WO2005039751A1

WO2005039751A1 - Elastomeric microplates, supporting reactive ligands and production method thereof

Info

Publication number: WO2005039751A1
Application number: PCT/FR2004/002678
Authority: WO
Inventors: Christophe Marquette; Loïc BLUM
Original assignee: Universite Claude Bernard Lyon I; Centre National De La Recherche Scientifique (C.N.R.S.)
Priority date: 2003-10-20
Filing date: 2004-10-20
Publication date: 2005-05-06
Also published as: FR2861178A1; FR2861178B1

Abstract

The present invention relates to microplates with two large faces, one of said faces, called the active face, supporting reactive ligands on localised sites, which are accessible for subsequent reactions. Said microplate is characterised in being substantially made of an elastomer, preferably PDMS, which partially encloses particles of micrometre scale in the region of the active face, the reactive ligands being immobilised on the surface or inside thereof. The invention also relates to the production method thereof.

Description

LIGAND CARRYING ELASTOMER MICROPLATES

REAGENTS, AS WELL AS THEIR PREPARATION PROCESS The present invention relates to the technical field of microplates, and more particularly to support for analysis cards, commonly called microplates. 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 the 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. As examples of application of analysis techniques, mention may be made of immunoassays, detection and / or quantification of nucleic acids, enzymatic tests, etc. In terms of implementation, these analysis cards can be presented under different shapes, for example square, rectangular or circular. These cards consist of a support and possibly a cover. The support, also called microplate, is often provided with a plurality of wells and / or channels in which the different analysis processes are implemented. The supports for analysis cards and microplates are generally produced by machining of materials chosen from glass, quartz, silicon, optionally coated with a layer of oxide or nitride or else in polymers of the polystyrene type or plastic materials of the polycarbonate or hydrocarbon polymer type such as polyolefins, having a hydrophobic surface. Most often, to allow the functionalization of the support with the desired biological ligands, it is necessary, beforehand, to physically or chemically modify the surface of the support. These modifications are very often cumbersome to implement and depend on the nature of the biological ligand which one wishes to graft. The grafting or immobilization step, which risks altering the biological ligands, it is often necessary to control the "quality" of the biological ligands on the final functionalized microplate. In this context, one of the objectives of the present invention is to provide an easy, inexpensive, easily industrializable microplate manufacturing process, adapting to different types of reactive ligands. Another objective of the invention is to provide a method allowing the microplate to be functionalized differently on certain localized sites. To achieve the above-mentioned objectives, the present invention relates to a process for the manufacture of an elastomer microplate having two large faces, one of which, called the active face, carries, on localized sites, at least at least one type of reactive ligand, and which comprises the following successive steps: a) depositing on an imprint intended to serve as a countermold for the large active face, according to localized sites, an aqueous solution containing: - an organic solvent whose temperature d boiling and viscosity are higher than those of water, - a salt, capable of co-crystallizing with the organic solvent during step b) later, and - micrometric particles on the surface or inside of which reactive ligands are immobilized; b) evaporating the aqueous solution, at a temperature T sufficient but lower than the boiling temperature of the organic solvent and the degradation temperature of the reactive ligands, so as to obtain on the localized sites a co-crystallization of the organic solvent and the salt around micrometric particles; c) polymerizing the elastomer on the impression serving as a countermould for the microplate, so that it covers the localized sites and conforms to the shape of the impression; d) demolding the microplate into the elastomer obtained, the micrometric particles being partially trapped on the active face of the microplate, at the localized sites, at least part of the reactive ligands being accessible for a subsequent reaction. According to another of its aspects, the present invention relates to a microplate having two large faces, one of which, called the active face, carries, on localized sites, reactive ligands accessible for subsequent reactions. This microplate essentially consists of an elastomer which partially traps, at the level of the active face, micrometric particles on the surface or inside of which the reactive ligands are immobilized. The description below, with reference to the appended figures, will allow a better understanding of the invention. Fig. 1 is a diagram illustrating steps (A), (B) and (C) of the manufacture of a microplate with a low density network of localized sites. (D) is the micrograph of the network revealed by chemiluminescence. Fig. 2 illustrates different variants of microplates according to the invention, functionalized with different biological ligands. Fig. 3 is a diagram illustrating steps (A), (B) and (C) of the manufacture of a microplate with a network of localized active fluid sites. (D) is the micrograph of the network revealed by chemiluminescence. Fig. 4 illustrates the performance of microplates according to the invention, functionalized with different biological ligands. The microplates, produced according to the process of the invention, can be of different shapes: square, rectangular, in the form of a disc. They have two large faces: one called inert which is generally planar and one called "active" on which are fixed reactive ligands accessible for subsequent reactions. The first step of the process, called step a), consists in depositing on an imprint and on localized sites, an aqueous solution containing microbeads carrying reactive ligands. The impression will serve as a counter-mold for the face

"Active": it can therefore be flat or non-planar and include, in particular, the negative footprint of wells or channels. Preferably, this imprint will be made of a material having good wettability, such as PNC. The deposited solution contains an organic solvent and a salt intended to co-crystallize around the microbeads and thus protect them during the subsequent stages of the process. The organic solvent must have a boiling point and a viscosity higher than those of water. Preferably, the organic solvent has a boiling temperature, at atmospheric pressure, greater than 150 ° C. Its viscosity is high and more particularly, its Brookfield viscosity measured at 20 ° C. will be greater than 100 cps (centipoise). As organic solvent suitable for the process of the invention, mention may be made of the tween® series, in particular tween®20 (polyoxyethylene sorbitan monolaurate), tween®80 (polyoxyethylene sorbitan monooleate), the series of triton® ( alkylarylpolyether alcohol), in particular triton®X100, polyethylene glycol diacids, glycerol, etc. Glycerol is preferably used as organic solvent and sodium chloride (ΝaCl) as salt. Advantageously, a glycerol / ΝaCl molar ratio of between 0.05 and 0.1 is used. By micrometric particles is meant particles whose average diameter is less than or equal to 1000 μm. The micrometric particles used have, for example, an average diameter of less than 500 μm, preferably between 1 and 150 μm. The expression “micrometric particles” includes particles of nanometric size, in particular of diameter less than 1 μm. These micrometric particles are advantageously made of a polymer, such as latex, polysaccharides, or any other material, such as glass for example. The particles can be of any shape, the particles of spherical shape, called beads or microbeads are, however, preferred. Reactive ligands are immobilized on the surface or inside these particles. Certainly, preferably, the reactive ligands are biological ligands. However, it is possible to envisage using other ligands of the pesticide, heavy metal, polymer, synthetic receptor type, etc. 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 of polynucleotides, antigens, antibodies, polypeptides, proteins, haptens and active living cells. 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, at the level of the skeleton. 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, a ribosomal RNA, a messenger AKN, a transfer RNA, a nucleic acid obtained by an enzymatic amplification technique. By "polypeptide" is meant a chain of at least two 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, enzymes. Within the meaning of the invention, the term “immobilized” designates any type of interaction, such as electrostatic interactions, Van interactions Der Waals, hydrogen bonds, hydrophobic bonds, complexations, covalent chemical bonds, molecular reactions, physical adsorptions, allowing immobilization. Immobilization can therefore be carried out by any means. The immobilization can be carried out so that the reactive ligands remain on the surface of the particle or else are therein, for example trapped in the pores while remaining accessible for subsequent reactions. The reactive ligands can in particular be chemically grafted onto the particles, by conventional coupling, optionally preceded by an activation step. In this case, before coupling, the particles carry functional groups such as carboxylic, amino, thiol, aldehyde, hydroxyl, tosyl, hydrazine, epoxide or anhydride groups capable of grafting at least one reactive ligand. In the case of enzymes, immobilization on the particles is most often carried out by electrostatic bonding, but can also be of a covalent nature. We can in particular refer to Biosens. & Bioelectron. (2000), 15, 125-133; Anal. Lett. (2000), 33 (9), 1779-1796 and Sensors and actuators B (2003), 90 (1-3), 112-117 which describe examples of such functionalized particles. Functionalization therefore takes place directly on the particles and not in the final stage on the microplate. As a result, it is possible to carry out a variety of chemistry upstream and to better control the nature and quality of the reactive ligands present on the surface of the microplate. The deposition of the solution containing the particles is done simply by depositing drops on the desired localized sites. In general, when the imprint is not flat, the deposits are made, advantageously, at the level of the counterprints of the wells or microchannels. It is possible to deposit a solution containing different particles, for example, carrying different reactive ligands. Another variant also consists in depositing a solution containing particles carrying certain reactive ligands on certain localized sites and another solution containing other particles carrying other reactive ligands at other localized sites. The second step of the process, called step b), consists in heating the surface of the imprint on which the drops are deposited, in order to evaporate the phase aqueous and obtain the co-crystallization of the solvent and the salt. The latter surround the particles, thus ensuring their protection, especially during the next step of the process. The heating temperature must be sufficient to obtain this co-crystallization, but lower than the boiling point of the organic solvent and the degradation temperature of the reactive ligands, under the pressure conditions used. In general, this temperature will be of the order of 70-80 ° C, for heating carried out at atmospheric pressure. A Tungsten lamp could, for example, be used for heating. The third step, called step c), consists in molding the elastomer on the surface of the imprint functionalized with the particles. An elastomer can be defined as a macromolecular material which, at room temperature, is able to recover its initial shape and size after being deformed by a stretching force. Its molding on a three-dimensional impression makes it possible to obtain high form factors. Mention may be made of elastomers of the polyurethane (in particular marketed by A.SMA), polyether (in particular marketed by NUTEC), polyester (in particular marketed by Harvest), SU-8 photoresin or polydimethylsiloxane (PDMS) type. PDMS is particularly preferred. The polymerization is most often carried out by deposition on the imprint of a solution containing a prepolymer of the elastomer, in combination with a polymerization catalyst, followed by heating. Such solutions include commercially available: one can, for example, cited in the case of PDMS, SYLGARD ^® 184 marketed by Dow Corning. In the case of PDMS, this polymerization is carried out by heating at a temperature between 70 and 90 ° C and for a period between 20 and 30 min. Heating is preceded by degassing under reduced pressure. The conditions are chosen by a person skilled in the art to obtain the polymerization of the chosen elastomer. By polymerizing, the elastomer, and in particular PDMS, partially traps the particles. The solvent and the salt dissolve in the PDMS. Once the polymerization is complete, it is cooled to harden the elatomer and allow release from the mold. Thus, after demolding, a microplate is obtained having two large faces, one of which, called the active face, is a carrier, on localized sites, of ligands reagents accessible for subsequent reactions. This microplate essentially consists of an elastomer which partially traps, at the level of the active face, micrometric particles on the surface of which the reactive ligands are immobilized. In general, the relief of the non-planar microplates according to the invention may include a fluid circuit based on elements such as channels or microchannels, valves or intersections of channels (T-shaped, shaped Y or cross-shaped), parts of the circuit which allow incubation (wells or meandering channels) or separation (lengths of channels equivalent to columns for example). These microplates constitute another aspect of the invention. Their thickness preferably varies between 1 and 5 mm. Advantageously, the micrometric particles emerge, for the most part, from the active face of the microplate at least 10% of their diameter. The method according to the invention makes it possible to prepare compartmentalized microplates. Indeed, it is possible to deposit particles of different nature, that is to say carriers of different reactive ligands, on certain localized sites, depending on the intended application for the microplate. In the field of diagnostics, the method according to the invention makes it possible to manufacture microplates capable of carrying out different analyzes on a sample. The examples below illustrate the invention without, however, limiting it. Anti-human IgG antibodies (specific for the γ chain) labeled with biotin, d (T) ₂ oligonucleotides labeled with biotin, glucose oxidase (Gox, quality I, EC 1.1.3.4, from Aspergillus niger), IgG human, luminol (3-aminophthalhydrazide), streptavidin labeled with peroxidase, Sepharose beads carrying human IgG, Sepharose beads bearing d (A) ₂ were purchased from Sigma (France). DEAE (diethylaminoethyl) Sepharose Fast Flow and IDA (imidodiacetic acid) Sepharose Fast Flow were obtained from Pharmacia. The PDMS precursor and the curing agent (Sylgard®184) were supplied by Dow Corning. All buffers and aqueous solutions were prepared with distilled deionized water. Example 1: Preparation of bioactive microbeads la) Microbeads carrying glucose oxidase (Gox) were prepared by interaction of IDA-Sepharose loaded with Ni with Gox modified with histidine. The grafting of histidine on Gox was carried out using a method described previously for modifying horseradish peroxidase (T NC Safack, CA Marquette, F. Pizzolato and LJ Blum, Biosens. & Bioelectron., 15 (2000) 125 ). Briefly, 5 mg of Gox dissolved in 200 μl of distilled water and 80 μl of a 0.1 M solution of ΝaIO ₄ were added and mixed with stirring for 20 min. Then, 40 μl of a 0.2 M solution of histidine in 0.1 M carbonate buffer, pH 9, and 480 μl of 0.1 M carbonate buffer, pH 9, were added to the activated glucose oxidase and incubated for 2 h with shaking. At this time, the reaction was complete and 20 μl of 0.1 M NaBH solution in freshly prepared water were added and mixed for 15 min to stabilize the binding of histidine to the activated Gox. The Gox-His was then separated from the unreacted species by removing the salts on a column of Sephadex G25-M. Then, 500 μl of IDA-Sepharose microbeads were equilibrated with 30 mM Neronal buffer, 30 mM KC1, pH 8.5 and perfused with a 100 mM ΝiCl ₂ solution. On the Ni-IDA-Sepharose column thus obtained, 5 mg of Gox-His freed from the salts as described above were deposited. The column was then washed with 10 ml of equilibration buffer and stored at 4 ° C. lb) Luminol was immobilized on DEAE-Sepharose microbeads by electrostatic interactions. 500 μl of DEAE-Sepharose suspended in ethanol were introduced into a plastic column, equilibrated with 10 ml of 30 mM Neronal buffer, 30 mM KC1, pH 8.5 and perfused with 1 ml of 10 mM luminol solution in balancing buffer. The column was then washed with 25 ml of equilibration buffer and stored at 4 ° C.

Example 2: Preparation of microplates 2a) Microplates were prepared by depositing, on localized sites forming a network, drops of 0.3 μl of deposition solutions. The deposition solutions were prepared by mixing 45 .mu.l of an aqueous solution composed of 2.5% glycerol, 0.05 M ΝaCl with 5 .mu.l of microbeads wet laying d (A) _2, 5 .mu.l of microbeads carrying human IgG or 5 μl of microbeads carrying glucose oxidase with the addition of 5 μl of microbeads carrying luminol. These different types of functionalization, as well as their mode of disclosure are illustrated in FIG. 2. (A) A Sepharose bead carrying an oligonucleotide emerges from the surface of the PDMS. A biotinylated tracer is hybridized to the immobilized nucleic acid and is revealed with streptavidin labeled with peroxidase. (B) A Sepharose ball modified with a protein is included in the PDMS polymer. An antibody labeled with biotin reacts with the immobilized antigen and is revealed with streptavidin labeled with peroxidase. (C) A ball of IDA-Sepharose carrying glucose oxidase (Gox) and a ball charged with luminol emerge from the surface of the PDMS. Gox oxidizes the glucose present in the reaction medium, producing H ₂ O which, for its part, reacts with the immobilized luminol to produce light. 2b) To form a low density network, drops of 0.3 μl were deposited on a flat PNC substrate every 3 mm and dried under a tungsten lamp for 30 min. The presence of glycerol in the deposition solution makes it possible to obtain, on localized sites, deposits of homogeneous and uniform dry microbeads. The dry microbead deposits were then transferred to the PDMS interface by pouring a mixture of precursor and curing agent onto the PNC substrate and curing for 90 min under a tungsten lamp. The preparation of the microplates was then completed by separating the PDMS polymer. These different stages are illustrated in FIG. 1 (A), (B) and (C). After removal of the solidified PDMS, an emergence of the microbeads appeared on the surface of the polymer in coincidence with the localized sites. No residue of microbeads was observed on the PNC matrix after the removal of the PDMS layer, which shows the high efficiency of the transfer of microbeads at the PDMS interface. These islands of microbeads are therefore capable of anchoring bioactive molecules at the interface, by means of Sepharose microbeads, as illustrated in FIG. 2. 2c) In a similar manner, microplates comprising channels, for a fluidic application, hereinafter called active fluid microplate, were prepared by depositing drops of 0.3 μl of different deposition solutions (see paragraph 2a)) on a PNC matrix composed of 12 channels (width: 2.5 mm, height: 1 mm, length: 15 mm), prepared by fixing different together PNC parts with epoxy adhesive, and following the same protocol as that presented in paragraph 2b). Separating the PDMS substrate and covering it with a flat PVC surface allows 12 active channels to be obtained. These different stages are illustrated in FIG. 3 (A), (B) and (C).

EXAMPLE 3 Test for Microplates Functionalized with Nucleic Acids A pair of d (T) ₂₂ -d (A) ₂₂ was used as a model of the oligonucleotide hybridization method. The low density microplates with active fluidics were tested according to the same general protocol. Briefly, the microplate or a particular channel was saturated for 30 min with a buffer solution A (Neronal (diethyl barbiturate) 30 mM, pH 8.5 containing 30 mM KC1, 0.2 M ΝaCl, 0, 1% Tween and 1% BSA), then incubated for 1 h with d (T) ₂₂ biotinylated at a particular concentration in buffer A. The microplate or a particular channel was then washed 10 min with buffer B (Neronal 30 mM, pH 8.5 containing 30 mM KC1, 0.2 M ΝaCl), then saturated with buffer A for 20 min and then incubated with streptavidin labeled with peroxidase at a concentration of 1 μg / ml for 30 min. The microplate or a particular channel was then washed for 20 min with buffer B after which it was ready for measurement.

Example 4 Test for Microplates Functionalized with Proteins In a similar manner to that used for the microplate functionalized with nucleic acids, the microplates functionalized with proteins were saturated for 30 min with a solution of buffer A, then incubated for 1 h with a biotinylated anti-IgG antibody at a particular concentration in buffer A. The microplates were then washed for 10 min with buffer B, saturated with buffer A for 20 min, then incubated with streptavidin labeled with peroxidase at a concentration of 1 μg / ml for 30 min. The microplates were then washed for 20 min with buffer B after which they were ready for measurement. Example 5: Measurement on the Microplates The microplates with nucleic acid and proteins ready for the measurement were brought into contact with a buffered solution (Neronal 30 mM, KC1 30 mM, pH 8.5) containing in addition 200 μM of luminol, 500 μM of hydrogen peroxide and 20 μM of jσαra-iodophenol. At this time, the microplates were introduced into the CCD (Intelligent Dark Box II, Fuji Film) light measurement system and the emitted light was integrated for 3 min. The enzyme substrate microplate was used in a similar manner: a buffered solution (Neronal 30 mM, KC1 30 mM, pH 8.5) containing a particular concentration of glucose and 200 μM of triggering agent (1.3 -dichloro-

5,5-dimethylhydantoin) was injected into each channel just before the introduction of the microplate into the measurement system, and the signal was integrated for 3 min. The microplate images obtained were quantified with a FUJIFILM image analysis program (Image Gauge 3.12).

Example 6: Results 6a) Low density microplates The network of localized low density sites was used to design microplates functionalized with proteins or oligonucleotides based on Sepharose microbeads carrying a human IgG or a homooligonucleotide d (A). Fig. 1 (D) presents a micrograph of a network of 100 d (A) _{22 points} revealed by chemiluminescence, with a streptavidin-peroxidase conjugate after hybridization of the microplate with a d (T) ₂₂ labeled with biotin. An 8% standard deviation of the chemiluminescent signal was found in the network. Similar results were obtained when the protein network, composed of immobilized human IgG, was incubated with anti-human IgG antibodies labeled with biotin.

It then appeared that the present process of microplate preparation allows to obtain a homogeneous network of the two most studied types of biomolecules. In addition, a modification of the properties of the microbeads used could lead to immobilization of biomolecules on a larger scale. Thus, an ohgosaccharide (S.

Fukui, T. Feizi, C. Galustian, A.M. Lawson and W. Chai, Nature Biotech., 20 (2002)

1011) or an inase substrate (BT Houseman, JH Huh, SJ Kron and M. Mrksich, Nature Biotech., 20 (2002) 270) could be immobilized easily and used as analytical tools. The non-specific signal produced by the non-specific adsorption on the PDMS or Sepharose material was evaluated by preparing a microplate with unmodified microbeads (carrying no biological ligand) and by carrying out the test on a protein or a nucleic acid in the presence a maximum amount of labeled antibody or labeled nitric acid (100 μg and 7 pmol respectively). An extraordinarily weak non-specific signal, close to the instrument sensitivity limit, was observed. With the microplates according to the invention) ^' , there is therefore a small standard deviation and a substantially non-existent background noise.

6b) Quantitative measurement with active fluidic microplates A first study was carried out to assess the ability of the microplate to quantitatively detect a nucleic acid. Samples with different concentrations of biotin-labeled d (T) ₂ were injected into each particular channel, hybridized and revealed. Fig. 4 (A) shows the evolution of the chemiluminescent signal obtained as a function of the amount of oligonucleotide introduced into each channel. The measurements on the microplates were carried out for 3 min and the image was quantified with a FUJIFILM quantification program. B ₀ represents the light intensity obtained in the absence of free IgG and B represents the light intensity obtained in the presence of a particular concentration of IgG. In fact, the nucleic acid microplate allows the quantitative detection of quantities of biotinylated oligonucleotides in the range of 58 pM-23 nM. In relation to the detection limits obtained with fluorescence systems (D. Trau, TMH Lee, AIK Lao, R. Lenigk, I. Hsing, NY Ip, MC Caries and NJ Sucher, Anal. Chem., 74 (13) ( 2002) 3168 and JR Epstein, M. Lee and DR Walt, Anal. Chem., 74 (8) (2002) 18366) or based on radiolabelling (H. Salin, T. Vujasinovic, A. Mazurie, S. Maitrejean, C. Menini, J. Mallet and S. Dumas, Nucl. Acids Res., 30 (2002) el7), generally involving very complex microarray arrangements, the present method has a slightly lower sensitivity but a wider detection domain. . The present detection limit of 58 pM corresponds to an amount of 10 ¹⁰ molecules in a test volume of 300 μl. However, when the microplate is used in a low density array format, a sample volume of 3 μl can be used, which reduces the number of nucleic acid molecules detected to 10 ⁸ , which is close to the best results obtained in previous work. In a later study, the performance of a microplate functionalized with proteins to detect a free antigen in a competition format was evaluated. Samples containing human IgG at different concentrations were incubated in a particular channel, together with anti-IgG antibodies labeled with biotin. The chemilumiscent reading of the biochip leads to the obtaining of the competition curve, which is a linearization by the "logit" function, represented in FIG. 4 (B). lo & t ≈ lnB / βo-B) where B is the signal obtained at a fixed free antigen concentration and B ₀ is the signal obtained in the absence of free antigen. The protein-functionalized microplate allows the detection of a free antigen with a detection limit of 12 ng / ml and a detection domain which spans at least four decades. This amount corresponds to 1.5 x 10 ⁹ molecules in the active fluid format and could be lowered, as above, to 1.5 x 10 molecules in the low density format. To demonstrate all the potential of active fluid microplates, an enzyme-based system in which chelating microbeads carrying glucose oxidase (S. Fukui, T. Feizi, C. Galustian, AM Lawson and W. Chai, Nature Biotech ., 20 (2002) 1011) were co-trapped at the PDMS interface with luminol-loaded microbeads, was designed. In the following reactions, glucose oxidase catalyzes the oxidation of glucose and the production of hydrogen peroxide which reacts in the presence of a triggering agent (Z. Rao, X. Zhang and WRG Baeyens, Talanta, 57 (2002) 993) with the luminol immobilized to produce light. It has been shown previously that this configuration, with a different immobilization system, makes it possible to produce a sensitive biosensor network without the problem of contamination between the different points. Samples containing glucose at different concentrations were injected into the microplate and the intensity light produced was measured simultaneously on the entire microplate. The calibration curve obtained is presented in FIG. 4 (C). Again, the performance of the microplate of the invention appeared to be competitive, taking into account the sensitivity and the linear domain presented in various articles concerning the chemiluminescent detection of an enzyme substrate (T NC safack, CA Marquette, F. Pizzolato and LJ Blum, Biosens. & Bioelectron., 15 (2000) 125; LJ Blum, Bio- Chemi-Luminescent Sensors, World Scientific, Singapore, 1997; I. Moser, G. Jobst and GA Urban, Biosens. & Bioelectr. , 17 (2002) 297 and CA Marquette and LJ Blum, Anal. Chi. Acta, 381 (1999) 1). In addition, in a previous study, it was demonstrated that it was possible to use six different oxidase enzymes at the same time under the same experimental conditions, which makes it possible to obtain a biosensor with six parameters. The present invention is also applicable for the design of a PDMS-based enzyme substrate biochip for the detection of multiple parameters.

Claims

CLAIMS 1 - Method for manufacturing an elastomer microplate having two large faces, one of which, called the active face, carries, on localized sites, at least one type of reactive ligands, comprising the following successive steps: a) deposit on an imprint intended to serve as a countermold for the large active face, according to localized sites, an aqueous solution containing: - an organic solvent whose boiling point and viscosity are higher than those of water, - a salt, capable of co-crystallizing with the organic solvent during step b) later, and - micrometric particles on the surface or inside of which reactive ligands are immobilized; b) evaporating the aqueous solution, at a temperature T sufficient but lower than the boiling temperature of the organic solvent and the degradation temperature of the reactive ligands, so as to obtain on the localized sites a co-crystallization of the organic solvent and the salt around micrometric particles; c) polymerizing the elastomer on the impression serving as a countermold for the microplate, so that it comes to cover the localized sites and conform to the shape of the impression; d) unmolding the elastomer microplate obtained, the micrometric particles being partially trapped on the active face of the microplate, at the localized sites, at least part of the reactive ligands being accessible for a subsequent reaction. 2 - Process according to claim 1 characterized in that the elastomer is polydimethylsiloxane (PDMS). 3 - Method according to claim 1 or 2, characterized in that in step a), the solvent used has a boiling temperature above 150 ° C and a Brookfield viscosity at 20 ° C greater than 100 cps. 4 - Method according to one of claims 1 to 3, characterized in that in step a), glycerol is used as organic solvent and sodium chloride (NaCl) as salt, preferably in a glycerol / NaCl molar ratio of between 0.05 and 0.1. 5 - Method according to one of claims 1 to 4, characterized in that the micrometric particles used have an average diameter between 1 and 150 microns. 6 - Method according to one of claims 1 to 5, characterized in that the micrometric particles used are made of a polymer, preferably latex. 7 - Method according to one of claims 1 to 6, characterized in that the imprint used is flat. 8 - Method according to one of claims 1 to 6, characterized in that the imprint used is non-planar, so as to give the reactive surface of the microplate a relief, for example formed of wells and / or channels. 9 - Method according to one of claims 1 to 8, characterized in that at least one localized site carries reactive ligands different from those present on the other sites. 10 - Method according to one of claims 1 to 9, characterized in that the reactive ligands are biological ligands. 11 - Process according to claim 10, characterized in that the biological ligands used are chosen from polynucleotides, antigens, antibodies, polypeptides, proteins, haptens and active living cells. 12 - Microplate having two large faces, one of which, called the active face, carries, on localized sites, reactive ligands accessible for subsequent reactions, characterized in that the microplate consists essentially of an elastomer which partially traps , at the active face, micrometric particles on the surface or inside which the reactive ligands are immobilized. 13 - Microplate according to claim 12, characterized in that, characterized in that the elastomer is polydimethylsiloxane (PDMS). 14 - Microplate according to claim 12 or 13, characterized in that, for the majority, the micrometric particles emerge, from the active face, at least 10% of their diameter. 15 - Microplate according to one of claims 12 to 14, characterized in that the micrometric particles have an average diameter between 1 and 150 microns. 16 - Microplate according to one of claims 12 to 15, characterized in that the active face has a shaped relief, in particular, of wells and / or channels. 17 - Microplate according to one of claims 12 to 16, characterized in that at least one localized site carries reactive ligands different from those present on the other sites. 18 - Microplate according to one of claims 12 to 17, characterized in that the reactive ligands are biological ligands. 19 - Microplate according to claim 18, characterized in that the biological ligands are chosen from polynucleotides, antigens, antibodies, polypeptides, proteins, haptens or active living cells.