 5 and a camera 6 associated with a goniometer for measuring Î ̧, not shown, a computer 7 comprising program means for determining the profile and the respective contact angles of the drops 3 by extracting an average value of Î ̧, to take into account the environmental factors that influence the static contact angle.

According to one aspect of the invention, the step of regulating the hydrophilicity / hydrophobia of the surface of a support of a biosensor involves a polymerization step of monomers capable of conferring respective contributions to the hydrophilicity of the polymer, which depends on the molar ratio among the monomers. The polymerization effect is shown in tables 1-3 for three classes of synthetic polymers, in order CMS-styrene polymers, polymers containing GMA and at least one monomer chosen from HEMA and MMA, and polymers containing PEGMA, MPEGMA and an alkyl methacrylate, in this case n-BuMA. The tables show the values of the static contact angle Î ̧ (figure 1) measured with the method indicated above (figure 2), with reference to a polymeric film obtained by spin coating.

As shown in table 1, in the case of copolymers based on CMS and styrene, as the ratio in moles CMS / styrene increases, the hydrophilicity of the polymer increases, the angle Î ̧ passing with substantial regularity between the value of polystyrene (about 98 °) to the value that belongs to polychloromethylstyrene, close to 86 °. This trend depends on the greater polarity and, therefore, on the greater hydrophilicity of the units deriving from CMS compared to the units deriving from styrene.

Table 1

Static contact angle values measured on film

from CMS-styrene copolymer

CMS polymer / Styrene moles Static contact angle

PS 0/100 98 °

S1 1/99 96.2 ° ± 0.6

S2 2/98 93.4 ° ± 1.5

S4 4/96 90.4 ° ± 0.9

S7 7/93 92.7 ° ± 0.3

S10 10/90 88.0 ° ± 0.9

PCMS 100/0 85.9 ° ± 1.0

Table 2 shows the values of the static angle angles measured on films from terpolymer GMA / HEMA / MMA (G1) and from copolymer GMA / MMA (G2). These polymers exhibit static angle values lower than those of CMS-styrene polymers due to the more hydrophilic character of the monomers used.

Table 2

Static contact angle values measured on film from GMA / HEMA / MMA terpolymer and GMA / MMA copolymer

Polymer GMA moles HEMA moles MMA moles Static contact angle

G1 10 40 50 66.1 ° ± 1.5

G2 10 0 90 72.2 ° ± 0.5

Table 3 shows the static contact angle values measured on PEGMA / MPEGMA / BuMA terpolymer film. The static angle increases as the content in units deriving from n-BuMA increases. The presence of increasing fractions of PEGMA and MPEGMA significantly increases hydrophilicity, reducing the angle Î ̧, which for the n-BuMA homopolymer is about 85 °.

Table 3

Static contact angle values measured on film

from PEGMA / MPEGMA / BuMA terpolymer

PEGMA / MPEGMA / BuMA polymer Static contact angle

P4 8/32/60 64.7 ° ± 1.7

P5 6/22/72 72.8 ° ± 1.6

P6 5/14/81 73.1 ° ± 0.8

The synthesis of the polymers referred to in Tables 1, 2 and 3 was carried out according to procedures well known to a person skilled in the art.

According to another aspect of the invention, the regulation of hydrophilicity occurs through a step of introducing hydrophilic spacer groups as side chains of the polymer that forms the surface of the support. An example of this introduction is given by the use in the reaction mixture that leads to the polymer, of monomers provided with such spacer groups. In the case of the polymers of PEGMA and MPEGMA, mentioned above, these hydrophilic spacer groups consist of oxyethylene sequences. In other cases, as will be shown below, the introduction of such spacer groups occurs at the same time as the creation, within the polymer of the surface of the support, of functional groups suitable for combining with reactive groups of the biomolecules, or for the modification, i.e. ™ activation of pre-existing functional groups within the polymer.

According to yet another aspect of the invention, the hydrophilic regulation step involves the creation or transformation of functional groups in the polymer that forms the surface of the support, suitable for combining with biomolecules, in particular with proteins.

For example, a naturally hydrophilic polymer such as cellulose can be provided with carboxylic groups capable of combining with biomolecules such as proteins by treating cellulose sheets at 60 ° C for about 3 hours with alternating copolymer solutions of maleic anhydride (AM ) with methyl vinyl ether (MeVAM) or with isobutyl vinyl ether (BuVAM) in tetrahydrofuran, and subsequent cooling and washing with the same solvent. The sheets thus obtained, unlike the original ones, are essentially water repellent. The cellulose thus functionalized exposes carboxylic groups and unreacted rings deriving from AM. These rings constitute a function suitable for combining by ionic bond with the amino groups of proteins; the carboxylic groups, on the other hand, are susceptible of further modification, or activation, by means of two-stage treatment with EDC / NHS, to form reactive ester groups, according to processes known to a person skilled in the art.

In the case of polyesters such as PET or PBT, carboxylic groups can be created by controlled hydrolysis, i.e. partial hydrolysis of the ester bonds in the presence of common inorganic acids such as sulfuric acid or even hydrochloric acid, for a few hours and at moderate temperatures and concentrations, according to well-defined processes. known to a person skilled in the art. These groups too can be subsequently activated by reaction with carbodiimides and NHS as described above. Table 4 shows the results obtained in terms of static contact angle, while Table 4a shows the luminous intensity values corresponding to the residual activity of the deposited antibodies.

Table 4

Static contact angle and activity values measured on PET film treated with inorganic acid solutions

Sample Acid Time of Activity Angle inorganic treatment contact (0-255) static

PET - - 80.2 ° ± 0.9 84 ± 25 PET HCl 3.65 M 1 h 79.7 ° ± 1.6 81 ± 14 PET HCl 3.65 M 3 h 76.9 ° ± 2.5 n.d. PET H2SO43.22 M 1 h 68.3 ° ± 0.7 n.d. PET 1) H2SO43.3 M 1h n.d. 255

2) CDI / NHS

Bioriented PET - - 75.8 ° ± 2.1 87 ± 16 Bioriented PET H2SO43.22 M 1 h 68.1 ° ± 1.8 255 Bioriented PET 1) H2SO43.3 M 1h n.d. 255

2) CDI / NHS

In the case of methacrylic polymers such as PMMA, carboxylic groups can be created by controlled hydrolysis of the lateral methyl ester groups in an alkaline medium, typically in a sodium hydroxide solution, according to procedures well known to a person skilled in the art, for at least 16-24 hours. . Such groups can be subsequently activated by reaction with carbodiimides and NHS as described above. Table 5 shows the results obtained in terms of static contact angle.

Table 5

Static contact angle and activity values measured on PMMA film treated with sodium hydroxide solutions

Treatment time Static contact angle Activity (0-255)

- 75.3 ° ± 1.1 107 ± 9

8 h 75.5 ° ± 1.2 n.a.

16 h 72.8 ° ± 2.7 n.d.

24 h 70.4 ° ± 2.1 89 ± 21

24 h <a)> n.d. 132 ± 35

a) After further treatment with EDC / NHS

The CMS polymers are endowed with a chloromethyl group suitable for combining with biomolecules such as proteins, binding them to the support in a stable way. It is also advantageous to modify this chloromethylene group by means of reactions with polyfunctional or bifunctional reagents capable of providing further functional groups, in particular amino, aldehyde or carboxylic groups, suitable for combining with the biomolecules. To regulate hydrophilicity, bifunctional reagents capable of providing also spacer groups are preferable, for example an Î ±, Ï ‰ -diamine with a portion of hydrophilic linear molecular chain of predetermined length, so that the biomolecules are bound to a distance such as to substantially exclude other forms of interaction with the substrate surface, leaving the biomolecule substantially free to arrange itself in a conformation as close as possible to the natural one, thus best expressing its activity as a sensitive element towards the analytes. Table 6 shows the static contact angles referred to CMS polymers treated for 48 hours with a 100: 1 excess of diamDEG on the CMS units, in an ethanol solution at room temperature.

Table 6

Static contact angle and activity values measured on CMS-styrene copolymer film before and after treatment with diamDEG.

copolymer Static contact angle Activity (0-255)

(see table 1) <Before> After Before After treatment treatment treatment treatment

S1 96.2 ° ± 0.6 89.0 ° ± 1.3 101.7 ± 4.9 118.8 ± 10.3

S2 93.4 ° ± 1.5 85.4 ° ± 3.3 130.5 ± 6.7 170.3 ± 18.5

S4 90.4 ° ± 0.9 86.8 ° ± 0.9 120.8 ± 8.6 118.0 ± 10.1

S7 92.7 ° ± 0.3 85.5 ° ± 1.6 106.2 ± 4.8 140.0 ± 12.6

S10 88.0 ° ± 0.9 80.4 ° ± 4.9 117.8 ± 7.9 119.2 ± 7.5

PCMS 85.9 ° ± 1.0 78.3 ° ± 1.0 139.7 ± 7.2 163.8 ± 7.7

As table 7 shows, even more sensitive reductions in the static contact angle, and therefore more marked increases in hydrophilicity, are obtained by treating the same copolymers with diamPEG, i.e. by increasing the length of the hydrophilic spacer chain of the reagent used, without prejudice to all other operating conditions. The decrease in the static contact angle is in all cases at least 15%.

Table 7

Static contact angle and activity values measured on CMS-styrene copolymer film before and after treatment with diamPEG.

polymer Static contact angle Activity (0-255) (see table 1) Before treatment After treatment After treatment

S1 96.2 ° ± 0.6 68.1 ° ± 3.1 126.1 ± 4.3

S2 93.4 ° ± 1.5 n.a. 130.6 ± 12.6

S4 90.4 ° ± 0.9 65.7 ° ± 1.9 121.0 ± 2.8

S7 92.7 ° ± 0.3 66.6 ° ± 0.9 114.4 ± 10.6

S10 88.0 ° ± 0.9 65.2 ° ± 1.3 123.4 ± 9.6

PCMS 85.9 ° ± 1.0 49.7 ° ± 1.1 147.4 ± 7.1

Table 8 shows the static contact angle values still of polymers containing units derived from CMS after 5 hours of treatment at 55 ° C with an aqueous solution of PEI at 0.05%, i.e. with an excess of 6: 1 compared to CMS units. Only one polymer is reported as the angle is substantially independent of the proportion of CMS units, and very similar to the angle that competes with the PCMS homopolymer, probably because a uniform layer of PEI is formed which hides the surface of the polymer.

Table 8

Static contact angle and activity values measured on CMS-styrene copolymer film before and after treatment with PEI.

polymer Static contact angle Activity (0-255) (see table 1) Before treatment After treatment After treatment

S2 93.4 ° ± 1.5 64.6 ° ± 1.8 95.2 ± 5.1

PCMS 85.9 ° ± 1.0 62.8 ° ± 1.5 105.7 ± 10.5

Table 9 shows the static contact angles referring to CMS copolymers, previously treated with diamDEG or PEI, before and after 72 hours of treatment with a solution of glutaric aldehyde in absolute ethanol, at room temperature and in slight excess on CMS groups.

Table 9

Static contact angle and activity values measured on CMS-styrene copolymer film before and after treatment with glutaric aldehyde.

Copolymer / 1st treatment Static contact angle Activities (0-255) (see tables 1 and 6) Before After After After treatment treatment treatment

S2 / diamDEG 85.4 ° ± 3.3 77.3 ° ± 3.4 153.1 ± 12.6 PCMS / diamDEG 78.3 ° ± 1.0 69.0 ° ± 0.5 132.7 ± 11.7

Table 10 reports the static contact angle measured on polymer films containing glycidyl methacrylate, before and after treatment with diamDEG under the same conditions adopted for CMS polymers.

Table 10

Static contact angle and activity values measured on polymer films including GMA before and after treatment with diamDEG.

polymer Static contact angle Activity (0-255)

(see table 2) Before After After Before After treatment treatment treatment treatment

G1 66.1 ° ± 1.5 55.0 ° ± 1.5 113.4 ± 12.5 185.3 ± 15.5

G2 72.2 ° ± 0.5 59.4 ° ± 1.8 118.9 ± 5.4 184.6 ± 8.8

The polymers containing units derived from GMA were also treated with PEI, under the same conditions as the CMS polymers, obtaining the results of table 11. The values after treatment approximate the approximately 62 ° that compete with PEI, confirming what has been seen for the € ™ other class of polymers.

Table 11

Static contact angle and activity values measured on polymer films including GMA before and after treatment with PEI.

polymer Static contact angle Brightness (a.u.) (see table 2) Before After After After treatment treatment treatment

G1 66.1 ° ± 1.5 63.5 ° ± 1.0 142.0 ± 5.7

G2 72.2 ° ± 0.5 61.4 ° ± 0.9 125.6 ± 7.4

Table 12 shows the values of the static contact angle obtained by subjecting the PVC to a hydrophilic regulation phase similar to the modification or activation of the chlorine groups of CMS polymers, by immersion in a solution of diamDEG or PEI in ethanol at 50 ° C for 5 hours.

The various polymers obtained after treatment with diamPEG or PEI were treated at room temperature with a PEG-diisocyanate solution. This modification introduced isocyanate groups capable of reacting with the amino groups of the proteins. However, it was not possible to measure the contact angle of the products obtained, as the isocyanate groups react spontaneously with water, developing CO2 gas.

Table 12

Static contact angle and activity values measured on PVC polymer film

Activation Static contact angle Activity (0-255)

- 82.4 ° ± 3.2 n.d diam-DEG 62.7 ° ± 2.3 154.7 ± 10.5 diam-DEG â € “GD 76.4 ° ± 3.3 181.3 ± 9.2

PEI 54.6 ° ± 7.6 90.6 ± 6.0

PEI - GD 68.2 ° ± 2.1 50.6 ± 11.4

The step of depositing the polymeric material to form the surface of the support is advantageously carried out by means of a spin coating technique well known to a person skilled in the art. The technique consists in the preparation of a solution of a polymer in a solvent as indicated in table 13, preferably at a polymer concentration of 0.2% weight / volume. This solution is filtered through a 2 µm Teflon filter, then deposited in doses of 100 µl in the center of respective substrates consisting, in the specific case of the laboratory, of microscope glasses, rotated at 1000 RPM for 10 seconds, then maintaining the manufactured articles obtained in a dry atmosphere up to the moment of use.

The application phase of the biological interaction layer, in particular proteins or antibodies, can be carried out by means of a per se known ink jet printing technique, which allows the creation of microarrays of predetermined second patterns.

Table 13

Solvents used for the deposition of polymer films by means of the spin-coating technique

Polymer Solvent Styrene / CMS Blend Chloroform / Toluene GMA / HEMA / MMA Tetrahydrofuran PEGMA / MPEGMA / n-BuMA Chloroform

In particular, the deposition phase can be carried out according to a casting or dip-coating or spraying phase.

In particular, the deposition phase is carried out according to a spin coating phase, in which a dose of a solution of the polymer in a solvent is placed on the support, and the support is then rotated, in so that due to the evaporation of the solvent and the centrifugal force created by the rotation, a film of the polymer that forms the surface remains on the support.

To determine the validity of the materials produced, the samples were subjected to residual activity tests of an antibody deposited on the surface of the related biochips. The results of these tests are reported in tables from 4 to 12 together with the static angle values.

In these tests, the antibody is printed on the polymeric surface, possibly treated or activated appropriately, by means of ink jet printing according to a predetermined pattern, with a resolution of 300x150 dpi. The rest of the surface is blocked with a protein inactive towards the analytes of interest.

A biological sample is placed in contact with the surface so that the analyte it contains can bind to the deposited antibody. After suitable incubation times, the surface of the biochip is treated with a solution capable of producing a chemoluminescent reaction with the antibody-analyte complex. The intensity of the emitted radiation, measured on a 0-255 scale by a cooled CCD digital camera, is proportional to the residual activity of the deposited antibody.

The above description of specific embodiments is able to show the invention from a conceptual point of view so that others, using the known art, will be able to modify and / or adapt these specific embodiments in various applications without further research and without departing from the inventive concept, and, therefore, it is understood that such adaptations and modifications will be considered as equivalent to the specific embodiments. The means and materials for carrying out the various functions described may be of various nature without thereby departing from the scope of the invention. It is understood that the expressions or terminology used have a purely descriptive purpose and therefore not limitative.

Claims (10)

CLAIMS 1. A method for producing biosensors capable of interacting with a biological sample or with one of its components, each biosensor being equipped with: A support having a surface comprising a polymeric material; ï £ § a biological interaction material, said biological interaction material being applicable to said support to form a biological interaction layer able to create an interaction with said sample or with said component when one of said biosensors is placed in a proximity of said sample, said method including the steps of: ï £ § preparation of said support; ï £ § application of said biological interaction material on said surface of said support, said step of applying said biological interaction material on said surface of said support comprising a creation of a chemical bond between a functional group of said biological interaction material and a functional group of said polymeric material of said support, - regulation of hydrophilicity / hydrophobia of said surface of said support, so that the interaction created between said biological sample or said component and said biological interaction layer applied on said surface of said support has an intensity substantially equal to that interaction between said sample or said component and said free biological interaction layer, i.e. not bound to said support, in particular, said biological interaction layer comprising a molecular material of biological origin, comprising biomolecules, in particular said biomolecules being proteins, more specifically particular said proteins being selected from antibodies, antibody fragments, antigens, parts of antigen, enzymes, glycoproteins. 2. The method according to claim 1, wherein said polymeric material of said support is a synthetic polymer and said hydrophilic / hydrophobic regulation step of said surface of said support provides for a polymerization step of a plurality of monomers having respective hydrophilic according to a predetermined molar ratio so as to obtain a polymer having a predetermined hydrophilicity, dependent on said molar ratio. 3. The method according to claim 2, wherein said synthesis polymer comprises units of the styrene type, in particular said units of the styrene type being units derived from 4-chloromethylstyrene (CMS) and units derived from styrene, said hydrophilic regulation phase / hydrophobia of said surface of said support then providing a polymerization step of CMS with styrene, in which CMS and styrene have a predetermined molar ratio, in particular said CMS and styrene polymer having a content of units derived from CMS between 0 and 15% mol. The method according to claim 2, wherein said synthetic polymer comprises units of the (meth) acrylic type, in particular said units of the (meth) acrylic type being selected from: ï £ § units derived from glycidyl methacrylate (GMA) and a monomer selected from hydroxyethyl methacrylate (HEMA) and methyl methacrylate (MMA), or preferably a combination of HEMA and MMA, called hydrophilic / hydrophobic regulation phase of said surface said support then providing a polymerization step of GMA with HEMA and / or MMA, in which GMA and HEMA and / or MMA have a predetermined molar ratio; ï £ § units derived from a polyethylene glycol methacrylate (PEGMA) and a monomethoxy polyethylene glycol methacrylate (MPEGMA), called PEGMA and said MPEGMA comprising side chains derived from polyethylene glycol (PEG) having a predetermined molecular weight, in particular, an additional comonomer chosen from among the alkyl (meth) acrylates (AlMA), in particular MMA and n-butyl methacrylate (n-BuMA), or a combination of MMA and n-BuMA, said hydrophilic / hydrophobic regulation step of said surface of said support then providing a polymerization step of PEGMA and MPEGMA and possibly said AlMA in which said PEGMA, said MPEGMA and possibly said AlMA have a predetermined molar ratio, in particular, said polymer of said PEGMA having a content of units derived from said PEGMA comprised between 2 and 10% mol, and a content of units derived from said AlMA comprised between 30% and 80%. The method according to claim 1, wherein said step of regulating the hydrophilicity / hydrophobia of said surface of said support provides for a step of introducing hydrophilic lateral or terminal spacer groups with respect to the main chain of said polymer of said surface of said support , in particular said hydrophilic spacer groups comprise glycolic or polyglycolic units. The method according to claim 1, wherein said polymeric material of said surface of said support is selected from: ï £ § a polyester, in particular polyethylene terephthalate (PET); ï £ § a polyamide, in particular nylon; ï £ § a polyurethane - a poly (meth) acrylate, in particular polymethyl methacrylate (PMMA); ï £ § a polyolefin; ï £ § a vinyl copolymer, in particular a copolymer of ethylene with a comonomer chosen from ï £ § acrylic acid; ï £ § vinyl alcohol; ï £ § maleic anhydride; ï £ § polyvinyl chloride (PVC); ï £ § a polysaccharide, in particular cellulose: ï £ § a combination of such polymers. 7. The method according to claim 1, wherein said step of preparing said support provides a step chosen from: deposition of the polymeric material on a base so as to form said surface of said support, in particular, said base comprises a rigid material such as glass, silicon or a plastic or cellulosic material, on which said polymeric material is applied; to provide a support having a surface made of said polymeric material, in particular to produce a film made of said polymeric material. The method according to claim 1, wherein said step of regulating the hydrophilicity / hydrophobia of said surface of said support comprises a step of creating or transforming said functional groups of said polymeric material of said support adapted to combine with said functional groups of said biological interaction material, in particular said functional groups of said polymeric material being selected from: ï £ § halogens; ï £ § epoxides; ï £ § hydroxyls; ï £ § amines; ï £ § amides; ï £ § foreign; ï £ § carboxyls; ï £ § bunkers; ï £ § urethanes ï £ § a combination of such functional groups, said functional groups of said polymeric material being introduced into said polymeric material by means of controlled hydrolysis reactions, in particular, said step of creating or transforming functional groups of said polymeric material being carried out on said surface of said support. The method according to claim 1, wherein said step of regulating the hydrophilicity / hydrophobia of said surface of said support comprises a step of activating said functional groups in said polymeric material of said support, said activation step providing at least one step reaction with a reagent having at least two functional groups, suitable for introducing into said polymeric material by means of said reaction functional groups selected from: ï £ § primary or secondary amino groups; ï £ § aldehyde groups; ï £ § carboxyl groups, ï £ § epoxy groups ï £ § isocyanate groups in particular, said reagent is a homobifunctional reagent selected from: ï £ § an amine having at least two amino functional groups, said amine being able to react with a polymer provided with functional units derived from a compound selected from ï £ § a monomer comprising a halogen atom bonded to an alkyl group, in particular vinyl chloride and / or CMS, and / or ï £ § a monomer comprising an epoxy group, in particular GMA, in particular said amine being selected from ï £ § an Î ±, Ï ‰ -diamine containing a portion of a molecular chain consisting of a sequence of oxyethylene units; - polyethyleneimine; It is a polyisocyanate having at least two isocyanate functional groups, said polyisocyanate being able to react with a polymer provided with functional units selected from: ï £ § primary hydroxyl groups; ï £ § primary or secondary amino groups, in particular said polyisocyanate being selected from ï £ § 2,4-diisocyanatotoluene; ï £ § 1,6-diisocyanatohexane; ï £ § a diisocyanate containing a portion of a molecular chain consisting of a sequence of oxyethylene units, it is a dialdehyde suitable to react with a polymer comprising primary amino groups, in particular said dialdehyde being glutaric dialdehyde; A compound comprising a dicarboxylic acid, in particular said dicarboxylic acid being used in the form of the corresponding anhydride, in particular said compound being selected from succinic anhydride, It is a copolymer of maleic anhydride and an alkyl vinyl ether, in particular said activation step by providing a subsequent transformation of carboxyl groups of said polymeric material of said surface of said support into ester groups reactive with the biological interaction material, in particular said ester groups being able to combine with amino groups of proteins. 10. A biosensor obtained through the method described in any one of the preceding claims.