WO2003000748A1 - Analysis cards - Google Patents

Abstract

The invention relates to the use of a particular polymer for the production of analysis cards, said polymer having the following properties:- said polymer has a glass transition temperature less than 60 DEG C, - in the absence of any functionalisation said polymer is hydrophobic and, for the purpose envisaged, said polymer is mass functionalised by reactive hydrophilic functions, or reactive hydrophilic function pre-cursors. The application of said polymer permits the simple monitoring of the properties of the surface of said analysis card and, in particular, the support thereof and to modify said properties on the monitored areas. The invention further relates to analysis cards, the support of which is made from said polymer and a method for modifying the surface properties of said support.

Description

 ANALYSIS CARDS

The present invention relates to the technical field of polymer materials used for the manufacture of analysis cards. Analysis cards allow the analysis of one or more different liquid samples, in which one seeks to identify one or more analytes, according to all simple or complex analysis processes involving one or more different reagents depending on the nature chemical, physical or biological of the analyte (s) sought. Analysis cards are not, in general, limited to a particular analyte, the only requirement is that the analyte is distributed in the sample to be analyzed in suspension or in solution. In particular, the analysis process used can be carried out in homogeneous, heterogeneous or mixed form.

An analysis card can, for example, allow biological analysis, of one or more ligands, requiring for their detection and / or their quantification, the use of one or more anti-ligands. Also, in this case, the analysis cards present on the surface biological ligands having at least one recognition site allowing them to react with a target molecule of biological interest.

An example of the application of analytical techniques concerns immunoassays, whatever their format, by direct analysis or by competition.

Another example of application relates to the detection and / or quantification of nucleic acids comprising all the operations necessary for this detection and / or this quantification from any sample containing the target nucleic acids. Among these various operations, mention may be made of lysis, fluidification, concentration, the steps of enzymatic amplification of nucleic acids, the detection steps incorporating a hybridization step using for example a DNA chip or a labeled probe. The patent applications published under the numbers WO 97/02 357 and WO 00/40 590 explain the various steps necessary in the case of nucleic acid analysis. In terms of implementation, these analysis cards can take various forms, for example square, rectangular or circular. These cards consist of a support and possibly a cover, the support being provided a plurality of wells and / or channels in which the different analysis processes are implemented. The supports for analysis cards and biochips are generally produced by machining of materials chosen from glass, quartz, silicon, possibly coated with a layer of oxide or nitride (WO 93/09 668) or else in polymers of the polystyrene type (FR 2 790 686). It is also envisaged in the patent application published under the number WO 99/58 245 to use plastic materials of the polycarbonate or hydrocarbon polymer type such as polyolefins, having a hydrophobic surface. To control the fluidics of the samples to be analyzed, these materials are chemically or physically modified, for example, under the action of grafting or plasma treatment, to create hydrophilic zones and thus modify the surface characteristics of the material.

Indeed, for the above-mentioned applications, it is very important to control the hydrophilic / hydrophobic character of the channels present on the supports of these analysis cards, in particular: - to avoid the formation of bubbles during the transport of fluids in these channels,

- to avoid losses of liquid due to too strong interactions between the fluid and the nature of the surface of the channels,

- to avoid the adsorption on the walls of the biological molecules which one wishes to transport. Also, the present invention relates to the new use of a particular polymer material for the manufacture of analysis cards, characterized in that the polymer:

- has a glass transition temperature of less than 60 ° C, preferably less than 37 ° C, and preferably less than 20 ° C, - is hydrophobic in the absence of functionalization, and

- is functionalized in its bulk by reactive hydrophilic functions, or generating reactive hydrophilic functions.

The use of the intrinsic properties of this material makes it possible to simply modify the characteristics of its surface, by locally modifying its hydrophobic / hydrophilic character.

Another essential objective of the present invention is to provide analysis cards which are simple, economical and industrial. The present invention also relates to analysis cards, the support of which is made from such a polymer.

Another essential objective is to provide a simple and economical method making it possible to modify the surface of certain wells or channels of the support of said card, by circulating an appropriate fluid.

The present invention therefore relates to a method for modifying, at least in part, the surface of the polymer constituting the support of an analysis card according to the invention, this method implementing a step of treatment of said surface, carried out at a temperature higher than the glass transition temperature of the polymer, with a medium capable of creating acid-base interactions with the reactive hydrophilic functions, this treatment step causing the surface migration of the reactive hydrophilic functions present in the mass of the polymer and , possibly, their hydrolysis into other reactive hydrophilic functions.

Another essential objective of the present invention is to provide a simple method making it possible to locally create hydrophobic or hydrophilic zones, in certain wells or channels of the support of the analysis card.

According to another of its aspects, the invention therefore also relates to a method for locally modifying the surface of the polymer constituting the support of an analysis card according to the invention. This process consists in particular in producing: hydrophobic zones on the surface of the polymer by carrying out on the zones to be modified: (1) a step of treating said surface, carried out at a temperature higher than the glass transition temperature of the polymer, with a medium capable of creating acid-base interactions with the reactive hydrophilic functions, this treatment step causing the surface migration of the reactive hydrophilic functions present in the mass of the polymer and possibly, their hydrolysis to other reactive hydrophilic functions, followed, ( 2a) a step of chemical grafting, on the reactive hydrophilic functions present on the surface, of hydrophobic groups,

- hydrophilic zones on the surface of the polymer by performing on the zones to be modified: (1) a step of treating said surface, carried out at a temperature higher than the glass transition temperature of the polymer, with a medium capable of creating acid-base interactions with the reactive hydrophilic functions, this treatment step causing migration into surface of the reactive hydrophilic functions present in the mass of the polymer and optionally, their hydrolysis into other reactive hydrophilic functions, optionally followed, (2b) by a chemical grafting step, on the reactive hydrophilic functions present on the surface, of biological ligands . Various other characteristics will emerge from the description given below, with reference to the accompanying drawings.

Fig. 1 shows a top view of an analysis card holder provided with a plurality of round bottom wells.

Fig. 2A shows a top view of an analysis card support provided with parallel channels and FIG. 2B a side view in section.

Fig. 3 shows a schematic top view of a branch between channels.

Fig. 4 represents the colorimetric detection results in the form of histograms, obtained during an ELOSA test. The object of the invention relates to the use of a particular polymer for the manufacture of analysis cards, said polymer having the following properties:

this polymer has a glass transition temperature of less than 60 ° C., preferably less than 37 ° C., and preferably less than 20 ° C., in the absence of functionalization, this polymer is hydrophobic, and

- For the envisaged application, it is functionalized in its bulk by reactive hydrophilic functions or generating reactive hydrophilic functions.

The use of such a polymer makes it possible to easily control the surface properties of the analysis card and in particular of its support, and to modify these properties on controlled areas. Indeed, the polymers used are, in the absence of functionalization, hydrophobic polymers. In addition, at a temperature above their glass transition temperature, the chains of the polymers used have high mobility. This property is known and is already used, for example in adhesion phenomena. Also, at a temperature higher than their glass transition temperature, if the polymer surface is brought into contact with a medium capable of creating acid-base interactions with the reactive hydrophilic functions or generating reactive hydrophilic functions present in the polymer, these functions will migrate from the mass of the rather hydrophobic character to its surface. The hydrophilic groups, then present at the surface, come from the material itself and not from an external chemical or physical treatment. Thus, unlike the silicon or polystyrene type surfaces used up to now, which require fairly heavy treatments intended to promote their reactivity, in particular physical treatments such as plasma treatment, flame treatment, or chemical treatments such as grafting, the polymers used in the present invention have intrinsic properties which make it possible to simply modify their surface.

For the present invention, the reference technique for measuring the glass transition (Tg) is described in "Engineering technique", volume AM2, number AM 3274.

As an example of hydrophilic functions according to the invention, mention may be made of the hydrazino, imino, azido, thiophosphate, hydrazono, sulfamoyl, thiocarboxy, optionally substituted amide, triazano, triazeno, amido, semicarbazono, carbamoyl, cyano, alkylthio, arylthio, sulfate groups. , sulfonate, phosphate, anhydride, epoxide, carboxylic acid, hydroxyl or amino ...

The anhydride, epoxide, carboxylic acid (-COOH), hydroxyl (-OH) or primary amine (-NH ₂ ) type functions are preferred reactive hydrophilic functions. These functions have the advantage of subsequently allowing grafting of hydrophobic molecules or biological ligands. The use of mass functionalized polymers makes it possible on the one hand to have a greater potential for available functions than in the case of a simple surface functionalization and on the other hand to use a greater diversity of hydrophilic functions compared to physical treatments, such as plasma treatment.

The present invention also relates to analysis cards, the support of which is made from such a polymer. In general, the fluid circuit of the analysis cards according to the invention comprises elements such as channels or microchannels, valves or intersections of channels (T-shaped, Y-shaped or cross-shaped ), parts of the circuit which allow incubation (wells or meandering channels) or separation (lengths of channels equivalent to columns for example). Fig. 1 is a top view of a simple example of a support 1 for an analysis card provided with a plurality of wells 2 with a round bottom. This support, dimension 65x35x4 mm ^3, has wells in the shape of a hemisphere with a diameter of 5 mm and a depth of 2.5 mm.

Figs. 2A and 2B represent another simple variant of an support 1 for an analysis card provided with parallel channels 3. This support of dimension 65 × 35 × 4 mm ³ has channels in the form of a half cylinder of 2 mm in diameter and a depth of 1 mm .

Depending on the type of analysis performed on the card, the shape of the analysis cards may vary. The analysis cards according to the invention can use a principle of separation by capillary electrophoresis.

According to another alternative embodiment of the cards, the fluids are conveyed, no longer electrically but by an external or integrated pump system, vacuum, syringe pump, or by centrifugal force as in the system described, for example, in the patent application published under the number WO 99/58 245. Another example of an analysis card is that presented in the patents

US 5,766,553 and US 4,318,994. These cards consist of around sixty wells, all connected to a common input by a set of microchannels. These wells are all filled simultaneously by evacuation followed by return to atmospheric pressure. Analysis cards can be obtained by precision molding of these so-called polymers but any other manufacturing method and in particular those used in semiconductor techniques, such as those described in the applications. Patent published under the numbers WO 97/02 357 and WO 00/78 452, can be used for the manufacture of these analysis cards.

According to a particular embodiment of the invention, the polymers used to manufacture the analysis cards have a glass transition temperature below 20 ° C. Thus, the polymer is rubbery and its chains are mobile, at room temperature.

By hydrophobic polymer in the absence of functionalization, is meant for example polymers whose contact angle with water is greater than or equal to 90 °.

These contact angles are, for example, determined by depositing, using a microsyringe, a drop of water on the flat surface of the polymer to be studied, then the contact angle is measured from the dimensions of the gout, according to a method well known to those skilled in the art.

Functionalized polyolefins, and in particular polyethylene, polypropylene or functionalized polyethylene / polypropylene copolymers are examples of polymers used according to the present invention.

In particular, polyethylene or polypropylene functionalized with acrylic acid, or preferably maleic anhydride is used. Advantageously, the polymer used contains up to 2%, preferably up to 0.3% by weight of maleic anhydride. Other polymers such as polyethylene imine (PEI), polyethylene vinyl acetate (PEVA), terpolymers of ethylene, acrylic esters and glycidyl methacrylate or maleic anhydride, sold respectively by the company ATOFINA under the names LOTADER ^® and OREVAC ^® can also be used. The hydrophilic reactive functions advantageously represent up to

3%, preferably up to 2% and, preferably, up to 0.3% by weight of the polymer used according to the invention.

Furthermore, the polymer used does not swell, neither in the presence of a polar solvent, nor in the presence of an apolar solvent. The present invention also relates to a method for modifying, at least in part, the surface of the polymer constituting the support of an analysis card according to the invention. Said method comprises a step of treating said surface, carried out at a temperature higher than the glass transition temperature of the polymer, with a medium capable of creating acid-base interactions with the reactive hydrophilic functions, this treatment step causing the surface migration of the reactive hydrophilic functions present in the mass of the polymer and optionally, their hydrolysis to other reactive hydrophilic functions.

As medium capable of creating acid-base interactions with the reactive hydrophilic functions, mention may be made, without limitation, of glycerol or ethylene glycol, or preferably polar or hydrophilic media, such as, for example, water or a liquid containing at least 50% by weight of water, such as a water / organic solvent mixture such as dimethyl sulfoxide.

According to the present invention, it is therefore very simple to modify the hydrophobic / hydrophilic nature of the surface of the support for analysis cards, the treatment being carried out by circulating or bringing the surface into contact with a medium capable of creating interactions. acid-base with reactive hydrophilic functions, for 1 to several hours, for example 15 hours. Indeed, the simple contact of the surface of the polymer with a medium capable of creating acid-base interactions with the reactive hydrophilic functions, and in particular with a polar or hydrophilic medium, leads to the migration of the hydrophilic chains present in the mass of the polymer , on the surface of it. In the particular case of polyolefins grafted with maleic anhydride, when a polar liquid such as water is used, this migration is accompanied by a hydrolysis of certain anhydride functions into -COOH functions.

This treatment can be followed by a chemical grafting step on the reactive hydrophilic functions present on the surface, of biological ligands. This grafting step can be preceded by a step of activating the reactive hydrophilic functions present on the surface.

By biological ligand is meant a compound which has at least one recognition site allowing it to react with a target molecule of biological interest. By way of example, mention may be made, as biological ligands, of polynucleotides, antigens, antibodies, polypeptides, proteins and haptens.

The term "polynucleotide" means a chain of at least 2 deoxyribonucleotides or ribonucleotides optionally comprising at least one modified nucleotide, for example at least one nucleotide comprising a modified base such as inosine, methyl-5-deoxycytidine, dimethylamino-5-deoxyuridine, deoxyuridine, diamino-2,6-purine, bromo-5- deoxyuridine or any other modified base allowing hybridization. This polynucleotide can also be modified at the level of the internucleotide bond such as for example phosphorothioates, H-phosphonates, alkyl-phosphonates, at the level of the backbone such as for example alpha-oligonucleotides (FR 2 607 507), or PNA ( Egholm M. et al., J. Am. Chem, Soc., 1992, 114, 1895-1897), or 2-O-alkyl ribose, or LNA (from “Loked Nucleic Acids”, described in particular in the patent application published under the number WO 00/66 604). Each of these modifications can be taken in combination. The polynucleotide can be an oligonucleotide, a natural nucleic acid or its fragment such as DNA, ribosomal RNA, messenger RNA, transfer RNA, nucleic acid obtained by an enzymatic amplification technique. By "polypeptide" is meant a chain of at least two amino acids.

By amino acids is meant the primary amino acids which code for proteins, amino acids derived after enzymatic action such as trans-4-hydroxyproline and natural amino acids but not present in proteins such as norvaline, N-methyl- L-leucine, Stalin (Hunt S. in Chemistry and Biochemistry of the amino acids, Barett GC, ed., Chapman and Hall, London, 1985), amino acids protected by chemical functions usable in synthesis on solid support or in liquid phase and unnatural amino acids.

The term "hapten" designates non-immunogenic compounds, that is to say incapable by themselves of promoting an immune reaction by production of antibodies, but capable of being recognized by antibodies obtained by immunization of animals in known conditions, in particular by immunization with a hapten-protein conjugate. These compounds generally have a molecular mass of less than 3000 Da, and most often less than 2000 Da and can be, for example, glycosylated peptides, metabolites, vitamins, hormones, prostaglandins, toxins or various drugs, nucleosides and nucleotides. The term "antibody" includes polyclonal or monoclonal antibodies, antibodies obtained by genetic recombination and antibody fragments. The term "antigen" designates a compound capable of being recognized by an antibody whose synthesis it has induced by an immune response. The term "protein" includes holoproteins and heteroproteins such as nucleoproteins, lipoproteins, phosphoproteins, metalloproteins and both fibrous and globular glycoproteins.

The stage of treatment with a medium capable of creating acid-base interactions with the reactive hydrophilic functions, conferring a hydrophilic character on the surface, can also be followed by a stage of chemical grafting on the hydrophilic functions present on the surface, of groups hydrophobic. Such grafting then confers hydrophobic properties on the surface, close to its initial properties. Silane groups such as thiolsilanes, such as mercaptopropyltrimethoxysilane, alkylsilanes, such as dimethylethoxysilane (DMES), w-octadecyltriethoxysilane, ODDMS (octadecyldimethylmethoxysilane), MDES (methyldiethethysilane) and methyldiethoxysilane) TFDMCS (trifluoropropyldimethylchorosilane) are examples of usable hydrophobic groups.

The steps of chemical grafting of hydrophobic or hydrophilic groups are carried out according to techniques well known to those skilled in the art.

The present invention also relates to a method for locally modifying the surface of the polymer constituting the support of an analysis card according to the invention. This process consists in carrying out:

- hydrophobic zones on the surface of the polymer by performing on the zones to be modified:

(1) a step of treating said surface, carried out at a temperature higher than the glass transition temperature of the polymer, with a medium capable of creating acid-base interactions with the reactive hydrophilic functions, this treatment causing the surface migration of reactive hydrophilic functions present in the mass of the polymer and optionally, their hydrolysis into other reactive hydrophilic functions, followed, (2a) of a chemical grafting step, on the hydrophilic functions present on the surface, of hydrophobic groups, - hydrophilic zones are produced on the surface of the polymer by carrying out on the zones to be modified: (1) a step of treating said surface, carried out at a temperature higher than the glass transition temperature of the polymer, with a medium capable of creating acid-base interactions with the reactive hydrophilic functions, this treatment causing the migration on the surface of the reactive hydrophilic functions present in the mass of the polymer and optionally, their hydrolysis into other reactive hydrophilic functions, optionally followed, (2b) by a step of chemical grafting, onto the hydrophilic functions present on the surface, of biological ligands, for example, chosen from polynucleotides, antigens, antibodies, polypeptides, proteins and haptens.

To locally modify the surface of the polymer, the use of masks or the circulation of fluids may, for example, be implemented.

The use of masks makes it possible to put only the non-masked surfaces in contact with solutions and therefore to only modify these. On the other hand, when the circulation of water or other solutions is used, the structure of the card must be suitable for modification limited to the desired zones. By playing on the shape of the branches or intersections between channels, it is possible to create valves. Fig.3 shows an intersection 4 arranged on an analysis card support according to the invention and comprising a first channel 5 and a second channel 7 meeting to form a common channel 6. The circulation of a hydrophilic fluid, by for example water, in channels 5 and 6 in the fl direction allows the hydrophilic chains present in the polymer to migrate to the surface, thus making the surface of channels 5 and 6 hydrophilic. Also, if an aqueous solution is routed in channel 6, in the opposite direction Î2, it will preferentially choose at the level of branch 4, channel 5, rather than channel 7 which has remained hydrophobic. The present invention therefore also relates to the analysis cards according to the invention, the support of which has a modified surface, at least in part, by virtue of the above method.

It is very important to locally control the hydrophobic / hydrophilic nature of the analysis cards. For example, in the case of analysis cards intended for samples containing proteins, it is known that certain proteins preferentially adsorb on hydrophobic surfaces. Also, to avoid losing proteins on the surface of channels or wells, it is important to modify their walls, so as to make them hydrophilic, possibly uncharged.

Similarly, for nucleic targets, a negatively charged hydrophilic surface preferentially prevents these molecules from adsorbing by electrostatic repulsion. On the contrary, it can also be envisaged to adsorb proteins on specific walls or zones. It is then necessary to make these zones hydrophobic. However, the adsorption of proteins on hydrophobic walls changes the nature of these.

To demonstrate the adsorption of biological ligands on the surface of the analysis cards and in particular of DNA targets, it is possible, for example, to inject an initial solution of targets of known concentration and recover the solution at the outlet of the fluid circuit. These two solutions are then subjected to a PCR type amplification reaction and deposited on an electrophoresis gel. The quantification is then carried out by comparing the intensity of the electrophoresis bands obtained. Fluorescent markers intercalating into double stranded DNA can also be used. By way of nonlimiting examples according to the invention, support for analysis cards according to FIG. 1 or FIG. 2 are produced by molding at 200 ° C., either of polyethylene granules functionalized with 0.26% by weight of maleic anhydride (reference: PEgAM XA 255 from the company SOLVAY), either of polypropylene granules functionalized with 0.05% by weight of maleic anhydride (reference: PRIEX GP 12 of the company SOLVAY), or of granules of polypropylene functionalized with 0.08% by weight of maleic anhydride (reference: PRIEX GP 13 from the company SOLVAY). The polyethylene used has a transition temperature glassy of the order of -120 ° C and the polypropylenes used have a glass transition temperature close to the order of -20 ° C. These polymers are therefore rubbery at room temperature.

The flow and the formation of bubbles after several passages on the analysis card supports according to the invention are studied visually, for example, by means of a camera and a microscope.

For practical reasons, the contact angle with water measurements, highlighting the various hydrophilic / hydrophobic surface properties obtained by the process according to the invention, are carried out on flat supports and not on these supports provided with wells or channels.

The examples below therefore illustrate the surface modification process according to the invention and demonstrate the hydrophilic / hydrophobic surface properties obtained.

EXAMPLES

EXAMPLE 1

Flat supports of dimension 25x15x0.5 mm ³ of polyethylene functionalized with 0.26% by weight of maleic anhydride (EXAMPLE 1), or of polypropylene functionalized with 0.05% (EXAMPLE 2) or 0.08% by weight ( EXAMPLE 3) of maleic anhydride, are carried out as follows:

The functionalized polymer is introduced, in the form of granules, into a steel or polytetrafluoroethylene (PTFE) mold and the assembly is inserted into a press, the plates of which are maintained at 200 ° C. After 10 minutes of stay, the support obtained is cooled, in the presence of an applied mass, intended to limit any deformation. The support is then placed between two glass slides grafted with n-octadecyl-triethoxysilane (OTES) (obtained by immersion of glass slides for 15 minutes in a sulfuric acid / hydrogen peroxide mixture at 30 °, 70/30, v / v, followed by rinsing with ultrapure water and immersion for 15 hours in a solution of OES 2% in anhydrous toluene) and reintroduced in an oven thermostatically controlled at 180 ° C. A mass is applied to this assembly and the oven is placed under primary vacuum, in order to limit the oxidation phenomena. After 1 hour 30 minutes, the vacuum is broken and the assembly is cooled slowly. The support is then washed for two hours with chloroform, then dried.

For comparison, supports made of hyper high density polyethylene (reference: PE-hhd ELTEX from the company SOLVAY) (PE) or polypropylene (reference: PP 3120 from the company APPRYL) (PP) are prepared, according to the same procedure, replacing the glass slides grafted with OTES, with simple glass slides.

The contact angles obtained when wetting the surface of the supports of EXAMPLES 1 to 3 with ultra pure water (θ _a ) and when wetting the surface (θ _r ) were measured. The hysteresis Δθ = θ _a -θ _r accounts for the reorganization of the surface with water. The results obtained with EXAMPLES 1, 2 and 3 are compared with those obtained with PE and PP supports.

The contact angles given in TABLE 1 below are average angles calculated from all the measurements made on a surface. They are supplemented by a standard deviation reflecting the heterogeneity of the surface studied.

TABLE 1

The contact angles obtained for the non-functionalized polyolefins correspond to the values found in the literature.

The contact angles obtained for EXAMPLES 1, 2 and 3, both at wetting and dewetting, are lower than those obtained with non-functionalized polyolefins. This fairly significant decrease is attributed to the presence of polar groups of maleic anhydride type or -COOH present on the surface and this, despite low incorporation rates (0.26% maximum for EXAMPLE 1).

In addition, the increase in Δθ obtained with EXAMPLES 1, 2 and 3 compared to the corresponding non-functionalized polyolefins, proves the reorganization of the surface groupings of the supports under the action of a medium capable of creating acid-base interactions : under the drop of water, the maleic anhydride functions are attracted to the surface and move towards the liquid and some of these maleic anhydride functions are hydrolyzed to give acid -COOH functions.

EXAMPLE 4

On the support of EXAMPLE 1, pure water is circulated at room temperature, then this support is immersed, still at room temperature, for 15 hours, in a solution containing 2% by weight of dimethylethoxysilane (DMES ) in anhydrous toluene.

Before measuring the contact angles, the supports are rinsed with chloroform to guarantee the extraction of the polycondensates of silane physisorbed on the surface of the polymer.

The contact angles of the water obtained are presented in TABLE 2 below.

TABLE 2

These results show that the grafting of DMES makes it possible to obtain a surface having a hydrophobic character close to that obtained with a support made of unmodified polyethylene (PE). EXAMPLE 5

Five different treatments are carried out, at room temperature, on the support of EXAMPLE 1, with the aim of characterizing the adsorption and / or coupling of oligonucleotides on the functionalized polymer:

EXAMPLE 5 A: EXAMPLE 1 activated by carbodiimide

EXAMPLE 5B: EXAMPLE 1 treated by immersion in water for 15 hours

EXAMPLE 5C: EXAMPLE 1 treated by immersion in water for 15 hours and activated with carbodiimide.

EXAMPLE 5D: EXAMPLE 1 treated by immersion in an aqueous solution of potassium hydroxide (> 10 μM) for 15 hours.

EXAMPLE 5E: EXAMPLE 1 treated by immersion in an aqueous solution of potassium hydroxide (> 10 mM) for 15 hours and activated with carbodiimide.

EXAMPLES 5A to 5E are subjected to the ELOSA test (from the English "Enzyme-Linked OligoSorbent Assay", reference may be made to F. Mallet et al., J. Clin. Microbiol., 1993, 31 (6), 1444 -1449 and WO 91/19812), for detection of nucleic acids on solid support This test, based on molecular hybridization between oligonucleotides and target nucleic acids, is carried out, in the present case, to detect the presence or not of a "capture probe" on a solid support. A target nucleic acid having a sequence complementary to that of the oligonucleotide (capture ODN) is brought into contact with the analysis card. The complementarity of the sequences results in a hybridization of the two molecules. A detection ODN is then added. It hybridizes to a sequence complementary to the target, different from the first. The enzyme then catalyzes the transformation of a substrate into a detectable product, the concentration of which is proportional to the target concentration.

The target ODN used of SEQ ID no.1 includes 78 bases: SEQ ID no.1: 5 * -AAG TAT CCC CAT AAG TTT CAT AGA TAT ATT GTT CTA AGC TAT GGA GCC ATA TCC TAG GAA AAT GTC TAA CAG CTT CAC -3 'The capture ODN used in SEQ ID No. 2 comprises 20 bases and comprises an amino arm in position 5', as described in the patent application published under the number WO 91/19812: SEQ ID No. 2: 5 * - TAT GAA ACT TAT GGG GAT AC -3 'This test comprises four stages: a stage of immobilization of the capture ODN, a stage of incubation of the target on the immobilized capture ODN, a stage of incubation of the detection ODN on the target and a colorimetric analysis step, corresponding to the enzymatic reaction. This test is carried out as follows. A step of immobilizing the capture ODN corresponds to bringing the capture probe into contact with the polymer support. The capture probe used is terminated by a primary aliphatic amine function. In the case of coupling with an anhydride function at the surface, coupling is ensured by the direct reaction of this function with the primary amine function of the capture ODN. For the case, where the groups present on the surface are -COOH functions resulting from the hydrolysis of anhydrides, an activating agent such as l-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) is used.

Typically, 40 μl of a solution containing 0.05 pmole / μl of capture ODN, in the presence or not of 0.2 pmole / μl of EDC in 10 raM Phosphate buffer, pH 6.8 are placed in contact with the surface for 1 hour. The support is then washed with the washing buffer (0.05 M phosphate buffer, 0.15 M NaCl, 0.05% Tween 20, pH 7.2) in order to remove the weakly absorbed biomolecules. This pad is used in all washing steps.

Incubation of the target is carried out by depositing, for 1 hour, 40 μl of a solution containing 10 ¹² copies / ml of hybridization buffer (0.06 M phosphate, 0.9 M NaCl, salmon sperm DNA 50 mg / ml, milk powder 3% by weight, Triton 0.05%, pH 7.4). At the end of the incubation, the support is rinsed with the washing buffer. The detection ODN of SEQ ID No. 3 is a conjugate consisting of a 23 base nucleic acid fragment complementary to the target and grafted in position 5 ′ on an enzyme, Ηorse Radish Peroxidase "or HRP, as described in the patent application published under the number WO 91/19812: SEQ ID No. 3: 5'-GAA GCT GTT AGA CAT TTT CCT AG -3 '

Hybridization of this conjugate on the immobilized target is carried out by incubating, for 1 hour, 40 μl of a detection ODN solution at 0.05 pmol / ml of hybridization buffer. At the end of the incubation, the support is rinsed with the washing buffer. The colorimetric analysis is carried out as follows. The samples are placed in the presence of the colorimetric substrate of the HRP, ortho-phenylene diamine or OPD. Initially colorless, it is transformed by HRP into a colored product, orthonitroaniline. After 10 minutes of development, the enzymatic reaction is stopped by adding a solution of sulfuric acid IN in the medium. The optical density (OD) of the coloration, proportional to the quantity of enzyme, and therefore to the quantity of hybridized targets, is measured at 492 nanometers.

This test therefore makes it possible to characterize the adsorption and / or coupling of oligonucleotides on the polymer studied. It also makes it possible to determine the nature of the chemical functions present on the surface of the samples. The presence of the detection probe in fact reflects the prior presence of the capture probe on the surface (whether it is adsorbed or coupled), therefore the presence of a reaction site.

The results obtained after ELOSA test on EXAMPLE 1 and EXAMPLES 5A to 5E by colorimetric detection are presented in FIG. 4. These results prove that -COOH functions, present on the surface of the supports of EXAMPLES 5C and 5E, are coupled with biomolecules. These results also demonstrate that the treatment of the surface of the polymer functionalized with maleic anhydride with water or a potassium hydroxide solution attracts the functions of maleic anhydride from the volume to the surface and then hydrolyzes them into -COOH functions. .

Claims

1 - Use of a polymer for the manufacture of analysis cards, characterized in that the polymer: - has a glass transition temperature below 60 ° C, - is hydrophobic in the absence of functionalization, and - is functionalized in its bulk by reactive hydrophilic functions, or generating reactive hydrophilic functions. 2 - Use according to claim 1, characterized in that the polymer is functionalized in its mass by reactive hydrophilic functions, or generating reactive hydrophilic functions chosen from the anhydride, epoxide, carboxylic acid, hydroxyl or primary amine functions. ^{^} 3 - Use according to claim 1 or 2, characterized in that the polymer has a glass transition temperature below 37 ° C, preferably below 20 ° C. 4 - Use according to claim 3, characterized in that the polymer used is a functionalized polyolefin. 5 - Use according to claim 4, characterized in that the polymer used is a functionalized polyethylene. 6 - Use according to claim 4, characterized in that the polymer used is a functionalized polypropylene. 7 - Use according to any one of claims 1 to 6, characterized in that the polymer is functionalized with maleic anhydride. 8 - Use according to claim 7, characterized in that the polymer contains up to 2%, preferably up to 0.3% by weight of maleic anhydride. 9 - Analysis card whose support is made from a polymer, characterized in that this polymer: - has a glass transition temperature below 60 ° C, and - is hydrophobic in the absence of functionalization, and - is functionalized in its bulk by reactive hydrophilic functions, or generating reactive hydrophilic functions. 10 - Analysis card according to claim 9 characterized in that the polymer is functionalized in its mass by reactive hydrophilic functions, or generating reactive hydrophilic functions chosen from the anhydride, epoxide, carboxylic acid, hydroxyl or primary amine functions. 11 - Analysis card according to claim 9 or 10, characterized in that the polymer has a glass transition temperature below 37 ° C, preferably below 20 ° C. 12 - Analysis card according to claim 11, characterized in that the polymer used is a functionalized polyolefin. 13 - Analysis card according to claim 12, characterized in that the polymer used is a functionalized polyethylene. 14 - Analysis card according to claim 12, characterized in that the polymer used is a functionalized polypropylene. 15 - Analysis card according to any one of claims 9 to 14, characterized in that the polymer is functionalized with maleic anhydride. 16 - Analysis card according to claim 15, characterized in that the polymer contains up to 2%, preferably up to 0.3% by weight of maleic anhydride. 17 - Method for modifying, at least in part, the surface of the polymer constituting the support of an analysis card according to any one of claims 9 to 16, characterized in that it comprises a step of treatment of said surface , carried out at a temperature higher than the glass transition temperature of the polymer, with a medium capable of creating acid-base interactions with the reactive hydrophilic functions, this treatment step causing the migration on the surface of the reactive hydrophilic functions present in the mass of the polymer and optionally their hydrolysis to other reactive hydrophilic functions. 18 - Process according to claim 17, characterized in that the medium capable of creating acid-base interactions with the reactive hydrophilic functions is a liquid containing at least 50% by weight of water. 19 - Process according to claim 17 or 18, characterized in that the treatment step is followed by a chemical grafting step, on the reactive hydrophilic functions present on the surface, of biological ligands. 20 - Process according to claim 19, characterized in that the biological ligands are chosen from polynucleotides, antigens, antibodies, polypeptides, proteins, and haptens. 21 - Process according to claim 17 or 18, characterized in that the treatment step is followed by a chemical grafting step, on the hydrophilic functions present on the surface, of hydrophobic groups. 22 - Process according to claim 21, characterized in that the hydrophobic groups are chosen from thiolsilanes and alkylsilanes. 23 - Method for locally modifying the surface of the polymer constituting the support of an analysis card according to any one of claims 9 to 16, characterized in that: (1) a step of treating said surface, carried out at a temperature higher than the glass transition temperature of the polymer, with a medium capable of creating acid-base interactions with the reactive hydrophilic functions, this treatment step causing migration into surface of the reactive hydrophilic functions present in the mass of the polymer and optionally, their hydrolysis into other reactive hydrophilic functions, followed, (2a) of a chemical grafting step, on the reactive hydrophilic functions present on the surface, of hydrophobic groups, hydrophilic zones on the surface of the polymer by carrying out on the zones to be modified: (1) a step of treatment of said surface, carried out at a temperature above the glass transition temperature of the polymer, with a medium capable of creating acido interactions -basic with the reactive hydrophilic functions, this treatment step causing the surface migration of the reactive hydrophilic functions present in the mass of the polymer and possibly, their hydrolysis into other reactive hydrophilic functions, optionally followed, (2b) by a step of chemical grafting, on the reactive hydrophilic functions present on the surface, of biological ligands. 24 - Process according to claim 23, characterized in that in the treatment step (1), the medium capable of creating acid-base interactions with the reactive hydrophilic functions is a liquid containing at least 50% by weight of water. 25 - Analysis card according to any one of claims 9 to 16, characterized in that its support has a modified surface, at least in part, according to the method of any one of claims 17 to 24.